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https://openalex.org/W3006188116	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/EA7EBAE82DB878D83316F100BD41C883/S0884291420000217a.pdf/div-class-title-microstructure-study-of-u-35-wt-zr-alloy-after-quick-annealing-at-650-c-div.pdf	English	null	Microstructure study of U–35 wt.% Zr alloy after quick annealing at 650 °C	Journal of materials research/Pratt's guide to venture capital sources	2,020	cc-by	6,338	ª Materials Research Society 2020. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Microstructure study of U–35 wt.% Zr alloy after quick annealing at 650 °C Cullison1, Cheng Zhen1, Jeshua J. Olson-Gross1, Yi Xie2, Michael T. Benson2 1 ) y 1School of Nuclear Science and Engineering, Oregon State University, Corvallis, Oregon 97333, USA 2Department of Advanced Fuel Manufacturing and Development, Idaho National Laboratory, Idaho Falls, Idaho 83402, USA a)Address all correspondence to this author. e-mail: Tianyi.chen@oregonstate.edu This paper has been selected as an Invited Feature Paper. Received: 15 October 2019; accepted: 6 January 2020 Received: 15 October 2019; accepted: 6 January 2020 Received: 15 October 2019; accepted: 6 January 2020 Uranium–35 wt.% zirconium (U–35 wt.% Zr) alloy was annealed for 1 h and 24 h at 650 °C and characterized to understand the early-stage microstructure evolution. Dendritic microstructure with ﬁne (∼300 nm in length) a-U precipitates clustered between dendrite branches were observed in the 1-h annealed sample. After 24-h annealing at 650 °C, the a-U precipitates coarsened, and the dendritic microstructure disappeared because of microstructure homogenization. Furthermore, microchemical homogenization observed with energy-dispersive X-ray spectroscopy analysis suggests that a-U precipitates are approaching thermodynamic equilibrium in the 24-h annealed sample. The ﬁndings from this study have potential impacts on the manufacturing and computer modeling of metallic nuclear fuel. Dr. Chen is an assistant professor at the School of Nuclear Science and Engineering, Oregon State University. He has a wide range of research interests in nuclear materials and radiation effects, including the radiation tolerance of advanced alloys, ion irradiation as a surrogate for neutron irradiation, materials deformation mechanisms, and fuel-cladding chemical interactions. His studies cross the boundaries between radiation science, materials science, and mechanics and materials. Before joining the Oregon State University, Dr. Chen worked at several national laboratories including INL, ANL, and ORNL. Tianyi Chen proaches e pushed h region ation for . Firstly, ions are % Zr fuel h U–Zr such as Reactor llurgical rication, e.org/JMR 1095 j Journal of Materials Research j Volume 35 j Issue 8 j Apr 28, 2020 j www.mrs.org/jmr j Journal of Materials Research j Volume 35 j Issue 8 j Apr 28, 2020 j www.mrs.org/jmr https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Press EARLY CAREER SCHOLARS IN MATERIALS SCIENCE ANNUAL ISSUE Invited Feature Paper DOI: 10.1557/jmr.2020.21 Introduction The bright contrast of the a- Figure 1: U–Zr binary phase diagram replotted from Ref. 4. The blue dots represent prior microstructure study temperatures and compositions [8, 9, 12]. The red triangle represents this study’s annealing conditions. Figure 2: SEM-BSE micrographs of samples electrolytic polished at (a) roo temperature and (b) 20 °C. Invited Feature Pape Figure 1: U–Zr binary phase diagram replotted from Ref. 4. The blue dots represent prior microstructure study temperatures and compositions [8, 9, 12]. The red triangle represents this study’s annealing conditions. Invited Feature Paper Figure 2: SEM-BSE micrographs of samples electrolytic polished at (a) room temperature and (b) 20 °C. Figure 2: SEM-BSE micrographs of samples electrolytic polished at (a) room temperature and (b) 20 °C. the effects of electrolytic temperature in scanning electron microscopy (SEM) images. Three contrast initially seen in backscatter electron (BSE) micrographs were bright precip- itates, dark features, and a gray matrix. Bright precipitates and gray matrix in BSE micrographs along with the phase diagram in Fig. 1 qualitatively suggest that a-U phase precipitated out of a d-UZr2 or c phase matrix. The bright contrast of the a-U precipitated in BSE micrographs come from electrons being backscattered by the high Z–U atoms. Pits left behind from etched precipitates display as dark features in Fig. 2(a) after electrolytic polishing at room temperature. With all other conditions held the same, room temperature electrolytic polish- ing etched about half the a-U precipitates, whereas cold (20 °C) electrolytic polishing preserved a-U precipitates as seen in Fig. 2(b). Additionally, cold electrolytic polishing achieved minimal scratching and pitting while removing re- sidual oxidation. The rest of this study uses cold electrolytic polished samples for consistency. More sample polishing details are elaborated in the methods section. Figure 1: U–Zr binary phase diagram replotted from Ref. 4. The blue dots represent prior microstructure study temperatures and compositions [8, 9, 12]. The red triangle represents this study’s annealing conditions. Accurate phase equilibria determination uncertainties re- main [6]. This is especially true for the Zr-rich (aU, d), (aU, c), and (bU, c) boundaries. As an example, Ahn pointed out several discrepancies between experimental measurements and the existing phase diagram while studying the solid phase transformation behavior in the (bU, c) phase using differential scanning calorimetry and thermogravimetric analysis [7]. One of the many solid phase boundary determination challenges include intrinsic sluggish phase transformation kinetics pre- venting U–Zr alloy phase equilibrium. Introduction One U–35 wt.% Zr alloy sluggish kinetics example includes Basak [10] ﬁnding zirco- nium supersaturation in a-U precipitates after 500-h soaking at 630 °C and 72-h annealing at 600 °C. Earlier studies on metallic U–Zr sample preparation have not accounted for electrolytic polishing temperature’s signiﬁ- cant impact on sample preparation quality [17]. An electro- polishing potential of 3–4 V versus traditional 20–30 V [18] is suggested by Kelly [17] to prevent etching or pitting around inclusions. Suggested electrolytic polishing removed sample oxide layers and scratches typically found after sample storage and mechanical polishing. However, ﬁne a-U precipitate removal observed after room temperature electrolytic polishing suggests preferential a-U etching. The low sample dissolution rate associated with lower electrolytic polishing potential voltage led to nonsaturated sample surface uranium cation concentration. Under this scenario, the electrochemical re- action is chemical reaction dominate rather than mass transfer dominate [19]. A higher reaction rate of a-U phase over the matrix phase is expected because of a higher concentration of the more-active element uranium in a-U. Surface cation supersaturation and mass-transfer controlled mechanisms are favored by reducing the environment temperature from room temperature to 20 °C where the saturation concentration and In this study, we investigated a U-35 wt.% Zr alloy annealed at 650 °C which is in the fuel operation temperature range of sodium-cooled faster reactor. Early-stage microstruc- ture and chemical evolution from the as-cast conditions were assessed with short 1-h and 24-h annealing times. Besides providing scientiﬁc insights, this study also addresses the re- search need in understanding the homogenization process for cast metallic fuels [13, 14, 15, 16]. Microstructural evolution, microchemical evolution, and phase transformation from the as-cast condition with shorter annealing times than previously done by other researchers are investigated. https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Press https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Pre Introduction reactor fuels. Recent experimental and theoretical approaches by Basak, Ahn, Irukuvarghula, Xiong, and Bagchi have pushed the compositional boundary toward the zirconium-rich region up to U–50 wt.% Zr [6, 7, 8, 9, 10, 11]. The motivation for studying these zirconium-rich compositions is twofold. Firstly, uranium-lean (as much as U–50 wt.% Zr) compositions are found in the center region of high burnup U–10 wt.% Zr fuel rods. Secondly, uranium-lean and zirconium-rich U–Zr alloys are considered for advanced nuclear reactors such as the Korean subcritical Hybrid Power Extraction Reactor (HYPER) [12]. U–Zr alloy microstructure and metallurgical behavior are critical for fuel performance codes, fabrication, and core design. reactor fuels. Recent experimental and theoretical approaches by Basak, Ahn, Irukuvarghula, Xiong, and Bagchi have pushed the compositional boundary toward the zirconium-rich region up to U–50 wt.% Zr [6, 7, 8, 9, 10, 11]. The motivation for studying these zirconium-rich compositions is twofold. Firstly, uranium-lean (as much as U–50 wt.% Zr) compositions are found in the center region of high burnup U–10 wt.% Zr fuel rods. Secondly, uranium-lean and zirconium-rich U–Zr alloys are considered for advanced nuclear reactors such as the Korean subcritical Hybrid Power Extraction Reactor (HYPER) [12]. U–Zr alloy microstructure and metallurgical behavior are critical for fuel performance codes, fabrication, and core design. Metallic nuclear fuels such as U–Zr and U–Pu–Zr alloys have several advantages over ceramic fuels, such as superior heat conductivity, large prompt negative temperature coefﬁcient of reactivity, and better safety margins [1]. Historically, U–Zr alloy fuels were used in several types of test and research reactors including the Experimental Breeder Reactor-II. The research interests in the U–Zr binary alloy also come from the candidacy of U–Pu–Zr ternary alloys for future fast spectrum reactors [2, 3, 4]. Within the scope of U–Zr binary system whose equilibrium phase diagram is presented in Fig. 1 [5], most of the research efforts have been placed in the uranium- rich compositions driven by the interests in metallic ﬁssion- https://doi.org/10.1557/jmr.2020.21 Published online by Cambridg cambridge.org/JM 1095 the effects of electrolytic temperature in scanning electro microscopy (SEM) images. Three contrast initially seen i backscatter electron (BSE) micrographs were bright precip itates, dark features, and a gray matrix. Bright precipitates an gray matrix in BSE micrographs along with the phase diagram in Fig. 1 qualitatively suggest that a-U phase precipitated out o a d-UZr2 or c phase matrix. Results and discussion Quality sample surface ﬁnish was achieved by electrolytic polishing to allow reliable microstructure characterization. Samples electrolytically polished at room temperature and 20 °C, respectively, shown in Figs. 2(a) and 2(b) highlight cambridge.org/JMR 1096 cambridge.org/JMR ª Materials Research Society 2020 Figure 3: XRD patterns and reﬁnement for the 24-h annealed sample. Invited Feature Paper Invited Feature Paper diffusion coefﬁcient of uranium cations are lower. The resulted diffusion-limited electrolytic polishing [20] minimizes the re- action rate difference between a-U precipitates and matrix. Fine a-U precipitate preservation achieved is presented in Fig. 2(b). An X-ray diffraction (XRD) pattern for the 24-h annealed sample presented in Fig. 3 identiﬁes and labels characteristic peaks for the a, c, and d phases. The residual proﬁle in the bottom of Fig. 3 illustrates good ﬁtting with Rietveld re- ﬁnement for all the three phases. The weighted proﬁle residual between the calculated and experimental pattern is 15.2%, with an expected proﬁle residual of 11.1%, determines an acceptable goodness of ﬁt of 1.9 [21]. The three-phase weight fractions estimated following the Hill and Howard method [22] are listed in Table I. Figure 3: XRD patterns and reﬁnement for the 24-h annealed sample. It is known that Rietveld reﬁnement of XRD patterns provides a semiquantitative analysis of the phase fractions. The small fraction of a-U phase precipitates qualitatively agrees to the phase diagram shown in Fig. 1 and the microstructural analysis presented later in this article. Most of the matrix phase being d rather than c suggests phase transformation took place during sample cooling after annealing at 650 °C. During the phase transformation from c to d, a metastable x phase has been reported by Irukuvarghula when the sample was quenched from elevated temperatures [11]. The x phase has the same chemical composition with the d phase but with a disordered hexagonal structure. To distinguish x from d phase via XRD is challenging because of their structural and chemical similarities. In this study, the samples were cooled through nature convention inside of a glovebox with a rela- tively slow cooling rate. Thus, it is reasonable to believe that more ordered d phase formed through atomic diffusion compared to Irukuvarghula’s study [11]. Recent studies using transmission electron microscopy (TEM) also evi- denced the formation of d phase in a more rapid fashion than traditionally believed [2]. ª Materials Research Society 2020 Results and discussion The microstructure evolution reported herein shows that the dendrites can be annealed out in U–35 wt.% Zr system at 650 °C for about 24 h, suggesting that annealing within the temperature range of (aU, c) may be an effective strategy to homogenize the as-cast dendritic microstructures in U–Zr alloys. changing the area fraction was found by means of machine learning–based microstructure analysis. Sample elemental analysis was performed with energy dispersive X-ray spectroscopy (EDS). Sample EDS line scans for 1-h and 24-h annealed samples are presented in Fig. 6. Uranium Ma (3.171 kV), La (13.614 keV), zirconium La (2.044 keV), and Ka (15.775 keV) characteristic X-rays were excited and used for elemental quantiﬁcation. a-U clusters had more average uranium content than the dendrite branches evident in Fig. 6(a) a-U precipitate clusters have a U-rich center with U- lean compositions on the fringe. A wider range of Zr composi- tion was observed in the 1-h annealed sample as seen in Fig. 6(a) as compared with the 24-h annealed sample presented in Fig. 6(b). Matrix compositions are found to be U–28 6 3 wt.% Zr and U–29 6 2 wt.% Zr for 1-h and 24-h annealed samples, respectively. Improved local chemical homogeneity with annealing was indicated in the 24-h annealed sample by the smaller Zr composition standard deviation in the matrix phase. Figure 5 presents BSE micrographs obtained at medium magniﬁcations (3000–4000) in (a and b), with their contain- ing a-U phase identiﬁed by machine learning and presented in (c and d). Trainable Weka Segmentation [27] machine learning was used and is elaborated more in the Methods section. Machine learning micrograph magniﬁcation played a signiﬁ- cant role in precipitate recognition and was optimized. Machine learning classiﬁcation improved with high magniﬁ- cation; however, sample heterogeneity makes use of a small area associated with high magniﬁcation impractical for capturing bulk properties. Low-magniﬁcation micrograph machine learning errors caused by ﬁner precipitates missed are because of low image resolution. High-magniﬁcation micrographs decrease the machine learning phase identiﬁca- tion errors with the same image resolution because of precipitates having more pixels; however, missed larger features impact results signiﬁcantly. The area fractions of a-U phase in U–35 wt.% Zr 1-h and 24-h annealed samples are 12 6 2 % and 13 6 1%, respectively. Precipitate sizes were assessed by measuring more than 200 precipitates for each sample. Results and discussion Dendrite Zr compositions as high as 45 wt.% Zr are expected, a composition so rich that it is in the single c/d-UZr2 phase indicated by red dashed lines in Fig. 1. Molten U-rich alloy pockets trapped between dendrite branches become uranium rich. Secondly, when the sample cooled down, the decomposition of the high- temperature c phase primarily occurred within the interden- dritic region with uranium supersaturation, leading to a high local density of a-U precipitates seen in Figs. 2(b) and 4(b). The dendrites remain precipitate free because they remain in a single-phase solid solution. a-U precipitates heavily decorate grain boundaries because grain boundaries are the last molten region during solidiﬁcation. As shown in Fig. 1, the melting temperature of U–Zr system increases with Zr concentration, so the solidiﬁcation initialized with a higher Zr concentration in the solid phase than the liquid. Compositional gradients were observed with Zr-rich dendrite cores and Zr-lean dendrite boundaries. Dendrite Zr compositions as high as 45 wt.% Zr are expected, a composition so rich that it is in the single c/d-UZr2 phase indicated by red dashed lines in Fig. 1. Molten U-rich alloy pockets trapped between dendrite branches become uranium rich. Secondly, when the sample cooled down, the decomposition of the high- temperature c phase primarily occurred within the interden- dritic region with uranium supersaturation, leading to a high local density of a-U precipitates seen in Figs. 2(b) and 4(b). The dendrites remain precipitate free because they remain in a single-phase solid solution. a-U precipitates heavily decorate grain boundaries because grain boundaries are the last molten region during solidiﬁcation. Figure 5: Original SEM-BES micrographs of U–35 wt.% Zr annealed for (a) 1 h and (b) 24 h. Machine learning–based precipitate recognition of U–35 wt.% Zr annealed for (c) 1 h and (d) 24 h. Trainable Weka Segmentation [27] classiﬁed a-U precipitates as black and c/d-UZr2 as white. Figure 5: Original SEM-BES micrographs of U–35 wt.% Zr annealed for (a) 1 h and (b) 24 h. Machine learning–based precipitate recognition of U–35 wt.% Zr annealed for (c) 1 h and (d) 24 h. Trainable Weka Segmentation [27] classiﬁed a-U precipitates as black and c/d-UZr2 as white. Recently, dendrite formation in cast alloys of uranium– zirconium [23], uranium–zirconium–niobium [13, 14], and uranium–molybdenum [15, 16, 24, 25, 26] has been reported to prevent microstructural homogenizition after thermomechan- ical treatments such as hot rolling [13]. Results and discussion TABLE I: Rietveld reﬁnement results for the XRD data on the sample annealed at 650 °C for 24 h. TABLE I: Rietveld reﬁnement results for the XRD data on the sample annealed at 650 °C for 24 h. Fitting parameters Phase fractions (wt.%) Expected proﬁle residual 11.1% a 19.4 Weighted proﬁle residual 15.2% c 6.3 Goodness of ﬁt 1.9 d 74.2 Figure 4: Microstructure of the 1-h annealed sample in (a) optical micro- graph and (b) SEM-BSE micrograph; and 24-h annealed sample in (c) optical micrograph and (d) SEM-BSE micrograph. j Journal of Materials Research j Volume 35 j Issue 8 j Apr 28, 2020 j www.mrs.org/jmr Figure 4 presents the optical and SEM images for the samples annealed for 1 h in Figs. 4(a) and 4(b) and 24-h in Figs. 4(c) and 4(d). As seen in Fig. 4(a), the 1-h annealed sample displays a black dendritic microstructure characteristic of the solidiﬁcation process. The comparison between the op- tical micrograph [Fig. 4(a)] and the BSE image [Fig. 4(b)] shows that the dendrites are precipitate free while high-density a-U precipitate clusters between dendrite branches. Most a-U precipitates have an elongated cross-sectional shape, with some of them showing a polygonal shape as seen in Fig. 4(b) inset. The 3-dimensional morphology of these precipitates is thus implied to be either needle-like or disc-like. After 24 h of annealing, the microstructure was homogenized as presented in Figs. 4(c) and 4(d). The dendritic microstructure disappeared with the a-U precipitates coarsened considerably. https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Press https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Pres Figure 4: Microstructure of the 1-h annealed sample in (a) optical micro- graph and (b) SEM-BSE micrograph; and 24-h annealed sample in (c) optical micrograph and (d) SEM-BSE micrograph. Such complex U–Zr dendritic microstructure as seen in Fig. 4 has not been reported by previous studies whose focuses have been on the near-equilibrium conditions [10]. The complex microstructure formed through a two-step mecha- nism. Firstly, microsegregation took place during solidiﬁcation. cambridge.org/JMR 1097 ª Materials Research Society 2020 Invited Feature Paper As shown in Fig. 1, the melting temperature of U–Zr system increases with Zr concentration, so the solidiﬁcation initialized with a higher Zr concentration in the solid phase than the liquid. Compositional gradients were observed with Zr-rich dendrite cores and Zr-lean dendrite boundaries. ª Materials Research Society 2020 Results and discussion The average lengths were 0.3 6 0.2 lm and 1.1 6 0.7 lm for 1-h and 24-h annealed samples, respectively. Area fraction cannot determine volume fraction [28]; however, a-U precipitates coarsened with annealing without signiﬁcantly https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Press https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Pres Although the nominal composition of the samples is U–35 Zr wt.%, the measured Zr contents in the EDS line scans presented in Fig. 6 are systematically lower than 35 wt.%. Besides the experimental error from EDS qualiﬁcation, there are two possible explanations to the lower measured Zr content. Firstly, the impurity-stabilized a-Zr precipitates formed during the sample casting deplete Zr from the rest of the materials. Impurity-stabilized a-Zr is inevitable and often observed in uranium–zirconium metallic alloys [29]. In this study, the a-Zr inclusions are observed as the black features in BSE images in Figs. 5(a) and 5(c). A recent study by Hirsch- horn analyzing the chemical composition of U–Zr alloys using mass spectroscopy and EDS showed that the Zr content https://doi.org/10.1557/jmr.2020.21 Published online by Cambrid cambridge.org/JMR 1098 ª Materials Research Society 2020 Invited Feature Paper Figure 6: (a) SEM-BSE micrograph and (b) EDS line scan results of the 1-h annealed sample. (c) SEM micrograph and (d) EDS line scan results of the 24-h annealed sample. The arrows in SEM micrographs indicate line scan distance and direction. Gray semitransparent boxes in line scan results indicate a-U regions in the line scans. Figure 6: (a) SEM-BSE micrograph and (b) EDS line scan results of the 1-h annealed sample. (c) SEM micrograph and (d) EDS line scan results of the 24-h annealed sample. The arrows in SEM micrographs indicate line scan distance and direction. Gray semitransparent boxes in line scan results indicate a-U regions in the line scans. micrograph and (b) EDS line scan results of the 1-h annealed sample. (c) SEM micrograph and (d) EDS line scan results of the 24-h anneale EM micrographs indicate line scan distance and direction. Gray semitransparent boxes in line scan results indicate a-U regions in the lin wt.% of Zr determined by EDS with different electron energies on a a-U particle of 786 nm in width. The measured Zr content decreases with the electron energy. ª Materials Research Society 2020 Results and discussion To further illustrate the effect of electron energy, electron scattering Monte Carlo simulation [30] was conducted to present the theoretical activation volumes of 10 and 20 keV electrons in a-U phase in Figs. 7(c) and 7(d). Figures 7(e) and 7(f) provide two examples of EDS patterns obtained from the same particle of submicron width with 10 and 20 keV electron energies, respectively. The Zr characteristic X- ray is negligible for 10 keV electrons but signiﬁcant for 20 keV electrons. Considering the dependency of activation volume on electron energies, it is suggested that EDS composition measure- ment of a small particle with a lower electron energy tends to provide more accuracy because of the reduced inﬂuence from the surrounding matrix. For this study, an electron energy #10 keV is recommended. measured by EDS is lower by .4 at.% because of the impurity- stabilized Zr inclusions not being counted by EDS [29]. Secondly, the small samples received were sectioned from a pin alloy of the U-35 wt.% Zr composition before being remelted. The sectioned samples were not subjected to chemical analysis to determine its composition before this work. Although nuclear fuel pins fabricated using the same method at Idaho National Laboratory generally show very good agreement between the nominal and real chemical compositions, it is possible that the small section of the pin alloy has a composition that is slightly off the alloy composition because of the inhomogeneity in the pin alloy. Nevertheless, the limited difference in chemical composition between the measured and nominal values is tolerable when comparisons were made between samples of the same material with different annealing times to show the short-term micro- structural and microchemical evolution. https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Press https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Pre https://doi.org/10.1557/jmr.2020.21 Published online by Camb Figure 7(b) shows the wt.% of Zr in a-U particles of different sizes determined by EDS with #10 keV electrons. A trend of decreasing Zr content with increasing particle size is observed. It is believed that measurements from larger particles better reﬂect the true composition of the a-U phase because the The chemical composition of the a-U precipitates was measured using EDS technique and presented in Fig. 7. The accuracy in composition determination as inﬂuenced by the incident electron energy and particle size is investigated by separately controlling these two variables. Results and discussion j Journal of Materials Research j Volume 35 j Issue 8 j Apr 28, 2020 j www.mrs.org/jmr https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Press https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Pre The EDS-decided composition for a-U precipitates in this study qualitatively agrees with the equilibrium value (;0.5 wt.%) at 650 °C. Comparing with the results from Basak [10], who observed a-U precipitates having as much as 11.6 wt.% of supersaturated Zr in U–35 wt.% Zr alloy after thermal annealing at 630 °C for 500 h followed by 240 h at 600 °C, the result herein indicates that a thermodynamic equilibrium can be approached, if not reached, in U–35 wt.% Zr within 24 h at 650 °C. Basak ascribed the supersaturation of Zr in a-U phase to the sluggish reaction kinetics associated with local thermodynamic equilibrium conditions within the (aU, d) temperature range [10]. The sluggish kinetics has also ª Materials Research Society 2020 Results and discussion Figure 7(a) shows the cambridge.org/JMR 1099 Figure 7: (a) The Zr content decided by EDS point scan as a function of electron energy in an a-U precipitate of 786 nm in width. (b) The mean Zr content as a function of a-U particle width using #10 keV electron beams. (c, d) Monte–Carlo simulated activation volumes in a-U phase of 10 and 20 keV electrons, respectively. (e, f) EDS spectra of a submicron a-U particle obtained with 10 and 20 keV electron beams, respectively. Invited Feature Paper Invited Feature Paper Figure 7: (a) The Zr content decided by EDS point scan as a function of electron energy in an a-U precipitate of 786 nm in width. (b) The mean Zr content as a function of a-U particle width using #10 keV electron beams. (c, d) Monte–Carlo simulated activation volumes in a-U phase of 10 and 20 keV electrons, respectively. (e, f) EDS spectra of a submicron a-U particle obtained with 10 and 20 keV electron beams, respectively. been suggested by the absence of peaks from d phase in XRD patterns in some 15–20 wt.% Zr alloys, as reviewed in Ref. 2. However, recent investigations using TEM showed that the nucleation of d phase is actually fast [2]. Without TEM analysis, this study suggests a nonsluggish reaction at 650 °C in reaching the equilibrium composition of the a-U phase. The signiﬁcant coarsening of a-U precipitates during the course of annealing is an evidence of sufﬁcient diffusion. been suggested by the absence of peaks from d phase in XRD patterns in some 15–20 wt.% Zr alloys, as reviewed in Ref. 2. However, recent investigations using TEM showed that the nucleation of d phase is actually fast [2]. Without TEM analysis, this study suggests a nonsluggish reaction at 650 °C in reaching the equilibrium composition of the a-U phase. The signiﬁcant coarsening of a-U precipitates during the course of annealing is an evidence of sufﬁcient diffusion. activation volume of X-rays is more likely to be within the particle domains. Based on the trends shown in Figs. 7(a) and 7(b), the composition of the a-U phase was estimated by averaging multiple EDS point scans collected from particles with .1 lm using #10 keV electron energy. The Zr content in a-U precipitates in the 24-h annealed sample is determined accordingly to be 1.5 6 0.6 wt.%. Conclusion In this study, we investigated the crystallography, microstruc- ture, and microchemistry for U–35 wt.% Zr alloys annealed at 650 °C for 1 h and 24 h. A modiﬁed electrolytic chemical polishing method was applied to the samples to preserve the ﬁne a-U precipitates and reduce the pitting effects, while maintaining the surface smoothness, and remove sample surface oxidation. Two-phase microstructure of a-U and the matrix phase in both the 1-h and 24-h annealed samples was cambridge.org/JMR 1100 ª Materials Research Society 2020 Invited Feature Paper using a pyrite standard to have a nominal accuracy of 61.5% absolute maximum. EDS scans were performed with electron beam energies no higher than 10 kV to excite the M uranium and L zirconium characteristic X-rays while maintaining a min- imal excitation volume for maximum accuracy. Automatic background subtraction was applied to the EDS spectra followed by the Z-factor, Absorption factor, Fluoresce factor (ZAF) method to quantify the EDS results [31]. ZAF method uses Z- factor, absorption factor, and ﬂuorescence factor to adjust and correct for atomic number effects, X-ray absorption, and X-ray ﬂuorescence, respectively. Simulating activation volume for EDS was done with EISS-Electron Scattering Monte Carlo Simulation Software [30]. One thousand trajectories were simulated with different beam energies for uranium and zirconium. revealed by XRD, SEM, and EDS analysis. The uranium-rich precipitate phase was identiﬁed as a-U, whereas the matrix was identiﬁed to be d and c phases using Rietveld reﬁnement on XRD patterns. Dendritic microstructure in the 1-h annealed sample remnant from as-cast was observed. The dendrites were slightly Zr rich without a-U precipitation, and the interdendritic regions were U rich with high density of a-U precipitates. After a short annealing of 24 h at 650 °C, the microstructure was ho- mogenized accompanied by the coarsening of a-U precipitates and disappearance of the dendrites. The 24-h annealed sample showed a more homogeneous matrix chemical composition than the 1-h annealed sample. Careful EDS-based analysis of the a-U precipitates suggested a thermal equilibrium chemical composi- tion of these precipitates after 24 h of annealing at 650 °C. XRD was used to obtain crystallographic information of the samples. The XRD spectra were collected using a Bruker-AXS D8 Discover instrument with copper K-a1 X-rays. The scans were done with a step size of 0.01°, step dwelling time of 2 s, and within the 15°–80° 2h range. Methods U–35 wt.% Zr alloy samples were melt-casted from high-purity depleted uranium and zirconium using an arc-melting furnace. The materials were ﬂipped three times and remelted to ensure homogeneity. The samples were casted into 10-g buttons in a ceramic crucible and then cooled by natural convection in an argon-ﬁlled glove box. After that, the samples were wrapped with a Ta foil, placed inside an argon atmosphere furnace and annealed at 650 °C for 1 h and 24 h separately, and cooled again by natural convection in the glove box. The glove box contains less than 10 ppm of oxygen and water. Before sample characterization, standard metallographic preparations were adopted, and samples were mechanically polished to a mir- ror-like ﬁnish with 0.05-lm alumina suspension. Rietveld reﬁnement was conducted to obtain more in- formation from XRD result. Based on the reﬁnement results, the weight fractions of crystalline phases are calculated using the method proposed by Hill and Howard [21, 22]: Wk ¼ Sk ZMV ð Þk P n i¼1 Si ZMV ð Þi ; Wk ¼ Sk ZMV ð Þk P n i¼1 Si ZMV ð Þi Surface oxides and ﬁne scratches were removed from the samples by using electrolytic polishing technique. Recommen- dations of Kelly et al. [17] were followed with a modiﬁed electrolytic polishing temperature of 20 °C instead of room temperature. Electrolytic polishing conditions were 10% H3PO4 (phosphoric acid) methanol solution electrolyte, a 316 stainless steel cathode, the sample as an anode, and 4 V direct current, for 2–5 s. Before and after electrolytic polishing, the sample surface quality was examined using an IM-5000 metallurgical microscope to examine the removal of oxidation and scratches. Oxidation was minimized by analyzing samples within 30 min of being polished. When not used, the samples were stored in a vacuumed desiccator at 20 kPa gauge. where Wk is the weight fraction of phase k, S is the Rietveld scale factor, Z is the number of formula units per unit cell, M is the mass of the formula unit, and V is the unit cell volume. j Journal of Materials Research j Volume 35 j Issue 8 j Apr 28, 2020 j www.mrs.org/jmr https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Press Conclusion The X-ray illuminated area is around 0.6 mm2, which is enough to provide representative results, given the level of microstructural inhomogeneity presented in Fig. 4. The X-ray penetration depth was estimated to be 8 lm for a normal incident beam based on the attenuation of electromagnetic radiation in U–35 wt.% Zr. ª Materials Research Society 2020 Acknowledgments https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Pres This work was supported by Oregon State University startup funding and the U.S. Department of Energy, Ofﬁce of Nuclear Energy under DOE Idaho Operations Ofﬁce Contract DE-AC07-05ID14517. Accordingly, the U.S. Government retains and the publisher, by accepting the article for publica- tion, acknowledges that the U.S. Government retains a non- exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript or allow others to do so, for U.S. Government purposes. Microstructure characterization was conducted with an FEI QUANTA 600F SEM and an FEI QUANTA 3D SEM using backscattered electron (BSE) imaging and EDS. Elemental composition line scans were done with an EDAX EDS system ﬁtted to the FEI QUANTA 600F SEM. Elemental composition point scans were done with an EDAX EDS system ﬁtted to the FEI QUANTA 3D. The EDS systems were calibrated with standard samples including uranium. The accuracy was checked Special thanks to Peter Eschbach, Teresa Sawyer for pro- fessional and expert training on SEM, and Brady Gibbons and Kyle Grove for assistance on XRD. We acknowledge OSU Electron Microscopy Facility for the access to SEM, and X-ray cambridge.org/JMR 1101 ª Materials Research Society 2020 Invited Feature Paper Diffraction Facility for the access to XRD. MHC thanks Jill Josselyn for the ARCS award. CZ acknowledges Oregon State University Provost’s Distinguished Graduate Fellowship for ﬁnancial support. mechanical properties, microstructure and corrosion behavior of U–10Mo monolithic fuel. J. Nucl. Mater. 527, 151804 (2019). 16. X. Hu, X. Wang, V.V. Joshi, and C.A. Lavender: The effect of thermomechanical processing on second phase particle redistribution in U–10 wt% Mo. J. Nucl. Mater. 500, 270 (2018). 17. A.M. Kelly, D.J. Thoma, R.D. Field, E. Cerreta, and R.E. Hackenberg: Metallographic preparation techniques for uranium/uranium alloys. Microsc. Microanal. 18, 454 (2012). References 33, 225 (2018 8. C. Basak, G.J. Prasad, H.S. Kamath, and N. Prabhu: An evaluation of the properties of As-cast U-rich U–Zr alloys. J. Alloys Compd. 480, 857 (2009). 25. S. Chakraborty, G. Choudhuri, J. Banerjee, R. Agarwal, K.B. Khan, and A. Kumar: Micro-structural study and Rietveld analysis of fast reactor fuels: U–Mo fuels. J. Nucl. Mater. 467, 618 (2015). 9. C.B. Basak, N. Prabhu, and M. Krishnan: On the formation mechanism of UZr2 phase. Intermetallics 18, 1707 (2010). 26. S. Jana, L. Sweet, D. Neal, A. Schemer-Kohrn, C. Lavender, and V. Joshi: The role of ternary alloying elements in eutectoid transformation of U10Mo alloy part I. Microstructure evolution during arc melting and subsequent homogenization annealing in U9.8Mo0.2X alloy (X 5 Cr, Ni, Co). J. Nucl. Mater. 509, 318 (2018). 10. C.B. Basak: Microstructural evaluation of U-rich U–Zr alloys under near-equilibrium condition. J. Nucl. Mater. 416, 280 (2011). 11. S. Irukuvarghula, S. Ahn, and S.M. McDeavitt: Decomposition of the c phase in as-cast and quenched U–Zr alloys. J. Nucl. Mater. 473, 206 (2016). 27. I. Arganda-Carreras, V. Kaynig, C. Rueden, K.W. Eliceiri, J. Schindelin, A. Cardona, and H. Sebastian Seung: Trainable weka segmentation: A machine learning tool for microscopy pixel classiﬁcation. Bioinformatics 33, 2424 (2017). https://doi.org/10.1557/jmr.2020.21 Published online by Cambridge University Press 12. W.S. Park, U. Shin, S-J. Han, T.Y. Song, B.H. Choi, and C.K. Park: HYPER (hybrid power extraction reactor): A system for clean nuclear energy. Nucl. Eng. Des. 199, 155 (2000). 28. Standard Test Method for Determining Volume Fraction by Systematic Manual Point Count. ASTM E562-08 (2008). 13. N.W.S. Morais, D.A. Lopes, and C.G. Schön: Effect of thermo- mechanical processing on microstructure and mechanical properties of U–Nb–Zr alloys: Part 1—U–6 wt% Nb–6 wt% Zr. J. Nucl. Mater. 488(Suppl. 3), 173 (2017). 29. J. Hirschhorn, M. Tonks, A. Aitkaliyeva, and C. Adkins: Reexamination of a U–Zr diffusion couple experiment using quantitative phase-ﬁeld modeling and sensitivity analysis. J. Nucl. Mater. 529, 151929 (2020). 14. N.W.S. Morais, D.A. Lopes, and C.G. Schön: Effect of thermo- mechanical processing on microstructure and mechanical properties of U–Nb–Zr alloys: Part 2—U–3 wt% Nb–9 wt% Zr and U–9 wt% Nb–3 wt% Zr. J. Nucl. Mater. 502, 51 (2018). 30. D.C. Joy: Monte Carlo Modeling for Electron Microscopy and Microanalysis (Oxford University Press, New York, 1995). 31. J.I. Goldstein, D.E. Newbury, J.R. Michael, N.W.M. Ritchie, J.H.J. Scott, and D.C. References 1. T. Sofu: A review of inherent safety characteristics of metal alloy sodium-cooled fast reactor fuel against postulated accidents. Nucl. Eng. Technol. 47, 227 (2015). 18. K.H. Eckelmeyer, A.D. Romig, and L.J. Weirick: The effect of quench rate on the microstructure, mechanical properties, and corrosion behavior of U–6 wt% Nb. Metall. Trans. A 15, 1319 (1984). 2. D.E. Janney, C.A. Papesch, D.E. Burkes, J.I. Cole, R.S. Fielding, S.M. Frank, T. Hartmann, T.A. Hyde, D.D. Keiser, Jr., J.R. Kennedy, A. Maddison, R.D. Mariani, S.C. Middlemas, T.P. O’Holleran, B.H. Sencer, and L.N. Squires: Metallic Fuels Handbook (Idaho National Laboratory, Idaho Falls, ID, United States) (2017). rrosion behavior of U–6 wt% Nb. Metall. Trans. A 15, 1319 (198 19. R. Fujita, M. Enda, and T. Morisue: REDOX decontamination technique development, (I). J. Nucl. Sci. Technol. 26, 339 (1989). 20. G. Yang, B. Wang, K. Tawﬁq, H. Wei, S. Zhou, and G. Chen: Electropolishing of surfaces: Theory and applications. Surf. 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Morgan: Thermodynamic modeling of the U–Zr system—A revisit. J. Nucl. Mater. 443, 331 (2013). 24. S. Chakraborty, G. Choudhuri, P.S. Somayajulu, R. Agarwal, and K.B. Khan: Microstructure characterization and phase ﬁeld analysis of dendritic crystal growth of c-U and BCC-Mo dendrite in U–33 at.% Mo fast reactor fuel. J. Mater. Res. 33, 225 (2018). 7. S. Ahn, S. Irukuvarghula, and S.M. McDeavitt: Thermophysical investigations of the uranium–zirconium alloy system. J. Alloys Compd. 611, 355 (2014). U–33 at.% Mo fast reactor fuel. J. Mater. Res. References Joy: Scanning Electron Microscopy and X- Ray Microanalysis (fourth edition) (Springer, New York, NY 2017). 15. R. Prabhakaran, L. Gardner, V. Joshi, D. Burkes, and C. Lavender: Effect of homogenization and hot rolling on the cambridge.org/JMR 1102 ª Materials Research Society 2020
https://openalex.org/W2795357452	https://juniperpublishers.com/rapsci/pdf/RAPSCI.MS.ID.555588.pdf	English	null	Thermal Treatment of Syrian Shot Coke	Recent advances in petrochemical science	2,017	cc-by	4,247	Shot Coke basketballs) are usually made up of a fused aggregation of uniformly-sized smaller spheres [1]. Shot coke is an abnormal form of delayed petroleum coke. Physically it can be comprised of: y p Physically it can be comprised of: Figure 1: Shot coke. a) Individual spheres, which are hard, dense, vitreous, nonporous spheres that may or may not be fractured (Figure 1). Normally, most shot coke spheres are uniformly sized (1-2mm dia or 2-6 mm), but they can range from as small as buckshot to as large as basket balls. It is thought that smaller spheres are made when very high feed rates are used in the coker. Larger spheres (up to 20 mm) and more mixed sizing tend to occur when a coker just begins to produce shot coke. Very large spheres (up to the size of b) Agglomerated or clustered spheres, where individual spheres may form an aggregate consisting of smaller individual spheres of 1mm to 10mm in size, bonded together somewhat loosely. An aggregate may range in size from a few millimeters to as much as 30 or more centimeters. Figure 1: Shot coke. c) Bonded spheres or matrix shot. This appears as spheres of coke that melted together on formation. When it fractures, it will fracture across the spheres, rather than at the boundaries of the spheres as in agglomerated shot. The structure of shot coke is described by the use of Montage microphotographs, comparing the structure to that of sponge coke. The photographs reveal the fine-mosaic, high-density structure of shot coke [2]. Figure 1: Shot coke. Figure 1: Shot coke. a) Individual spheres, which are hard, dense, vitreous, nonporous spheres that may or may not be fractured (Figure 1). Normally, most shot coke spheres are uniformly sized (1-2mm dia or 2-6 mm), but they can range from as small as buckshot to as large as basket balls. It is thought that smaller spheres are made when very high feed rates are used in the coker. Larger spheres (up to 20 mm) and more mixed sizing tend to occur when a coker just begins to produce shot coke. Very large spheres (up to the size of Shot coke, while it may look like it is entirely made up of shot, is not necessarily so. Abstract Shot coke is a by-product of the delayed coking process, and can represent a significant part of the petroleum coke produced. It is comprised in general of two basic types, namely regular shot coke and clustered or agglomerated coke. Shot coke is, however, of little or no commercial value, particularly if it has a high sulphur content, as is becoming increasingly the case in most oil refineries. Upgrading the coke and reducing its sulphur content would add considerably to its economic value both as a fuel and/or for making aluminium anodes. Towards this end, samples of Syrian shot coke, produced by the delayed coking unit at the Homs Oil Refinery, were thermally treated at high temperatures and increased residence time. A coke of higher quality was thereby obtained, with reduced sulphur content (1%) and higher real density (1.9g/ cm3). The observed weight loss was minimal, and the decrease in the calorific value was less than the average expected for other types of petroleum coke. Keywords: Petroleum coke; Shot coke; Thermal treatment Recent Adv Petrochem Sci Recent Adv Petrochem Sci Copyright © All rights are reserved by Hassasn Al-Haj Ibrahim Hassasn Al-Haj Ibrahim* Department of Chemical Engineering, Al-Baath University, Syria Department of Chemical Engineering, Al-Baath University, Syria Submission: April 25, 2017; Published: July 17, 2017 Department of Chemical Engineering, Al-Baath University, Submission: April 25, 2017; Published: July 17, 2017 Submission: April 25, 2017; Published: July 17, 2017 *Corresponding author: Hassasn Al-Haj Ibrahim, Department of Chemical Engineering, Al-Baath University, Homs, Syria Email: Recent Advances in Petrochemical Science ISSN: 2575-8578 Recent Advances in Petrochemical Science Recent Advances in Petrochemical Science Shot coke is normally found at lower levels in a coke drum but may, on occasion, be found in the upper section of the drum. Occasionally it may also appear on top of sponge coke [1]. coke and of coarse-grained mosaic (5.0–10.0μm dia.) and small domains (10-60μm dia.). Centrally located stress cracks are thought to be due to shrinkage of the core structure. Scanning electron microscopy photos of shot coke spheres which had been partially fractured show that the outer skin must be a discrete layer since it is fractured in a different pattern than the core [1-3]. The formation of shot coke is favored in most instances by a shorter delayed coking cycle, higher superficial velocity, low coke drum pressures, low recycle ratios and high coke drum temperatures. Such conditions lead to the increase of the asphaltene content, relative reactivity of the coker feedstock and immediate coke precursors, as well as the degree of turbulence in the coke drum. Fluidization in the coke drum may also cause shot coke formation, and a coker feedstock high in oxygen content can produce shot coke [3]. Other types of coke, notably sponge coke, may also have some embedded shot coke in the coke structure. Shot coke is usually produced from very heavy, low API, feedstocks that have lower levels of aromaticity and higher levels of asphaltenes and heteroatoms, i.e. nitrogen, sulphur and oxygen and other associated metals. Presumably there exists a sufficiently high content of aliphatic carbon and hydrogen and this could lead to significant volatile evolution within the coking drum [4]. Reducing the coke drum temperature can be an effective means of eliminating shot coke formation, but normally increases coke yield. The additional coke is produced from the internal recycle or heavy gas oil constituents which alone would produce a well-ordered coke; in combination they dilute shot coke forming constituents [1]. Shot coke is produced as the oil flows into the coke drum. With the light ends flashing off, small spherical droplets of heavy tar are suspended in the flow. These tar droplets rapidly form coke due to the exothermic heat produced by asphaltene polymerization. Their formation is caused by the shearing action of a highly turbulent dynamic environment which results from high fluid velocities and vaporisation of existent and/or cracked products. Recent Advances in Petrochemical Science Rapid chemical reactions which occur within the droplets while they are suspended in the vapour phase increase the viscosity of the developing ball of immature coke. Before the droplets can return back to and become an integral part of the bulk liquid phase the viscosity has increased sufficiently to “fix” the macroscopic form and microstructure of the shot coke. The coke balls formed then fall back into the drum as discrete little spheres. In the main channel up through the drum, some of the spheres will roll around and stick together forming large balls as large as 25cm. When these large balls are broken, they are found to be composed of many of the smaller balls [1-3]. Shot coke should not be confused with fluid coke which is formed in the fluid coking process. This is a completely different process from delayed coking, in which the coke is produced by spraying the heated residuum into a fluidized bed of hot coke particles which are maintained at 1.4- 2.7 bars and 500°C. The feed vapours are cracked while forming a liquid film on the coke particles. The residuum is formed immediately into coke with complete disorientation of the crystals in the hot coke particle. The particles grow by layers until they are removed and new seed coke particles are added. Fluid coke has smaller diameter spheres than shot coke. Shot coke tends to be more isotropic than other types of coke and its optical texture is of fine-grained mosaic (~1μm dia.) [4]. The carbon in shot coke is in the form of ribbon and lenticular/granular anisotropic domains arranged in concentric patterns. Magnification of the surface fractures show whirled, or nonlinear structure in the isotropic shot coke [1]. Shot coke can only be produced in a highly turbulent dynamic environment such as that found in commercial delayed cokers. It is very difficult to produce in conventional pilot plants with low superficial velocity without specially designed modifications. Also, it cannot be produced in a laboratory pot coker; although cokes formed from a shot coke prone feed stock will exhibit the same microstructure and coefficient of thermal expansion regardless of whether they are generated in a commercial-scale delayed or laboratory pot coker. Such cokes produced in a laboratory pot coker will not however have the microscopic form of shot coke [1]. Shot coke is unreactive and of relatively low electrical conductivity. Shot Coke Shot coke is unique in that the small spheres have a slick shiny exterior coating of needle or acicular type carbon, but the inside of each sphere contains isotropic or amorphous type coke. A microscopic study by Marsh, Calvert and Bacha showed that the shot coke spheres consist of a fine- grained mosaic core (0.5-1.5μm dia.) surrounded by a thin, slick outer skin, 50μm thick, of more well-developed mesosphere 0047 Recent Adv Petrochem Sci 2(3): RAPSCI.MS.ID.555588 (2017) Recent Advances in Petrochemical Science Recent Advances in Petrochemical Science coke, this coefficient of thermal expansion is rather high. It is typically greater than 20 (cm/cm/ °C×10-7) [3] were classified and divided into five basic types, namely shot coke, clustered shot coke, porous sponge coke, continuous sponge coke and fines. Significant differences were observed in the properties of the five types of the Syrian petroleum coke considered, particularly ash and VM contents [8]. Shot coke can have low mineral impurities (metal and sulphur) but it is usually rich in such impurities because it is produced most often from feedstocks derived from high sulphur, high metals content crude oils [3]. Shot coke is in general of low or no commercial value; and although it may be used as a fuel, it is less desirable in this usage than sponge coke The chief drawback to its use as a fuel is its extreme hardness which increases the cost of grinding [1,5-7]. Thermal Treatment Thermal treatment of petcoke is the most promising process for the desulphurization of petcoke, and can be the only one possible when other techniques prove to be difficult or inefficient as was found in at least one case with Syrian petcoke [9,10]. By thermal treatment is meant the process whereby a fixed static bed of petcoke is heated under atmospheric pressure in an inert atmosphere to a specified temperature and then kept at that temperature for a specified period of time [9]. Thermal treatment of Syrian petcoke in general and sponge coke in particular has already been made [1,11-13] with encouraging results, but in view of the significant differences between the different coke types, separate treatment of shot coke was deemed of benefit as shot coke represents a major part of Syrian coke production. Shot coke cannot be used in making aluminium anodes. While low grindability cokes are preferred since such cokes yield calcined coke which exhibits a very high vibrated bulk density which is desirable in an aluminium prebaked anode, good bonding with binder coal tar pitch is prevented by the slickness of the outer layer of the shot spheres, their lack of permeability and porosity and dissimilarity of the outer needle coke layer of the shot sphere which has a very low coefficient of thermal expansion and the interior core of the sphere which, being isotropic, has a very high coefficient of thermal expansion. The higher coefficient of thermal expansion of the shot coke is also thought to cause thermal shock cracking of the anode when it is set in a hot cell [1-4]. Experimental Work For the present work, samples of Syrian shot coke were taken from the coke heaps stored to the west of the Homs Oil refinery. The coke samples were classified and divided into regular shot coke and clustered or agglomerated shot coke. Matrix shot or bonded spheres of coke that melted together on formation were discarded. (Tables 1 & 2) give the results of the proximate and ultimate analysis for the coke samples. On the other hand, the special properties of shot coke, including its isotropy, spherical shape, hardness, high coefficient of thermal expansion and high density make it useful for certain specialty applications, e. g, titanium dioxide manufacture, nuclear graphite (if boron content is low), and graphite mould stocks. Table 1: Proximate Analyses of Syrian shot coke, Air-dried basis. Property Regular shot coke Clustered shot coke Ash (wt. %) 0.2 0.1 Moisture (wt. %) 0.2 0.4 Fixed carbon (wt. %) 89.7 89.7 Volatile Matter (wt. %) 9.9 9.8 Sulphur (wt. %) 7.9 8 Gross Calorific Value (kJ/kg) 34.7 × 103 34.3 × 103 Real density (g/cm3) 1.4 1.37 Surface area (B.E.T.)(m2 /g) 0.23 0.21 Table 2: Ultimate Analyses of Syrian shot coke, Dry, ash-fbasis. Regular shot Coke Carbon 85 Hydrogen 4.3 Nitrogen 1.1 Oxygen 1.7 Sulphur 7.9 C/H (wt.) 19.8 For the manufacture of special graphite for nuclear reactors, gilsonite coke, i.e. shot coke produced from Gilsonite ore, may be used. Air blowing of a sponge coke precursor before delayed coking can also be used to generate a shot coke forming feedstock, the ultimate objective being production of an isotropic coke for nuclear graphite manufacture [1]. Recent Advances in Petrochemical Science It is characterized by its lack of permeability, low porosity, low Hardgrove Grindability Index (which may be as low as 2.7) and high density which makes it difficult to crush. When the Hardgrove Grindability index in a coke drops suddenly, it is an indication of the coke becoming isotropic and of a heightened potential for shot coke formation. The coefficient of thermal expansion is used sometimes to determine a quantitative value describing coke structure. The coke is calcined, ground to a flour, mixed with coal tar pitch, extruded to orientate particles into. The concentration of shot coke particles can vary from only a small zone in a coke drum to making up the entire coke mass. These particles are generally harder than sponge coke and can be a problem on cutting and grinding; and on opening the delayed coker they can pour uncontrolled from the drum. mm rods, baked to 850 °C and graphitized to 2900 °C, and then the difference in expansion at 0ºC and 50 °C is measured for coefficient of thermal expansion determination. For shot Hassasn A H I. Thermal Treatment of Syrian Shot Coke. Recent Adv Petrochem Sci. 2017; 2(3): 555588. DOI: 10.19080/ Sulphur Removal is derived from the decomposition of the thermally-stable sulphur compounds bound in side chains [11]. These results may indicate a significant difference between clustered coke and other types of coke in the amount and/or the nature of sulphur compounds bound in side chains, but further work is necessary before a definite conclusion is reached (Table 3 & 4). D it I Table 5: Average degree of desulphurization for shot and other types of Syrian petroleum coke. Table 5: Average degree of desulphurization for shot and other types of Syrian petroleum coke. of Syrian petroleum coke. Temperature range (K) Average degree of desulphurization(%) Regular shot Clustered Other types 300–1075 4 8 7 1075–1175 19 6 18 1175–1450 30 39 23 1450–1550 1 5 13 1550–1650 28 24 22 1650-1700 5 6 7 Recent Advances in Petrochemical Science Table 3: Conditions of Thermal Treatment. Average weight of treated sample 10 g Coke Size range 0.85- 1.60mm Rate of heating 3.5°C/min Gas atmosphere N2 Pressure Atmospheric Rate of nitrogen flow 0.5 l/min/g Residence time at the maximum temperature 180min Table 3: Conditions of Thermal Treatment. treatment was carried out in an electrical tube furnace heated by a SiC element fully covering the working tube. The outside diameter of the working tube is 59 mm, and the heated length is 250 mm. A PtRh-Pt thermocouple is placed in the centre of the heating zone and is lead to the temperature control unit. The conditions used in the treatment were such that were expected to lead to a maximum rate of desulphurization at moderately high temperatures [11]. Table 3 is a summary of the treatment conditions used. A summary of the results of the thermal treatment is shown in Table 4. Table 4: Results of the thermal treatment. Temperature K Regular shot coke Clustered shot coke Wt loss % CV× 103 kJ/kg S wt % DR10 – 20 Wt loss % CV × 103kJ/kg S wt % 300 - 34.7 7.9 1.4 - 34.3 8 500 0.6 34.7 7.9 1.4 0.6 34.2 8 775 4.1 34.5 7.9 1.45 3.9 34.5 7.8 875 4.1 34.8 7.8 1.48 3.7 33.8 7.9 975 8.2 32.4 7.3 1.57 7.8 32.4 7.4 1075 8.5 32.3 7.6 1.74 8.2 32.3 7.4 1175 9.1 31.9 6.1 1.82 7.3 32 6.9 1450 12.2 31.8 3.7 1.89 12.2 31.6 3.8 1550 14.1 31.8 3.6 1.75 14.2 31.9 3.4 1650 17.9 31.8 1.4 1.9 18.1 31.9 1.5 1700 18.6 32 1 1.92 20.7 32.5 1 Syrian Delayed Coke Syrian delayed coke is coke produced by the delayed coking unit at the Homs Oil Refinery. This unit was designed and built during the late sixties of last century for the purpose of maximizing gasoline and distillate yields using a feedstock of residue materials. The Petcoke produced is considered merely as a by-product of little commercial value. This is mainly because of its high sulphur content and the high percentage of fines produced. There are at least four basic types of delayed petcoke with different microstructures due to differences in operating variables and nature of feedstock. The basic coke types are: needle coke, honeycomb coke, sponge coke and shot coke. Of these four types, only the last two types, namely sponge and shot cokes are produced at the Homs Oil Refinery. In order to characterise Syrian green petroleum coke, samples of the coke The coke samples were thermally treated in an inert atmosphere of nitrogen at atmospheric pressure. The Hassasn A H I. Thermal Treatment of Syrian Shot Coke. Recent Adv Petrochem Sci. 2017; 2(3): 555588. DOI: 10.19080/ 049 How to cite this article: Hassasn A H I. Thermal Treatment of Syrian Shot Coke. Recent Adv Petrochem Sci. 2017; 2(3): 555588. DOI: 10.19080/ RAPSCI.2017.02.555588 0049 Recent Advances in Petrochemical Science References 1. Ellis PJ (1996) Shot coke, Light Metals, pp. 477-484. 2. Mochida I, Furuno T, Korai Y, Fujitsu H (1986) Studies reveal shot-coke microstructure, suggest ways to minimize its formation. Oil & Gas J 84(5). 3. Ellis S, Paul CA (1998) Tutorial: Delayed coking fundamentals, AIChE Spring National Meeting’s International Conference on Refinery Processes Topical Conference Reprints, LA, pp. 29. 4. March H, Calvert C, Bacha J (1985) Structure and formation of shot coke- A microscopic study. Journal of Materials Science 20(1): 289-302. 5. Figueiredo JL, Mouljin JA (1986) Carbon and coal gasification. In: (Eds.) Figueiredo JL, Mouljin JA, Martinues Nijhoff Publishers, Dordrecht, Netherlands. 6. Meyers, Robert A (1986) Handbook of petroleum refinery processes. (3rd Edn), McGraw-Hill Companies, New York, USA. 7. Tulsa, Okla, GL Farrar (1976) NPRA Q & A Session on refining and petrochemical technology. The Petroleum Publishing Company, USA. The calorific value was also observed to increase slightly in the temperature range 1650-1700K. This must be related no doubt to the decreased sulphur content, as the heat of combustion of sulphur (9420 kJ/kg) is considerably less than that of carbon (33820kJ/kg). A similar result was also obtained with other types of coke [11]. 8. Ibrahim HA (2005) Analysis of Syrian green delayed coke, Proceedings of the sixth Egyptian Syrian conference on chemical and petroleum engineering, Syria, p. 22-33. 9. Ibrahim H, Morsi BI (1992) Desulfurization of petroleum coke. Industrial and Engineering Chemistry Research volume 31(8): 1835- 1840. 10. El-kaddah N, Ezz SY (1973) Thermal desulphurization of ultra high sulphur petroleum coke. Fuel 52: 128-129. Weight Loss The observed weight loss at the conclusion of the thermal treatment varied between 19 and 21%. This is in agreement with the generally observed weight loss for other types of coke which is normally of the order of 20% or so. This amount corresponds to the moisture and volatile matter content of the coke as well as to the amount of sulphur removed. 11. Ibrahim H, Ali M (2004) The effect of increased residence time on the thermal desulphurization of Syrian petroleum coke. Periodica Polytechnica Ser Chem Eng 48: 53-62. 12. Ibrahim H, Ali MM (2005) Effect of the removal of sulphur and volatile matter on the true density of petroleum coke. Periodica Polytechnica Ser Chem Eng 49: 19-24. 13. Ibrahim H (2006) Thermal treatment of Syrian sponge coke. Journal of King Saud University. Engineering Sciences 8(2): 261-270. Calorific Value increased residence time (180 minutes). The treated coke has a low sulphur content (1%) and a high real density (1.9g/ cm3). The adverse effects normally associated with thermal Treatments at high temperatures were minimal. The calorific value decreases, in general, with increasing temperature of the thermal treatment. However, there were observed two exceptions to this rule, where the calorific value increased rather than decreased. A slight increase in the calorific value was observed towards the end of the first initial stage of thermal treatment (300-1200K). This is the overall effect of the evaporation and removal of moisture and volatile matter which take place during this stage, where the removal of moisture, as an inert material, has an opposite effect on the calorific value to that of removing the volatile matter. Whereas the removal of moisture is accompanied by an increase of the calorific value, the removal of the volatile matter tends to lower this value. The overall effect of the thermal treatment is therefore a factor of both volatile matter and moisture content. Since the volatile matter content of shot coke is lower, in general, than for other types of coke, the effect of the removal of VM on the calorific value is expected to be less also, with the result that the increase in the calorific value is more pronounced for shot coke. Density Increase The true density of petcoke is expected to increase continuously with increasing treatment temperature. The rate of this increase is different, however, at different temperature ranges. Three stages of density change were recognized [12]. a) An initial stage (300-800K), with minimal density increase due probably to the removal of moisture and some volatile matter in the coke. The density increase observed was 0.05g/cm3 in the case of regular shot coke. Table 5 shows the results of sulphur removal for both types of shot coke as compared with other types of Syrian coke in general. Similar trends are observed in both cases, with a notable exception in the temperature range 1450-1550K where a marked drop in the degree of desulphurization of shot coke was observed. At temperature ≥ 15 00K desulphurization is normally inhibited by the formation of thermally-stable metal sulphides [14]. This is mostly the case with shot coke, which is usually rich in metal impurities as was pointed out above. In the temperature range 1075–1175 K, the expected increased degree of desulphurization is not observed in the case of the clustered shot coke, with only 6% sulphur removal as compared to 19% for regular shot coke and 18% for other types of coke. Most of the sulphur removed during this stage b) A second stage (800-1200) characterized by rapid increase in density related to the evaporation of the volatile matter adsorbed on the coke surface or in the pores. For regular shot coke, with 9.9 VM (wt.%), the density increased during this stage by 0.47 g/cm3. c) A final stage (1200-1700), where the density increase may be related to the rate of sulphur removal. The density increased by 0.10g/cm3 for regular shot coke. Hassasn A H I. Thermal Treatment of Syrian Shot Coke. Recent Adv Petrochem Sci. 2017; 2(3): 555588. DOI: 10.19080/ How to cite this article: Hassasn A H I. Thermal Treatment of Syrian Shot Coke. Recent Adv Petrochem Sci. 2017; 2(3): 555 RAPSCI.2017.02.555588 0050 Recent Advances in Petrochemical Science This work is licensed under Creative Commons Attribution 4.0 Licens DOI: 10.19080/RAPSCI.2017.02.555588 Conclusion 14. Vrbanovic Z (1978) Possibility of using high temperature treatment of petroleum coke to desulphurize it. Nafta (Zagreb) 2: 80-93. 14. Vrbanovic Z (1978) Possibility of using high temperature treatment of petroleum coke to desulphurize it. Nafta (Zagreb) 2: 80-93. Effective desulphurization of shot coke was achieved by means of thermal treatment to a temperature of 1700K and Your next submission with Juniper Publishers will reach you the below assets • Quality Editorial service • Swift Peer Review • Reprints availability • E-prints Service • Manuscript Podcast for convenient understanding • Global attainment for your research • Manuscript accessibility in different formats ( Pdf, E-pub, Full Text, Audio) • Unceasing customer service Track the below URL for one-step submission https://juniperpublishers.com/online-submission.php Your next submission with Juniper Publishers will reach you the below assets • Quality Editorial service • Swift Peer Review • Reprints availability • E-prints Service • Manuscript Podcast for convenient understanding • Global attainment for your research • Manuscript accessibility in different formats ( Pdf, E-pub, Full Text, Audio) • Unceasing customer service Track the below URL for one-step submission https://juniperpublishers.com/online-submission.php Your next submission with Juniper Publishers will reach you the below assets • Quality Editorial service • Swift Peer Review • Reprints availability • E-prints Service • Manuscript Podcast for convenient understanding • Global attainment for your research • Manuscript accessibility in different formats ( Pdf, E-pub, Full Text, Audio) • Unceasing customer service Track the below URL for one-step submission https://juniperpublishers.com/online-submission.php 0051
https://openalex.org/W1603926126	https://ethnobiomed.biomedcentral.com/counter/pdf/10.1186/1746-4269-2-19	English	null	Ethnotaxonomy of mastofauna as practised by hunters of the municipality of Paulista, state of Paraíba-Brazil	Journal of ethnobiology and ethnomedicine	2,006	cc-by	5,572	Journal of Ethnobiology and Ethnomedicine 2006, 2:19 doi:10.1186/1746-4269-2-19 Journal of Ethnobiology and Ethnomedicine 2006, 2:19 doi:10.1186/1746-4269-2-19 This article is available from: http://www.ethnobiomed.com/content/2/1/19 © 2006 Mourão 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. Abstract It was aimed in the present work to report aspects related to identification, naming and categorization of the mastofauna species of the caatinga biome, according to hunters' knowledge of Northeast Brazil. The methods of free and semi-structured interviews and guided tours were applied here. The data obtained were analyzed under the emic/etic point of view by comparing the local people's knowledge to those reported in the literature. The inland hunters use some classification models of mammals living around them as for example the folk taxonomy, and a categorization based on the animal utility. Some species are preferably hunted for providing food while others are hunted for therapeutic ends. Hunt techniques were made evident by the informers. The retrieval and comprehension of the whole process related to such knowledge is very important to evaluate the human culture and the protection that people exert on local biodiversity, since these aspects have implications for the conservation and management of faunistic resources. of studies in several areas of natural sciences. This knowl- edge is the aim of ethnoscience studies, where the prefix ethno refers to the system of knowledge and cognition that are typical of each culture [4]. Journal of Ethnobiology and Ethnomedicine BioMed Central BioMed Central Open Access Ethnotaxonomy of mastofauna as practised by hunters of the municipality of Paulista, state of Paraíba-Brazil José S Mourão1, Helder FP Araujo2 and Fabiana S Almeida3 Address: 1Departamento de Biologia, Núcleo de Etnoecologia, Educação Ambiental e Gestão, Universidade Estadual da Paraíba. Campus Universitário do Bodocongó, Campina Grande, PB, Brazil, 2Programa de Pós-Graduação em Ciências Biológicas (Zoologia), Departamento de Sistemática e Ecologia, Universidade Federal da Paraíba, 58051-900 João Pessoa, PB, Brazil and 3Prefeitura de Serra Negra, RN, Brasil Email: José S Mourão - tramataia@uol.com.br; Helder FP Araujo - hfparaujo@yahoo.com.br; Fabiana S Almeida - fabiologia2@hotmail.com * Corresponding author Received: 21 November 2005 Accepted: 07 April 2006 Published: 07 April 2006 Methodological procedures We performed 20 free and open interviews, in Portuguese language, with local hunters, inland herdsmen, in period of 2003 and 2004. The hunters has a low literary degree, however know to read and write. The biodiversity of the caatinga, (vegetation consisting of thorny shrubs and stunted trees situated in the interland of Northeast Brazil) have long been deemed as rather low [10-12]. However, recent studies revealed a number of animals expressively higher than the formerly reported ones, tearing down the myth of the poor caatinga's biodi- versity [13-15]. The figures currently reported, however, may even be increased, since 41% of the entire caatinga area [larger than 800,000 km2 in Northeast Brazil] has never been scientifically investigated, and 80% of the studied areas are rather small representative sampling effort [16]. We asked about the mastofaunistic composition known to them, followed by semi-structured direct forms on spe- cific topics of the mastofauna, as nomination, identifica- tion and use [20-22]. Only the most experienced hunters were interviewed. Their own choice followed the 'snow ball' technique, that consists of the indication in sequence of recognized informers, exactly, for the degree of experience [23,24]. The names of mammals known to the informers were obtained from them by applying the free listing tech- nique, that looks specific informations on a cultural domain of the studied community. The hunters were requested to list the names of known mammals [21]. The limitations of free listing technique were overcome by adopting the 'non-specific prompting' and 'reading back' techniques. The first consists of questions made to the informers when they declare not to remember of more elements, however those inductions should be sentences formulated that do not generate answers yes or not. The second is a technique used soon after, where the researcher reads the supplied list slowly and allowed the citation, by informers, of items not listed previously [21,25]. The dwelling places of inland herdsmen of the state of Paraíba extend from the 'agreste' (a transitional microre- gion between the littoral woods and the caatinga ecosys- tems of the 'carirí', a quite semi-arid microregion) to 'sertão' (the caatinga ecosystems microregion on the northwest) [17]. Such people grow crops and raise cattle, besides carrying out hunting activities as means of subsist- ence. The inland dweller is primarily a hunter, fisherman, or grower, and only after that, he is able to carry out another task [18]. The study area can be illustrated as follows: (i) nine species of non-sting bees (Meliponinae) were discovered thanks to the Kayapó Indian's ethnoentomology (in the Amazon basin) [6]; (ii) the number of fishes forming lunar reproductive aggre- gates, which are known to biologists, has more than dou- bled as a consequence of ethnoichthyology of fishermen from the Pacific islands [7]; (iii) the feeding behaviour of marmosets of the genus Callithrix was evaluated with the help of woodmen's ethnoethology of the State of Alagoas, Northeast Brazil [8,9]. can be illustrated as follows: (i) nine species of non-sting bees (Meliponinae) were discovered thanks to the Kayapó Indian's ethnoentomology (in the Amazon basin) [6]; (ii) the number of fishes forming lunar reproductive aggre- gates, which are known to biologists, has more than dou- bled as a consequence of ethnoichthyology of fishermen from the Pacific islands [7]; (iii) the feeding behaviour of marmosets of the genus Callithrix was evaluated with the help of woodmen's ethnoethology of the State of Alagoas, Northeast Brazil [8,9]. The municipality of Paulista is situated in a caatinga region designated as septentrional inland depression, whose geographic coordinates are at lat 6°35'58"S and long 37°37'27"W and at an altitude of 160 m (Figure 1). The Piranhas River, a permanent watercourse, is the main hydrologic resource of the municipality of Paulista. The climate of the region is typically semi-arid, with an average annual rainfall of 400 mm. The population of that municipality is of 15,349 [19]. Human being/animal is a quite ancient relation and an extremely important connection to human societies, because the latter depends very often on the former for their survival. Ethnozoology was the name given to this subfield of work by Mason in USA, in 1899, who defined it as 'the zoology of the region as actually told by the indigenous" [10]. Ethnozoology, as part of ethnobiology, is concerned with the revelations of traditional popula- tions' knowledge on wildlife, as well as their reciprocal relationship that is aimed at understanding the role of animals in the daily lives of human communities [9]. The natural environment, the caatinga, has the ground covered with herbaceous but non-grassy plants and the predominance of tropical xerophytes, which are tortuous thorny shrubs and stunted trees, having small and cadu- cous leaves [18]. Introduction h k l d The knowledge on natural world that peoples hand down orally through the generations are usually denominated as 'local' or 'traditional'. This knowledge is culturally transmitted through the generations by using symbols, phonetic, narratives, rituals, music, and dance [1], which are imprinted in people's mind as memes [2]. There is a variety of terms describing traditional knowledge and practices, e.g. the indigenous technical knowledge (ITK), traditional ecological knowledge (TEK), rural people knowledge (RPK), traditional botanical knowledge (TBK), and integrated knowledge system (IKS) [3]. Among the ethnosciences, the ethnobiology deserves spe- cial attention by involving the analysis of the classifica- tion system on Nature and for having a deep link with the subjects of botany, zoology, and ecology. Lévi-Strauss was one of the forerunners in this field of study, mainly for the emphasis he gave to the classification systems allowing to reach the logic of the native thought [5]. In the last years, traditional populations' wisdom has been deemed as extremely important for the development There have been great contributions of ethnobiological studies to the academic advancement of biology, which Page 1 of 7 (page number not for citation purposes) Page 1 of 7 (page number not for citation purposes) Journal of Ethnobiology and Ethnomedicine 2006, 2:19 http://www.ethnobiomed.com/content/2/1/19 Localizatio Figure 1 Localization of the municipality of Paulista, in the caatinga region of the state of Paraíba, Northeast- Brazil Figure 1 Localization of the municipality of Paulista, in the caatinga region of the state of Paraíba, Northeast- Brazil. p y , g g , g Localization of the municipality of Paulista, in the caatinga region of the state of Paraíba, Northeast- Brazil. Some nominations of generics are not classified in hierar- chical structure. However, they have a relation with cul- tural and/or economical importance. Some generic mentioned were also classified in an utilitarian way. In ethnotaxonomy, recongnized ethnobiological taxa are taxonomically distributed as members of six mutually exclusive ethonobiological ranks comparable in content to the ranks of Western zoology and botany [27]. The six ranks, in descending order of taxonomic inclusiveness, are the kingdom, life-form, intermediate, generic specific and varietal. The rank of kingdom is unique in that it but a sin- gle member. For ethnobiologically, the kingdom corre- sponds approximately to the biological taxons Plantae and Animalia. Taxa of the life-form mark a small number of highly distinctive morphotypes based on the recogni- tion of the strong correlation of gross morphological structure and ecological adaptation. Taxa of intermediate rank are found most commonly as members of life-form taxa, and are comprised of small numbers of folk generics that show marked perceptual similarities with one another. The most numerous taxa in folk biological taxon- omies will be taxa of generic rank. Roughly 80 percent of folk generic taxa in tipical folk systems are monotipic and include no taxa of lesser rank. Taxa of the rank of folk spe- cies partition folk generic taxa into two or more members; in those systems where they occur, folk varietals further subdivide folk species. Methodological procedures Despite the northeastern Brazilian region be a vast field for ethnobiological studies, the inlander's behaviour exploiting the natural resources has not been much investigated [17]. The morphological characteristics of some mammals they mentioned were obtained from the informers with the help of photographs and cards containing the animal's images, the scientific correspondents follow the nomen- clature of the list of the Caatinga biome [26]. It was aimed in the present work to obtain information about the regional mastofauna of Paraiban caatingas at the municipality of Paulista and its use by local inland herdsmen. Page 2 of 7 (page number not for citation purposes) Page 2 of 7 (page number not for citation purposes) Journal of Ethnobiology and Ethnomedicine 2006, 2:19 Journal of Ethnobiology and Ethnomedicine 2006, 2:19 http://www.ethnobiomed.com/content/2/1/19 Localization of the municipality of Paulista, in the caatinga region of the state of Paraíba, Northeast- Brazil Figure 1 Localization of the municipality of Paulista, in the caatinga region of the state of Paraíba, Northeast- Brazil. Results and discussion The hunters' information totalled 20 genera of the masto- fauna (Table I), which comprise 20 species of mammals out of 148 known species of the caatinga biome [15,24]. The hunters we interviewed have got a detailed knowledge of the mastofauna resource of caatinga ecosystems, which enables them to show a folk taxonomy through identifica- tion, denomination, and classification of organisms. This way, they utilize morphological, ecological, and behav- ioural criteria. Their ability for passing on information on the habitat, trophic relations, behaviour, and morpholog- ical details of animals, is a relevant feature of folk classifi- cation systems, an aspect also found among other Brazilian human groups reported elsewhere [27-36]. Biological classifications make up a general reference sys- tem on biological diversity and are actual storehouse of Page 3 of 7 (page number not for citation purposes) http://www.ethnobiomed.com/content/2/1/19 Journal of Ethnobiology and Ethnomedicine 2006, 2:19 Table 1: The generic folk of the mastofauna mentioned by the hunters of the municipality of Paulista, State of Paraíba, showing their respective scientific names, local habitats, and the habitats reported in the literature: 1 39 and 2 40 Habitats of the mammals Generic folk Corresponding taxon Macrohabitat Microhabitat Habitats/[literature] Artiodactyla Tayassuidae Collared peccary Pecari tajacu .....Rocky hill Den Den/[2] Carnivora Canidae Crab-eating fox Cerdocyon thous Sertão Burrow and den Thickets, tablelands/[2] Jaguarundi Felididae Herpailurus yaguarondi Everywhere Den and tree Rocky hill and tablelands/ [1] Margay Leopardus ........wiedii Everywhere Den and tree Rocky hill and tablelands [1] Greater grison Mustelidae Galictis vittata and/or G. Results and discussion cuja Rocky hill Burrow, den, tree (No reference) Crab-eaten raccoon 'Guará' Procyonidae Procyon cancrivorus (Unidentified species) 'Island'* Rocky hill, 'island' Den, tree Den Thickets/[1] (No reference) Didelphimorphia Skunk and 'timbu' (opossums) Didelphidae Rocky hill, 'island' Burrow, den tree Rocky hills, trees/[1] Primates Callithrichidae Common marmoset Callithrix jacchus Cebidae Sertão Slope, tree Trees [2] Tufted capuchin Cebus apella Rocky hill Tree Trees [2] Rodentia Caviidae Rock cavy Kerodon rupestris Rocky hill Cracks in the stones Caves, grottos, rocky hills/ [1] Spix's yellow- toothed cavy Galea spixii Everywhere Den and crevices in the rocks Rocky hills and grass fields/ [1] Rats Muridae Everywhere (No reference) Xenarthra Dasypodidae Nine-banded armadillo Dasypus novemcinctus Rocky hill, 'island' Burrow Burrows in rocky hills/[1] Six-banded armadillo Euphractus sexcinctus Rocky hill, 'island' Burrow, den Burrows in rocky hills/[1] Giant anteater Mymercophagidae Mymercophaga tridactyla Rocky hill, 'island', Woods Den, tree (No reference) Southern tamandua Tamandua tetradactyla Rocky hill, 'island', Woods Den, tree (No reference) * 'Island': a lowland area surrounded by stagnant water Table 1: The generic folk of the mastofauna mentioned by the hunters of the municipality of Paulista, Sta respective scientific names, local habitats, and the habitats reported in the literature: 1 39 and 2 40 evident that there is a great diversity of distinct kinds of mammals. The most related or similar kinds are generally grouped in one single category called 'family', as can be noticed from some parts of their interviews: information [37]. In this sense, ethnobiological classifica- tions are also a storehouse of information, since they con- tain immense richness of information on the biology, ecology, and ethology of several groups of animals and plants. • 'I think that they are of the same family: skunk, anteater, and opossum are of a same similarity'. The ethnobiological classification system is conceptually organized as a shallow hierarchical structure [27]. Thus, hunters categorize mammals hierarchically, as we can observe from their expressions: 'there are two kinds of ant- eaters'; 'there are many kinds of cats'. • 'The six- and nine-banded armadillos are quite alike: they must belong to the same group'. • 'Anteater and skunk have similar furs'. 'I believe that the crab-eating raccoon and crab-eating fox are of a single family; of a single species'. According to the hunters, a mammal is any sort of animal that has the habit of feeding its young on milk. * 'Island': a lowland area surrounded by stagnant water Journal of Ethnobiology and Ethnomedicine 2006, 2:19 • 'Monkey and marmoset are of the same species; some of them being larger than others'. useful for their identification. The generic folk rat (Muri- dae) has got a tail that makes it distinct from the rock cavy (K. rupestris). This latter and the Spix's yellow-toothed cavy (G. spixii) differ with respect to body length, the red hind back of the former and also with respect to their hab- itats; the informers say these are the most conspicuous features of each generic folk. The animals' habitats in the caatinga are classified by the hunters as 'macrohabitats', like (i) rocky hills, (ii) sertão, a plain covered by the native vegetation, and (iii) 'island', a lowland area surrounded by stagnant water; and 'microhabitats', like (a) burrow, (b) den, among rocks, (c) cave, (d) tree, (e) slopes, (f) tree hole, and (g) dark hiding places. In Table I the habitats of the generic terms mentioned here are presented, in com- parison with other denominations reported elsewhere. This kind of habitat categorization, also used for identify- ing organisms concerning their distribution and the areas they share, has been widely reported in the ethnoecologi- cal literature on several human populations [8,42-44]. Those groupings do not indicate hierarchical relations in taxonomy folk, but they are made with morphologic sim- ilarities and allow a comparison with the biological tax- onomy. Within the scientific taxonomy, the rock cavy (Kerodon rupestris) and the Spix's yellow-toothed cavy (Galea spixii), and rats (several species of Muridae) are spe- cies of the same order Rodentia, which were grouped in the same ethnobiological taxon by the informers. The hunters also use another form for identifying the animals, through ecological criterion, i.e. their feeding habits, as in the generic folk cat (several species of Felidae) and the crab-eating raccoon (Procyon cancrivorus), which are carni- vores (order Carnivora]. The semantic and ecological dimensions of animal identification show how important it is to recognize it for our comprehension on popular tax- onomy [22]. Nominations that indicate hierarchical and no-hierarchical relations were also recorded in a study of classification ethnozoological of Nage, in western Indone- sia [38]. In the same way of the generic folk above-mentioned, the informers say that the crab-eating raccoon (P. carnivorus) and the 'guará', an unidentified species of the family Cani- dae, differ with respect to size and colour: this latter is a smaller and more dark-coloured animal. Journal of Ethnobiology and Ethnomedicine 2006, 2:19 The generic 'ticaca' and 'timbu' (both species are some kind of opos- sum, family Didelphidae) differ by their morphology, like type and coloration of fur, as said by one of the informers: 'the "timbu" has a naked tail and the "ticaca" is black, with white stripes on its back, which are not present on the "timbu's" back'. Besides the hierarchical categorization, hunters use other classification systems. They classify mammals, for exam- ple, according to their utility (for human purposes): 'hunting animals' and just 'animals'. In the former cate- gory they include the nine-banded armadillo (Dasypus novemcinctus) and the six-banded armadillo (Euphractus sexcinctus) which are of the same generic folk and are the most hunted animals. Such classification criteria have also been used by other peoples of aural tradition, like the Montagnais ethnozoological classification [42], which categorize the animals as edible (red meat and white meat) and non-edible. Several studies focusing the folk taxonomy have reported that Indian people and fishermen, besides using morpho- logical classification criteria, also use, for example, ecolog- ical and behavioural criteria for denominating and identifying animal groups [34,36,22]. In studies on the avifauna classification used by the Wayampi, an Amazo- nian native people of Brazil, it was observed the prepon- derance of such criteria, added up of characteristics from their religious belief [30]. The inland hunters of the State of Paraíba identify and name mammals by taking in account their colour, body shape, size, or a typical characteristic of any part of the animal. As an example, the generic 'red cat' or margay (Leopardus wiedii) is so called because 'it is entirely red'. They also justify naming some mammals for their analogy with objects, like the anteater called by them as 'taman- duá-bandeira' literally meaning 'flag anteater', because of its fury tail looking like a flag (a designation widely known in Brazil for this animal). Another anteater is called by the hunters as 'leather-tailed' anteater because of its naked tail, or as they say: 'it's smooth; it's really a long leathery tail'. The same criteria they use for naming mam- mals are generally used for identifying them. http://www.ethnobiomed.com/content/2/1/19 Journal of Ethnobiology and Ethnomedicine 2006, 2:19 Results and discussion This is a purely biological-based classification criterion, where the taxon of the life-form is known as animals that suck or that possess mammary glands. Despite they recognize this category as bearing similar characteristics, they also make • 'The cat, despite its different fur, is also an animal that feeds on the same kind of food that is preferred by the crab-eating rac- coon and crab-eating fox'. • 'The cat, despite its different fur, is also an animal that feeds on the same kind of food that is preferred by the crab-eating rac- coon and crab-eating fox'. Page 4 of 7 (page number not for citation purposes) Page 4 of 7 (page number not for citation purposes) (page number not for citation purposes) http://www.ethnobiomed.com/content/2/1/19 Page 5 of 7 (page number not for citation purposes) http://www.ethnobiomed.com/content/2/1/19 from January to March, we would kill one or more individuals because the poor pups are in the cave waiting that their parents feed them'. since they provide variations for a further selection by their peers. Ecological history makes use of the study of changes in the environmental language to infer that his- tory, not evolutionary events, is responsible for the chief changes in the relations between the human society and its close environment [45]. This is the way the hunters have to manage their local ani- mal resources, by sparing the females and their pups' lives. However, hunting and deforestation are still the main fac- tors threatening those animal species in the wild, in the region we studied. That paradox suggests a deeper investi- gation, where can generate quantitative informations about the abundance of the resources mastofaunistics and of the respective hunt products. Like this, showing to real relation resource/hunt of the area. The use of mastofauna Food is not simply implicit in the category denominated 'animals', say the inland hunters, who consider as such, just the 'hunting animals'. These are: nine-banded arma- dillo (D. novemcinctus), six-banded armadillo (E. sexcinc- tus), Spix's yellow-toothed cavy (G. spixii), and rock cavy (K. rupestris), which are also used for other purposes. The armadillos are their favourite generic folk, being the nine- banded armadillo's meat the most appreciated by the hunters. Such preference is confirmed in other northeast- ern Brazilian states [46], like in Alagoas where 100% of hunters elected this animal's meat as the one with the best pleasant taste, being then one of the most hunted animals [47]. In the Paraiban interland the rock cavy is used as food and its curd is utilized for making cheese. Acknowledgements We are grateful for the hunters of municipality of Paulista, since our research was founded on their knowledge. We are also grateful to CAPES (Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior). 17. Diegues AC, Arruda RS: Saberes tradicionais e biodiversidade no Brasil Brasília: Ministério do Meio Ambiente. São Paulo: USP; 2001. Journal of Ethnobiology and Ethnomedicine 2006, 2:19 http://www.ethnobiomed.com/content/2/1/19 http://www.ethnobiomed.com/content/2/1/19 Journal of Ethnobiology and Ethnomedicine 2006, 2:19 Such identi- fication criteria are purely morphological, like the ones used for the generic folk Spix's yellow-toothed cavy (Galea spixii) and the rock cavy (Kerodon rupestris), and also for rats (several species of Muridae), whose body shapes and existent or non-existent body parts and even their size are Cultural and biological diversity are partly responsible for the richness of names recorded in the folk taxonomy. This is one of the reasons concerning the importance of ethno- biological studies of the denominations that local human populations give to the flora and fauna. This makes easier our understanding on human/Nature relations and can be useful for testing the possible effects of cultural evolution for maintaining certain denominations [22]. Fishermen in the State of Alagoas use fishing nomenclature com- posed of synonymies, emergent names, and the most commonly used names [8]. The emergence and localiza- tion of ichythyonymic denominations may be used as an evidence of cultural evolution of fishermen communities, Page 5 of 7 (page number not for citation purposes) Page 5 of 7 (page number not for citation purposes) Journal of Ethnobiology and Ethnomedicine 2006, 2:19 References 1. Toledo MP: What is ethnoecology? Origins, scope and impli- cations of a rising discipline. Etnoecológica 1992, 1:5-21. 1. Toledo MP: What is ethnoecology? Origins, scope and impli- cations of a rising discipline. Etnoecológica 1992, 1:5-21. 2. Dawkings R: O gene egoísta São Paulo; 1979. 3. Martin GL: Ethnobotany: methods manual London: Campamam & Hall; 1995. 3. Martin GL: Ethnobotany: methods manual London: Campamam & Hall; 1995. All the other animals are of secondary importance for them, being only used if captured by the hunters. Cats' furs for example, are only used as ornament; and other animals can be caught and kept in captivity as pets. 4. Campos MD'O: Etnociência ou etnografia de saberes, técnicas e práticas. In Métodos de Coleta e Análise de Dados em Etnobiologia, Etnoecologia e Disciplinas Correlatas Edited by: Amorozo MCM, Ming LC, Silva SP. Rio Claro: UNESP/CNPq; 2002. q 5. Begossi A: Ecologia Humana: Um enfoque das relações homem-ambiente. Interciência 1993, 18:121-131. 6. Posey D: Introdução-Etnobiologia: Teoria e Prática. In Suma Etnológica Brasileira Edited by: Ribeiro B. Petrópolis: Vozes; 1986:15-25. The traditional populations of the Paraiban interland also use local faunistic resources for medical purposes in the same way they use the phytotherapeutical resources [48]. When asked about that, the hunters said the generic folk Spix's yellow-toothed cavy (G. spixii), rock cavy (K. rupes- tris), and skunk (Didelphidae) are the most hunted ani- mals, whose meat and its broth are used for treating rheumatism, weakness, and loss of appetite. 7. Johannes RE: The utilitarian factor in folk biological classifica- tion. American Antropologist 1981. 8. Marques JGW: Aspectos ecológicos na Etnoictiologia dos Pes- cadores do Complexo Estuarino – Lagunar Mundaú – Mangu- aba. In Tese Universidade Federal de Campinas, Campinas – SP; 1991. 9. Sanches RA: Caiçaras e a estação ecológica da Juréia-Itatins (litaoral sul – SP): uma abordagem etnográfica e etnoecológ- ica para o estudo da relação homem e meio ambiente. In Dis- sertação Universidade de São Paulo, São Paulo-SP; 2000. http://www.ethnobiomed.com/content/2/1/19 http://www.ethnobiomed.com/content/2/1/19 18. Mendes BV: Biodiversidade e Desenvolvimento Sustentável do Semi-árido Fortaleza: SEMACE; 1997. 43. Thé APG: Etnoecologia e produção pesqueira dos pescadores da represa de Três Marias-MG. In Dissertação Universidade Fed- eral de São Carlos, São Carlos -SP; 1999. 19. Projeto Alvorada:: Relação dos municípios do estado da Paraíba. [http://www.assistenciasocial.gov.br/relcrys/alvorada/ docs/PB.pdf.]. Accessed in: 3.II.2005. 44. Costa-Neto EM: Etnoictiologia, Desenvolvimento e Sustenta- bilidade no Litoral Norte Baiano. Um Estudo de Caso entre pescadores do Município de Conde. Dissertação de Mestrado- UFAL- Maceió – AL; 1998. 20. Bernard HR: Research Methods in Cultural Anthropology Newbury Park: Sage Publication; 1998:520. 45. Balée W: Biodiversidade e os índios amazônicos. In Amazônia etnologia e historia indígena FAPESP/NHII; 1994:385-393. g 21. Albuquerque UP, Lucena RF: Métodos e técnicas para coleta de dados. Métodos e técnicas na pesquisa etnobotânica 2004:37-62. 46. Paiva MP: Fauna do Nordeste do Brasil Conhecimento Científico e Pop- ular. Fortaleza – BNB; 1995. 22. Montenegro SCS: A Conexão Homem/Camarão (Macrobra- chium acanthurus e M. carcinus) no Baixo São Francisco Alagoano: uma abordagem etnoecológica. In Tese Universi- dade Federal de São Carlos, São Carlos – SP; 2002. 47. Silva GA: Mamíferos de importância cinegética na Várzea de Marituba e na Fazenda Boa Vista, Alagoas: espécies caçadas e métodos de caça. In Dissertação Universidade Federal de Alagoas, Maceió; 1993. 23. Bailey K: Methods of social reached 4th edition. New York: The Free Press; 1994:588. 48. Bonifácio KM: Pescadores e Peixes: um estudo sobre o con- hecimento ecológico tradicional dos pescadores do Açude Bodocongó, Campina Grande -PB. Simpósio Brasileiro de Etnobi- ologia, 4. Resumos 2002. 24. Albuquerque UP, Lucena RF: Seleção e escolha dos informantes. In Métodos e técnicas na pesquisa etnobotânica Edited by: Albuquerque UP, Lucena RF. Recife: Livro Rápido; 2004:19-35. 25. Brewer DD: Supplementary interviewing techniques to max- imize output in free listing tasks. Field Methods 2002, 14(1):108-118. ( ) 26. Oliveira JA, Gonçalvez PR, Bonvicino CR: Mamíferos da Caatinga. In Ecologia e conservação da Caatinga Edited by: Leal IR, Tabarlli M, Silva JMC. Editora Universitária, Universidade Federal de Pernambuco, Recife; 2003:275-302. 27. Berlin B: Ethnobiolocal Classification: Principles of categorization of plants and animals in traditional societies Princepton, Princepton University Press; 1992. 28. MaranhÃo T: Naútica e classificação ictiológica em Icaraí, Ceará: um estudo em antropologia cognitva. Dissertação de Mestrado, UNB, Brasília- DF. 1975. 29. Silva GO: Tudo que tem na terra tem no amar. The hunter's collection techniques and their implications 10. Vanzolini PE, Ramos-Costa AMM, Vitt LJ: Répteis das Caatingas Academia Brasileira de Ciências. Rio de Janeiro; 1980. The hunter's collection techniques and their implications The hunters themselves report that these are sporting and subsistence hunting, and for both kinds they utilize shot- gun during the day for capturing mammals, birds, and reptiles. During the night they make use of dogs for cap- turing armadillos and skunk. A hunter said: '... we arrive at the hill around 10 pm; we loose the dogs and sit down waiting for them; they come back and we move to another place walking for 10 or 15 minutes; the dogs find the animal's burrow and we dig the animal out and capture it'. 11. Andrade-Lima D: Present-day Forest refuges in northeastern Brazil. In Biological diversification in the tropics Edited by: Prance. Columbia Univ. Press, New York; 1982:123-135. 12. Prance GT: Vegetation. In Biogeography and Quaternary history in tropical America Edited by: Whitmore TC, Prance GT. Oxford Science Publications; 1987:28-45. 13. Leal IR, Tabarlli M, Silva JMC: Ecologia e conservação da Caatinga Edi- tora Universitária, Universidade Federal de Pernambuco, Recife; 2003. 14. Silva JMC, Tabarelli M, Fonseca MT, Lins L: Biodiversidade da Caatinga: áreas prioritárias para conservação Ministério do Meio Ambiente, Brasília; 2004. 15. Leal IR, Silva JMC, Tabarelli M, Lacher TE: Changing the course of biodiversity conservation in the Caatinga of Northeastern Brazil. Conservation Biology 2005, 19(3):701-706. The interviewers report that they do not like to hunt from December to March because of the females' reproductive period or because they are caring after their pups. The hunters said: 'We do not hunt in December and January because the females are pregnant; and we do not hunt during the pups' birth period because if we kill the pregnant female we would kill "one plus four individuals"; and if we kill an animal 16. Tabarelli M, Vicente A: Conhecimento sobre plantas lenhosas da Caatinga: lacunas geográficas e ecológicas. In Biodiversidade da Caatinga: áreas prioritárias para conservação Edited by: Silva JMC, Tabarelli M, Fonseca MT, Lins L. Ministério do Meio Ambiente, Brasília; 2004:101-111. 17. Diegues AC, Arruda RS: Saberes tradicionais e biodiversidade no Brasil Brasília: Ministério do Meio Ambiente. São Paulo: USP; 2001. Page 6 of 7 (page number not for citation purposes) Page 6 of 7 (page number not for citation purposes) (page number not for citation purposes) Journal of Ethnobiology and Ethnomedicine 2006, 2:19 http://www.ethnobiomed.com/content/2/1/19 A classificação dos seres vivos entre os trabalhadores da pesca em Piratin- inga. Rio de janeiro, FUNARTE/Instituto Nacional do Folclore 1988. g j 30. Jensen AA: Sistemas indígenas de classificação de aves: aspectos compar- ativos, ecológicos e evolutivos Belém, Museu Paraense Emílio Goeldi; 1988. 31. Begossi A, Garavello JC: Notes on the ethnoichthyology od fish- ermen from the Tocantins river (Brazil). Acta Amazônia 1990, 20:341-118. 32. Paz VA, Begossi A: Ethnoichthyology of Gamboa Fishermen of Septiba Bay, Brazil. J Ethnobiol 1996, 16(2):157-168. 33. Costa Neto E: Etnoictiologia, desenvolvimento e sustentabili- dade no litoral norte baiano. Um estudo de caso entre pes- cadores do município do Conde. In Dissertação Universidade Federal de Alagoas. Maceió – AL; 1998. g 34. Mourão JS, Nordi N: Principais critérios utilizados por pes- cadores artesanais na taxonomia folk dos peixes do estuário do rio Mamanguape, Paraíba-Brasil. Interciência 2002, 27(11):607-612. ( ) 35. Mourão JS, Nordi N: Comparações entre as taxonomias folk e científica para peixes do estuário do rio Mamanguape, Paraíba-Brasil. Interciência 2002, 27(12):664-668. ( ) 36. Araujo HFP: Composição da avifuana e etnoornitologia em complexos estuários-manguezais no estado da Paraíba – Brasil. Dissertação. Universidade Federal da Paraíba. João Pessoa-PB 2002. 37. Hennig W: Phylogenetic Sistematic Urbana III. University of Tlenois Press; 1966. 38. Forth G: Ethnozoological Classification and Calssificatory Language among the Nage of Eastern Indonesia. J Ethnobiol 1995, 15(1):45-69. http://www.ethnobiomed.com/content/2/1/19 Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 7 of 7 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge ( ) 39. Varella-Freire , et al.: Zoogeografia do Rio Grande do Norte III. Lista preliminar dos mamíferos da Estação Ecológica do Seridó, SNN-RN. In Caderno Norte-riograndense de Temas Geográfi- cos Universidade Federal do Rio Grande do Norte, Natal – RN; 1997 37. Cabrera A, Yepes J: Mamíferos Sud. Americanos – Vidas, Costum- bres y Descipicion. Buenos Aires: Compañia Argentinas de Editores; 1995. 40. Ihering RV: Dicionário dos Animais do Brasil São Paulo: Editora Univer- sidade de Brasília;; 1968. 41. Clément D: Why is taxonomy utilitarian? 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https://openalex.org/W2792337751	https://europepmc.org/articles/pmc5796891?pdf=render	English	null	Restless Legs Syndrome/Willis–Ekbom Disease Is Prevalent in Working Nurses, but Seems Not to Be Associated with Shift Work Schedules	Frontiers in neurology	2,018	cc-by	5,828	R Siri Waage1,2, Ståle Pallesen3, Bente Elisabeth Moen1,4 and Bjørn Bjorvatn1,2 1 Department of Global Public Health and Primary Care, University of Bergen, Bergen, Norway, 2 Norwegian Competence Center for Sleep Disorders, Haukeland University Hospital, Bergen, Norway, 3 Department of Psychosocial Science, University of Bergen, Bergen, Norway, 4 Centre for International Health, University of Bergen, Bergen, Norway Insomnia and excessive sleepiness are among the most commonly reported sleep problems related to shift work. Sleep-related movement disorders have, however, received far less attention in relation to such work schedules. The objective of this study was to investigate the association between different shift work schedules and the prevalence of Restless legs syndrome/Willis–Ekbom disease (RLS/WED) in a large sample of Norwegian nurses. Our hypothesis was that shift working nurses would report higher prevalence of RLS/WED compared to day workers. A total of 1,788 nurses with different work schedules (day work, two-shift rotation, night work, three shift rotation) participated in a cohort study, started in 2008/2009. Four questions about RLS/WED based on the diagnostic criteria were included in wave 4 (2012). RLS/WED prevalence rates across different shift schedules were explored by the Pearson chi-square test. Logistic regression analysis was used to assess the association between RLS/WED and work schedules and shift work disorder (SWD) with adjustment for sex, age, marital status, smoking, and caffeine use. In total, 90.0% of the nurses were females, mean age 36.5 years (SD = 8.6, range 25–67). The overall prevalence of RLS/WED was 26.8%. We found no significant differences between the prevalence of RLS/WED across the different shift schedules, ranging from 23.3% (day work) to 29.4% (night work). There was a significant difference (p < 0.001) in the prevalence of RLS/WED between nurses having SWD (33.5%) compared to nurses not having SWD (23.8%). SWD remained significantly associated with RLS/WED in the adjusted logistic regression analysis (1.56, CI: 1.24–1.97). This study did not support the hypothesis. RLS/WED was associated with SWD, which might indicate that nurses vulnerable to shift work also are sensitive to other complaints related to a misalignment of the biological clock. Keywords: RLS, WED, sleep, shift work disorder, shift work, night work Citation: Waage S, Pallesen S, Moen BE and Bjorvatn B (2018) Restless Legs Syndrome/Willis–Ekbom Disease Is Prevalent in Working Nurses, but Seems Not to Be Associated with Shift Work Schedules. Original Research published: 29 January 2018 doi: 10.3389/fneur.2018.00021 Edited by: Thomas Pollmächer, Klinikum Ingolstadt, Germany Reviewed by: Thomas Christian Wetter, University of Regensburg, Germany Fabrizio De Carli, Istituto di Bioimmagini e Fisiologia Molecolare (CNR), Italy Reviewed by: Thomas Christian Wetter, University of Regensburg, Germany Fabrizio De Carli, Istituto di Bioimmagini e Fisiologia Molecolare (CNR), Italy Correspondence: Siri Waage siri.waage@uib.no *Correspondence: Siri Waage siri.waage@uib.no Specialty section: This article was submitted to Sleep and Chronobiology, a section of the journal Frontiers in Neurology Received: 08 November 2017 Accepted: 11 January 2018 Published: 29 January 2018 Edited by: Thomas Pollmächer, Klinikum Ingolstadt, Germany INTRODUCTION Shift work is associated with impaired health, whereof sleep problems are among the most commonly reported complaints (1, 2). Work schedules that involve night work are in particular associated with sleep problems, i.e., sleep onset and maintenance difficulties, reduced sleep duration, and exces- sive sleepiness during work (3). About 95% of shift workers working nights report sleep problems January 2018 \| Volume 9 \| Article 21 Frontiers in Neurology \| www.frontiersin.org 1 RLS/WED Associated with Shift Work Waage et al. most of the nurses in Norway. The initial sample comprised five equal strata based on the numbers of years since graduation from nursing school (0–11 months, 1–3 years, 3.1–6 years, 6.1–9 years, and 9.1–12 years). A total of 2,059 (response rate = 38.1%) com- pleted the questionnaire at the first wave (2008/2009). In order to increase the study population, an additional sample of 906 newly educated nurses (response rate = 33.1%) was recruited in 2009. The total sample in wave 1, therefore, included 2,965 nurses. The nurses who responded to the first wave were invited to participate in annual follow-ups by receiving questionnaires sent by postal mail with prepaid envelopes for returning the completed forms. Up to two reminders were sent for each wave to those who did not respond. For each wave, the nurses received information about a lottery performed upon completing and returning the questionnaires, where 25 individuals would win a gift card with a value of 500 NOK (~70 US $). The present study reports findings based on data from the fourth (2012) wave. In 2012, 2,198 nurses completed the questionnaire, yielding a response rate of 74.1%. Nurses who reported that they were no longer working as nurses in wave 4 were excluded from the analyses, leaving a total study population of 1,788 nurses. compared to 40% of day workers (4). Night workers are on duty during their biological resting phase and try to sleep during their biological active phase which has been proposed as a cause to sleep and health problems (5). Even though insomnia and excessive sleepiness have been widely studied in relation to shift work, other sleep disorders, like for instance sleep-related movement disorders, have received little attention in this type of working population. Shift Work Disorder (SWD)t Shift work disorder was measured with three previous used ques- tions (15, 16) based on the minimal criteria from the Diagnostic and Coding Manual from the second edition of the International Classification of Sleep Disorders (ICSD-2) (17). The questions were: (a) do you experience either difficulties sleeping or experi- ence excessive sleepiness? (yes or no), (b) is the sleep or sleepiness problem related to a work schedule that makes you work when you normally would sleep? (yes or no), and (c) have you had this sleep or sleepiness problem related to the work schedule for at least 1 month? (yes or no). Participants were classified as having SWD when they responded “yes” to all three questions. Instruments Demographicsh Despite the fact that the occurrence of RLS/WED seems to be influenced by a circadian component, few studies have investigated the relationship between shift work and this disorder. The few existing studies on this topic show inconsistent results (11–13). Both sleep deprivation and fatigue might trigger RLS/WED symptoms; however, the impact of chronic circadian disruption on RLS/WED is unclear. Shift work is associated with reduced sleep duration (3), which may aggravate RLS/WED. Sleeping on a regular schedule, following proper sleep hygiene, eliminating caffeine before sleep, no smoking, exercising, and losing weight are some of the most common behavioral recommendations to ease and prevent symptoms of RLS/WED (7). Still, many of these recommendations are difficult to adhere to when working shifts. Notably, shift work is associated with higher BMI, sleeping at irregular times, use of caffeine to prevent sleepiness at work and the like (14). Against this backdrop, it is conceivable that shift work may be associated with increased rates of RLS/WED. Therefore, the present study aimed to investigate the association between different shift work schedules and the prevalence of RLS/ WED in a population of Norwegian nurses. Our main hypothesis was that shift working nurses would report higher prevalence of RLS/WED compared to day workers. The demographic variables were collected in wave 4, except for age and sex that were collected in wave 1. The questionnaire included data on the sociodemographic variables; marital status and caretaker responsibility for children in the household, work- related variables; work schedule (day only, evening only, two-shift including day and evening, night only, three-shift schedule including day, evening and night, and other schedules), type and percentage of full time position, and life-style variables; daily smoking (yes/no), daily number of cups of caffeine and body mass index. Due to low numbers, the nurses who were working “evening shift only” (n = 8) and “other schedules involving night shifts” (n = 57) were excluded from the analysis. Also nurses who did not report their work schedule (n = 19) were excluded. INTRODUCTION One common sleep-related movement disorder is Restless legs syndrome/ Willis–Ekbom disease (RLS/WED), which is characterized by unpleasant sensations in the legs represented by crawling, pulling and creeping feelings, and with an urge to move the legs (6). The diagnosis is also described as a circadian disorder of sensory- motor integration (7). The symptoms show a clear circadian rhythmicity and worsen during evening and night or while at rest. The symptoms typically occur at the transition between waking and sleep, disrupting sleep onset, or the return to sleep (8). The symptoms are relieved, at least for a short time, when moving, walking, rubbing, and massaging the legs (6). Prevalence rates of RLS/WED in Europe and the USA are reported to be between 2.4 and 10.8% (9). In the general population in Norway, a prevalence of 14.3% was reported, in which about half reported symptoms of moderate to very severe degree (10). Studies have shown that RLS/ WED is more prevalent among females and increases with age (9). MATERIALS AND METHODS Procedure and Participantsh p The data stemmed from the ongoing longitudinal cohort study “SUrvey of Shift work, Sleep and Health (SUSSH)” among Norwegian nurses. The first data collection was conducted during winter 2008/2009 (wave 1) when a sample of 5,400 nurses was ran- domly selected from the Norwegian Nurses Organisation’s (NNO) membership roll and invited to participate. The NNO includes Restless Legs Syndrome/Willis–Ekbom Disease Restless Legs Syndrome/Willis–Ekbom Disease RLS/WED was assessed with questions based on the ICSD-2 (17), and in line with the current third edition of this diagnostic system (8). Four questions were used to make a diagnosis of RLS/ WED: (a) during the past 3 months, have you had an urge to January 2018 \| Volume 9 \| Article 21 Frontiers in Neurology \| www.frontiersin.org 2 RLS/WED Associated with Shift Work Waage et al. Table 1 \| Demographic characteristics of Norwegian nurses included in the study. Sex (n = 1,787): female 90.0% Age in years; mean and SD (n = 1,784) 36.5 (8.6) Married/cohabiting (n = 1,762) 74.2% Children living at home (n = 1,744): yes 54.5% Smoking (n = 1,778): yes 8.9% Cups of caffeine daily; mean and SD (n = 1,784) 3.5 (2.5) Body mass index; mean and SD (n = 1,771) 25.1 (4.5) Table 1 \| Demographic characteristics of Norwegian nurses included in the study. move the legs or arms, usually accompanied with discomfort or unpleasant sensations in the legs or arms? (b) If yes, does this urge start or increase when you are resting, such as when lying or sitting? (c) Is the urge to move or the unpleasant sensation partially or completely relieved when you are moving, such as when walking or stretching? (d) Is the urge to move or unpleasant sensation worse late in the day or at night compared with the rest of the day? All four questions were rated “yes” or “no.” Only those who answered yes to all four diagnostic criteria were defined as suffering from RLS/WED. RESULTS Demographic characteristics are presented in Table 1. In total, 1,608 (90.0%) of the nurses were females and 179 (10.0%) were males. Mean age in 2012 was 36.5 years (SD = 8.6, range 25–67). Altogether, 12.5% were day only workers, 34.6% were two-shift workers, 8.1% were night only workers, and 44.8% were three- shift workers. Mean age of the day only workers was 39.1 years, 37.2 years among the two-shift workers, 36.0 years among the night only workers, and 35.3 years among three-shift workers, respectively. Close to three quarters (74.2%) of the nurses were married/cohabiting, and 54.5% had children living at home.h Statistics IBM SPSS Statistics 23 for Windows was used for the statistical analyses. The prevalence of RLS/WED in relation to the different types of shift schedules was explored by the Pearson chi-square test. In addition, logistic regression analyses were used to assess RLS/WED as the dependent variable with work schedule (day only, two-shift, night only and three-shift, day shift as reference) and SWD as predictors and with sex, age, marital status, smoking, daily cups of caffeine use as covariates. In the total sample of nurses, 30.7% (n = 532) were classified as having SWD. There was a significant difference (p < 0.01) in the prevalence of RLS/WED between nurses having SWD (33.5%) compared to nurses not having SWD (23.8%). Table 3 reports the results from the logistic regression analyses with two predictors and five covariates where RLS/WED com- prised the dependent variable. Only age and having SWD were positively associated with RLS/WED. Table 4 reports similar results from the adjusted logistic regression analyses with the same predictors and covariates where severe and frequent RLS/ WED comprised the dependent variable. Ethicsh The study was approved by the Regional Committee for Medical and Health Research Ethics of Western Norway (REK-West, no 088.08). Informed consent in written form was obtained from all participants. the comparisons of severe RLS/WED and severe and frequent RLS/WED across the different work schedules, also showing non-significant associations. In terms of RLS/WED prevalence, there were no significant differences when comparing day only to the two-shift schedule (p = 0.17), day only compared to night only (p = 0.21), and day only compared to the three-shift schedule (p = 0.32).i Frontiers in Neurology \| www.frontiersin.org MATERIALS AND METHODS To assess the severity of symptoms, we also included a question about how much the nurses were bothered by the symptoms, rated on a five point scale ranging from “not bothered,” “mildly bothered,” “moderately bothered,” “quite bothered” to “very bothered,” and one question pertaining to how often they experienced the symptoms, rated on a five point scale ranging from “never,” “1 day or less per week,” “2–3 days per week,” “4–5 days per week,” and “6–7 days per week.” Severe RLS/ WED was defined as answering “yes” on all four diagnostic crite- ria and being from moderately bothered to very bothered. Severe and frequent RLS/WED was defined as having severe RLS/WED and at least having these symptoms 2 days or more per week. Table 2 \| Prevalence of RLS/WED among nurses in different types of shift work schedules. alence of RLS/WED among nurses in different types of shift work Table 2 \| Prevalence of RLS/WED among nurses in different types of shift work schedules. Day only Two- shift Night only Three- shift p-Value RLS/WED % (n) 23.3 (49) 28.2 (163) 29.4 (40) 26.8 (201) 0.51 Severe RLS/WED % (n) 11.7 (25) 14.2 (84) 11.6 (16) 11.9 (91) 0.57 Severe and frequent RLS/WED % (n) 9.4 (20) 9.8 (58) 5.1 (7) 8.0 (146) 0.28 RLS/WED: experienced RLS during the last 3 months. Severe RLS/WED: experienced RLS during the last 3 months and being at least moderately bothered. Severe and frequent RLS/WED: experienced RLS during the last 3 months, being at least moderately bothered and with a frequency of 2 days or more per week. DISCUSSION Age and cups of caffeine were entered as continuous variables. bAdjusted for sex and age. cAdjusted for sex, age, marital status, smoking, and use of caffeine. Significant values are shown in bold. RLS/WED, restless legs syndrome/Willis–Ekbom disease. bAdjusted for sex and age. cAdjusted for sex, age, marital status, smoking, and use of caffeine. Significant values are shown in bold. RLS/WED, restless legs syndrome/Willis–Ekbom disease. Table 4 \| Logistic regression analyses with severe and frequent RLS/WED as the dependent variable among shift working nurses in Norway (n = 1,788). Independent variables (n in crude analyses) Crudea OR (95% CI) Adjustedb OR (95% CI) n = 1,700–1,728 Adjustedc OR (95% CI) n = 1,650–1,696 Sex Female (1,579) 1.00 Male (176) 0.77 (0.42–1.41) Age (1,752) 1.04 (1.03–1.06) Married/cohabiting No (451) 1.00 Yes (1,301) 1.11 (0.75–1.64) Smoking No (1,590) 1.00 Yes (156) 2.00 (1.24–3.23) Cups of caffeine daily (1,752) 1.12 (1.06–1.19) Shift work disorder No (1,192) 1.00 Yes (531) 1.93 (1.37–2.72) 1.94 (1.37–2.73) 1.94 (1.37–2.74) Work schedule Day only (210) 1.00 1.00 1.00 Two-shift (577) 1.06 (0.62–1.80) 1.11 (0.65–1.92) 1.06 (0.61–1.83) Night only (136) 0.52 (0.21–1.25) 0.60 (0.24–1.46) 0.50 (0.20–1.23) Three-shift (751) 0.84 (0.49–1.42) 0.98 (0.57–1.68) 0.92 (0.54–1.59) aIn the crude regression analyses, each independent variable was entered separately. Age and cups of caffeine were entered as continuous variables. bAdjusted for sex and age. cAdjusted for sex, age, marital status, smoking, and use of caffeine. Significant values are shown in bold. RLS/WED, restless legs syndrome/Willis–Ekbom disease. Independent variables (n in crude analyses) Crudea OR (95% CI) Adjustedb OR (95% CI) n = 1,700–1,728 Adjustedc OR (95% CI) n = 1,650–1,696 aIn the crude regression analyses, each independent variable was entered separately. Age and cups of caffeine were entered as continuous variables. bAdjusted for sex and age. cAdjusted for sex, age, marital status, smoking, and use of caffeine. Significant values are shown in bold. RLS/WED, restless legs syndrome/Willis–Ekbom disease. WED among the nurses across the different shift schedules. Very few studies have explored the relationship between shift sched- ules and symptoms of RLS/WED, and the few results reported to date have been inconsistent. The present results were at odds with a cross-sectional study among 780 automobile manufactory a unique diagnostic feature (18). Rotating shift work and night work generally cause circadian rhythm misalignment and sleep problems including shortened sleep duration. Thus, such work hours were expected to accentuate sleep disturbances like RLS/ WED. DISCUSSION The present study showed that nurses working in different shift work schedules reported similar prevalence of RLS/WED as compared to nurses working regular day work. This finding did not support our hypothesis that shift working nurses would report higher prevalence of RLS/WED compared to day workers. However, the overall RLS/WED prevalence of 26.8% was higher than expected when compared to the prevalence reported in the general population. In addition, nurses with SWD reported more RLS/WED compared to nurses without SWD. The overall prevalence of RLS/WED was 26.8% (n = 470). In the total sample of nurses, the prevalence of severe RLS/WED was 12.4% and the prevalence of severe and frequent RLS/WED was 8.4%, respectively. There were no significant differences between the prevalence of RLS/WED and the different shift work schedules, ranging from 23.3% among day only workers to 29.4% among the night only workers (Table 2). Table 2 also presents Symptoms of RLS/WED worsen in the evening and during the night, and the circadian variation of symptoms constitutes January 2018 \| Volume 9 \| Article 21 3 RLS/WED Associated with Shift Work Waage et al. Table 3 \| Logistic regression analyses with RLS/WED as the dependent variable among shift working nurses in Norway (n = 1,788). I d d i bl ( i d l ) C d OR (9 % CI) b Table 3 \| Logistic regression analyses with RLS/WED as the dependent variable among shift working nurses in Norway (n = 1,788). Independent variables (n in crude analyses) Crudea OR (95% CI) Adjustedb OR (95% CI) n = 1,670–1,718 Adjustedc OR (95% CI) n = 1,640–1,686 Sex Female (1,579) 1.00 Male (176) 0.76 (0.52–1.10) Age (1,752) 1.03 (1.01–1.04) Married/cohabiting No (451) 1.00 Yes (1,301) 1.05 (0.82–1.34) Smoking No (1,590) 1.00 Yes (156) 0.78 (0.55–1.12) Cups of caffeine daily (1,752) 1.02 (0.98–1.06) Shift work disorder No (1,192) 1.00 Yes (531) 1.61 (1.29–2.02) 1.60 (1.28–2.01) 1.56 (1.24–1.97) Work schedule Day only (210) 1.00 1.00 1.00 Two-shift (577) 1.29 (0.90–1.87) 1.34 (0.92–1.94) 1.33 (0.91–1.93) Night only (136) 1.37 (0.84–2.23) 1.46 (0.89–2.40) 1.42 (0.86–2.35) Three-shift (751) 1.20 (0.84–1.72) 1.32 (0.92–1.90) 1.32 (0.91–1.90) aIn the crude regression analyses, each independent variable was entered separately. Age and cups of caffeine were entered as continuous variables. Independent variables (n in crude analyses) Crudea OR (95% CI) Adjustedb OR (95% CI) n = 1,670–1,718 Adj aIn the crude regression analyses, each independent variable was entered separately. n analyses, each independent variable was entered separately. Age and cups of caffeine were entered as continuous variables. DISCUSSION However, some of the variations in preva- lence rates of RLS/WED across studies could partly be explained by methodological differences in the assessment of the disorder. Not surprisingly, when we looked at severe RLS/WED and at severe and frequent RLS/WED, the prevalence rates dropped. In fact, when assessing only severe and frequent RLS/WED, the prevalence rates were lowest in the night only group (although not significant), in contrast to the finding for the diagnosis of RLS/WED. This result is difficult to interpret and further studies on this topic are warranted. Still, the results might reflect the healthy shift worker effect (24) or some methodological artifacts, such a low number of observations within some cells. t The increase of RLS/WED in the evening and during the night is explained by several factors. First, the symptoms increase with the increase of sleepiness in the evening. A second contributing factor is the decrease in motor activity in the evening compared with the daytime (19). Nurses working in the evening and during the night are likely to report less sleepiness at these hours since they need to be physical active during their work hours due to work tasks and work demands. On the other hand, studies have demonstrated that the circadian oscillation in RLS/WED may occur independently of activity or sleep deprivation (18). Still, the urge to move the legs and any accompanying unpleasant sensation begin or worsen during periods of rest or inactivity such as lying down or sitting, and the symptoms are relieved by movement, such as walking or stretching, at least as long as the activity continues (9). Hence, it is possible that the workers in the present study suffering of RLS/WED would report less complaints when they work during the evening or night, since their work- related activity could reduce their symptoms. Thus, the specific occurrence of RLS/WED symptoms in shift workers should, therefore, be studied in more detail, for example, by sleep and RLS/WED diaries. Also, studies with longitudinal designs should be performed. In our study, the presence of a healthy worker effect is likely to be present and could hamper the ability to detect a relationship between RLS/WED and shift work. Furthermore, it should also be taken into account that the vast majority of the nurses in the present study were females working more than 50% of full position in addition to having child and family obligations. DISCUSSION However, we found no differences in the prevalence of RLS/ January 2018 \| Volume 9 \| Article 21 Frontiers in Neurology \| www.frontiersin.org 4 RLS/WED Associated with Shift Work Waage et al. etc.) between these and other sleep-related disorders (22). It is also possible that people suffering of sleep-related problems like RLS/WED and SWD could be prone to associate their sleep problems to shift work.t workers in Iran where a significant higher prevalence of RLS/ WED in rotational shift workers compared to permanent day workers was reported. The highest prevalence rate in that study was found among rotating shift workers, mostly working night shifts. The authors conclude that rotational shift work may act as a risk or exacerbating factor for RLS/WED (13). The present findings are, however, in accordance with another Iranian study among textile factory workers where no significant difference in the prevalence of RLS/WED was found when comparing 210 shift workers to 204 non-shift workers (12). Similarly, one Greek epidemiological study also reported no association between RLS/ WED and shift work (11).h pt Despite that our hypothesis about shift workers experiencing more RLS/WED compared to day workers was not supported, the overall prevalence of RLS/WED was high in the present population of nurses. Prevalence rates of RLS/WED throughout the Western part of the world vary between 2.4 and 15% (9, 23). In Norway, a mean overall prevalence rate of RLS/WED in the general population of 14.3% has been reported (10). RLS/WED is, however, more frequent in females compared to males (8), and females older than 35 years of age are twice as likely to suffer from RLS/WED compared to males (9). The mean age of the day workers was slightly higher than in the shift working groups. This may be one possible explanation why there was no difference in the prevalence of RLS/WED between day workers compared to the other shift working groups, as the prevalence of RLS/WED increase with age. It should, however, be noted that age was controlled for as a possible confounder in the analyses. Among females in two Scandinavian countries (Denmark and Norway together), a prevalence rate of 13.4% was reported (10). About 90% of the nurses in the present study were females, and in addi- tion their mean age was 36.5 years, which can be one reason for the high prevalence. DISCUSSION Hence, most of the sample represented a healthy working popula- tion of “double-working women.” Frontiers in Neurology \| www.frontiersin.org REFERENCES a comparison between shift workers and nonshift workers. Sleep Disord (2014) 2014:870320. doi:10.1155/2014/870320 1. Costa G. Shift work and occupational medicine: an overview. Occup Med (2003) 53:83–8. doi:10.1093/occmed/kqg045 t 1. Costa G. Shift work and occupational medicine: an overview. Occup Med (2003) 53:83–8. doi:10.1093/occmed/kqg045 13. Sharifian A, Firoozeh M, Pouryaghoub G, Shahryari M, Rahimi M, Hesamian M, et al. Restless legs syndrome in shift workers: a cross sec- tional study on male assembly workers. J Circadian Rhythms (2009) 7:12. doi:10.1186/1740-3391-7-12 qg 2. Harma M, Kecklund G. Shift work and health – how to proceed? Scand J Work Environ Health (2010) 36:81–4. doi:10.5271/sjweh.2902 qg 2. Harma M, Kecklund G. Shift work and health – how to proceed? Scand J Work 2. Harma M, Kecklund G. Shift work and health – how to proceed? Scand J Work Environ Health (2010) 36:81–4. doi:10.5271/sjweh.2902 t Environ Health (2010) 36:81–4. doi:10.5271/sjweh.2902 t Environ Health (2010) 36:81–4. doi:10.5271/sjweh.2902 14. Pallesen S, Bjorvatn B, Mageroy N, Saksvik IB, Waage S, Moen BE. Measures to counteract the negative effects of night work. Scand J Work Environ Health (2010) 36:109–20. doi:10.5271/sjweh.2886 3. Akerstedt T. Shift work and disturbed sleep/wakefulness. Occup Med (2003) 53:89–94. doi:10.1093/occmed/kqg046 tfifi 4. Wedderburn AI. Shiftwork and Health. Luxenbourg: Office for Official Publications of the European Communities (2000). 15. Flo E, Pallesen S, Mageroy N, Moen BE, Gronli J, Hilde Nordhus I, et al. Shift work disorder in nurses – assessment, prevalence and related health problems. PLoS One (2012) 7:e33981. doi:10.1371/journal.pone. 0033981 Publications of the European Communities (2000). 5. Smith CS, Robie C, Folkard S, Barton J, Macdonald I, Smith L, et al. A process model of shiftwork and health. J Occup Health Psychol (1999) 4:207–18. doi:10.1037/1076-8998.4.3.207 model of shiftwork and health. J Occup Health Psychol (1999) 4:207–18. doi:10.1037/1076-8998.4.3.207 16. Waage S, Moen BE, Pallesen S, Eriksen HR, Ursin H, Akerstedt T, et al. Shift work disorder among oil rig workers in the North Sea. Sleep (2009) 32:558–65. doi:10.1093/sleep/32.4.558 i 6. Allen RP, Picchietti D, Hening WA, Trenkwalder C, Walters AS, Montplaisi J, et al. Restless legs syndrome: diagnostic criteria, special considerations, and epidemiology. A report from the restless legs syndrome diagnosis and epidemiology workshop at the National Institutes of Health. Sleep Med (2003) 4:101–19. doi:10.1016/S1389-9457(03)00010-8 17. American Academy of Sleep Medicine. International Classification of Sleep Disorders, Diagnostic and Coding Manual. Westchester, Illinois: American Academy of Sleep Medicine (2005). 7. Wijemanne S, Jankovic J. Limitations and Strengthsh The present study was based on a large and homogeneous sample of nurses, which limits the influence from possible confounding variables. In addition, the study used a standardized and a well- validated instrument to diagnose RLS/WED. As a limitation, the response rate in the first wave was low, which may make the interpretation of the data and conclusions less generalizable. Still, it is an asset that the response rate in the wave where RLS/ WED was assessed was as high as 74.1%. Another limitation of the present study was the fact that all data exclusively were based on self-reports, and not supplemented by, e.g., clinical interviews. Such interviews would strengthen the validation of the diagnosis and could also provide additional information about the onset of symptoms, family history of RLS/WED, possible use of medica- tion, and so on. In addition, the symptoms that were included in the questions to assess RLS/WED are similar to symptoms of other medical and behavioral conditions such as myalgia, leg edema, leg cramps, and others, and could, therefore, be confused with RLS/WED (18). This could as such have contributed to an overestimation of the RLS/WED prevalence rate. However, The prevalence of RLS/WED is reported to increase with increasing age, and the mean age of onset is during the third or fourth decade (20). Our findings were in line with this, as we found that RLS/WED was positively associated with age. In addition, we found an association between RLS/WED and SWD. SWD is a circadian rhythm sleep disorder that is due to a work schedule that overlaps with the individuals’ habitual sleep period. Main symptoms are insomnia during the sleep period and/or excessive sleepiness during the wake period (21). The prevalence of SWD has previously been reported to be high in Norwegian nurses (15). The fact that SWD was associated with RLS/WED in these nurses might indicate that nurses having one sleep disorder associated to shift work are more sensitive and are at greater risk to also experience other complaints related to their work schedule. Future studies should, therefore, investigate whether there might be some common vulnerability (e.g., genetic, health behavior, January 2018 \| Volume 9 \| Article 21 Frontiers in Neurology \| www.frontiersin.org 5 Waage et al. RLS/WED Associated with Shift Work ETHICS STATEMENT since our aim was to study differences in prevalence across shift schedules, such an overestimation will likely not influence com- parisons. Still, it should be noted that the RLS/WED diagnosis, according to the diagnostic criteria, is based on subjective assess- ment (8, 17). The overall prevalence of RLS/WED in the present study was high, but we found no difference between day workers and the shift working groups. A limitation of this study was that information about former shift work was not collected. Thus, it is likely to assume that the day worker group also consisted of some former shift workers. Furthermore, although the prevalence of RLS/WED was high, the number of persons with this diagnosis in some of the shift work groups was low, which may have reduced the statistical power of the study. The study was approved by the Regional Committee for Medical and Health Research Ethics of Western Norway (REK-West, no 088.08). Informed consent in written form was obtained from all participants. FUNDING The results of the present study did not support the hypothesis that shift workers report higher prevalence of RLS/WED com- pared to day workers. Still, the overall prevalence of RLS/WED in this population of Norwegian nurses was high. Furthermore, RLS/WED was associated with SWD, which might indicate that nurses vulnerable to shift work also are sensitive to other com- plaints related to a misalignment of the biological clock. The study received a grant for practical administration and data collection from The Western Norway Regional Health Authority (grant number 911386, no personal payment/salary). The Norwegian Nurses Organization has provided grants to cover some of the running expenses of the SUSSH study. The study was further partly funded from Nordforsk, Nordic Program on Health and Welfare (74809). The study received a grant for practical administration and data collection from The Western Norway Regional Health Authority (grant number 911386, no personal payment/salary). The Norwegian Nurses Organization has provided grants to cover some of the running expenses of the SUSSH study. The study was further partly funded from Nordforsk, Nordic Program on Health and Welfare (74809). AUTHOR CONTRIBUTIONS SW: design of the study, collecting the data, data analysis, inter- pretation of the results, and drafting the paper. SP, BM, and BB: design of the study, interpretation of the results, and critical review of the paper. All authors have approved the final manuscript. 24. Knutsson A. Methodological aspects of shift-work research. Chronobiol Int (2004) 21:1037–47. doi:10.1081/CBI-200038525 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. REFERENCES Restless legs syndrome: clinical presentation diag- nosis and treatment. Sleep Med (2015) 16:678–90. doi:10.1016/j.sleep.2015. 03.002 18. Allen RP, Picchietti DL, Garcia-Borreguero D, Ondo WG, Walters AS, Winkelman JW, et al. Restless legs syndrome/Willis-Ekbom disease diagnostic criteria: updated International Restless Legs Syndrome Study Group (IRLSSG) consensus criteria – history, rationale, description, and significance. Sleep Med (2014) 15:860–73. doi:10.1016/j.sleep.2014.03.025 8. American Academy of Sleep Medicine. International Classification of Sleep Disorders. 3rd ed. Darien IL, editor. Darian: American Academy of Sleep Medicine (2014). 9. Garcia-Borreguero D, Williams AM. An update on restless legs syndrome (Willis-Ekbom disease): clinical features, pathogenesis and treatment. Curr Opin Neurol (2014) 27:493–501. doi:10.1097/WCO.0000000000000117 19. Michaud M, Dumont M, Selmaoui B, Paquet J, Fantini ML, Montplaisir J. Circadian rhythm of restless legs syndrome: relationship with biological markers. Ann Neurol (2004) 55:372–80. doi:10.1002/ana.10843 20. Picchietti DL, Van Den Eeden SK, Inoue Y, Berger K. Achievements, chal- lenges, and future perspectives of epidemiologic research in restless legs syndrome (RLS). Sleep Med (2017) 31:3–9. doi:10.1016/j.sleep.2016.06.007 t 10. Bjorvatn B, Leissner L, Ulfberg J, Gyring J, Karlsborg M, Regeur L, et al. Prevalence, severity and risk factors of restless legs syndrome in the general adult population in two Scandinavian countries. Sleep Med (2005) 6:307–12. doi:10.1016/j.sleep.2005.03.008 21. Zee PC, Goldstein CA. Treatment of shift work disorder and jet lag. Curr Treat Options Neurol (2010) 12:396–411. doi:10.1007/s11940-010-0090-9 h 11. Hadjigeorgiou GM, Stefanidis I, Dardiotis E, Aggellakis K, Sakkas GK, Xiromerisiou G, et al. Low RLS prevalence and awareness in central Greece: an epidemiological survey. Eur J Neurol (2007) 14:1275–80. doi:10.1111/j.1468-1331.2007.01966.x 22. Thun E, Le Hellard S, Osland T, Bjorvatn B, Moen B, Mageroy N, et al. Circadian clock gene variants and insomnia, sleepiness, and shift work disorder. Sleep Biol Rhythms (2016) 14:55–62. doi:10.1007/s41105-015-0023-9 12. Yazdi Z, Sadeghniiat-Haghighi K, Loukzadeh Z, Elmizadeh K, Abbasi M. Prevalence of sleep disorders and their impacts on occupational performance: 23. Einollahi B, Izadianmehr N. Restless leg syndrome: a neglected diagnosis. Nephrourol Mon (2014) 6:e22009. doi:10.5812/numonthly.22009 January 2018 \| Volume 9 \| Article 21 Frontiers in Neurology \| www.frontiersin.org 6 Waage et al. RLS/WED Associated with Shift Work Copyright © 2018 Waage, Pallesen, Moen and Bjorvatn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Copyright © 2018 Waage, Pallesen, Moen and Bjorvatn. 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. RLS/WED Associated with Shift Work January 2018 \| Volume 9 \| Article 21 Frontiers in Neurology \| www.frontiersin.org REFERENCES 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. January 2018 \| Volume 9 \| Article 21 Frontiers in Neurology \| www.frontiersin.org
https://openalex.org/W4315698768	https://rua.ua.es/dspace/bitstream/10045/122794/6/CultCuid62_20.pdf	es		Cuidando en el hogar a una persona con enfermedad cerebrovascular: del desconocimiento a la satisfacción	Cultura de los cuidados	2,022	cc-by	7,018	294 Cuidando en el hogar a una persona con enfermedad cerebrovascular: del desconocimiento a la satisfacción Caring a Person with Cerebrovascular disease at home: from the lack knowledge to satisfaction Cuidando de uma pessoa com doença cerebrovascular em casa: da ignorância à satisfação Fredy Tamayo-Botero1, Mauricio Arias-Rojas2, Stefany Ortega-Pérez3 1 Magister en Enfermería. Estudiante de Doctorado en Enfermería. Profesor asociado, Facultad de Enfermería, Universidad de Antioquia. Medellín, Colombia. Orcid: https://orcid.org/00000003-2828-9216. Correo: duvan.tamayo@udea.edu.co 2 Doctor en Enfermería. Profesor asistente, Facultad de Enfermería, Universidad de Antioquia. Medellín, Colombia. Orcid: https://orcid.org/0000-0003-2096-1792. Correo: emarias@udea.edu.co 3 Doctora en Enfermería. Profesora asociada, Departamento de Enfermería, Universidad del Norte. Barranquilla, Colombia. Orcid: https://orcid.org/0000-0003-3058-5161 Correo: srortega@uninorte.edu.co Correo electrónico de contacto: duvan.tamayo@udea.edu.co Correspondencia: Calle 64 no. 53-09, Oficina 323. Medellín, Colombia. Para citar este artículo: Tamayo-Botero, F. Arias-Rojas, M. Ortega-Pérez, S. (2022). Cuidando en el hogar a una persona con enfermedad cerebrovascular: del desconocimiento a la satisfacción. Cultura de los Cuidados, 26(62). Recuperado de http://dx.doi.org/10.14198/cuid.2022.62.20 Recibido:02/11/2021 Aceptado: 02/02/2022 ABSTRACT Introduction: cerebrovascular diseases generate disability and dependence. It is the family caregiver who assumes responsibility for the daily care of the patient with sequelae of this disease. Assuming this new role is not an easy activity and requires preparation and accompaniment. Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 295 Objective: to know the experience of the family caregiver when they first take care of a person with sequelae of a stroke at home. Methodology: qualitative study with a grounded theory approach. 16 family caregivers participated. The information was collected through semistructured interviews. The data analysis was done with initial, focused, axial and theoretical coding. Results: five categories were identified: Taking care of my family: a new experience, Changing the environment: making the experience easier, Support from others: feeling company in care, Feeling satisfaction: the result of care well done and Living care: between fear, faith and love. The central category was called Caring at home: from lack of knowledge to satisfaction. Conclusions: the experience of caring for the first time represents a challenge for the family caregiver to create new strategies that favor caring for the person. Nursing professionals have the responsibility of designing nursing interventions that meet the specific needs of this population. Key words: Carer; Caregivers; Stroke; Home Nursing Care, Grounded Theory RESUMEN Introducción: las enfermedades cerebrovasculares generan discapacidad y dependencia. Es el cuidador familiar quien asume la responsabilidad del cuidado diario de la persona con secuelas de esta enfermedad. El asumir este nuevo rol no es una actividad sencilla y requiere de preparación y acompañamiento. Objetivo: comprender la experiencia del cuidador familiar cuando asume por primera vez el cuidado en el hogar de una persona con secuelas de un accidente cerebrovascular. Metodología: estudio cualitativo con enfoque de teoría fundamentada. Participaron 16 cuidadores familiares. Se recolectó la información por entrevistas semiestructuradas. El análisis de los datos se hizo con codificación inicial, focalizada, axial y teórica. Resultados: se identificaron cinco categorías: Asumiendo el cuidado de mi familiar: una nueva experiencia, Cambiando el entorno: haciendo más fácil la experiencia, El apoyo de otros: sintiendo compañía en el cuidado, Sintiendo satisfacción: el resultado de un cuidado bien hecho y Viviendo el cuidado: entre el miedo, la fe y el amor. La categoría central se denominó Cuidando en el hogar: desde la falta de conocimiento a la satisfacción. Conclusiones: la experiencia de cuidar por primera vez representa para el cuidador familiar un reto para crear nuevas estrategias que favorezcan el cuidado de la persona. Los profesionales de enfermería tienen la responsabilidad de diseñar intervenciones de enfermería que atiendan las necesidades específicas de esta población. Palabras clave: Cuidadores; accidente cerebrovascular; cuidados de enfermería en el hogar; teoría fundamentada. RESUMO Introdução: as doenças cerebrovasculares geram incapacidade e dependência. É o cuidador familiar que assume a responsabilidade pelo cuidado diário ao pessoa com sequelas desta doença. Assumir este novo papel não é uma atividade fácil e requer preparação e acompanhamento. Objetivo: conhecer a vivência do cuidador familiar ao cuidar pela primeira vez de uma pessoa com sequela de AVE no domicílio. Metodologia: estudo qualitativo com abordagem teórica fundamentada. Participaram 16 cuidadores familiares. As informações foram coletadas por meio de entrevistas semiestruturadas. A análise dos dados foi realizada com codificação inicial, focada, axial e teórica. Resultados: foram identificadas cinco categorias: Cuidar do meu familiar: uma nova experiência, Mudar o ambiente: Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 296 tornar a experiência mais fácil, O apoio dos outros: sentir-se companhia no cuidado, Sentir-se satisfeito: o resultado de um cuidado bem feito , Viver o cuidado: entre o medo, a fé e o amor. Conclusões: a experiência de cuidar pela primeira vez representa um desafio para o cuidador familiar criar novas estratégias que favoreçam o cuidar da pessoa. Os profissionais de enfermagem têm a responsabilidade de projetar intervenções de enfermagem que atendam às necessidades específicas desta população. Palavras chave: Cuidadores; acidente vascular cerebral; cuidados de enfermagem no domicílio; teoria fundamentada. INTRODUCCIÓN El accidente cerebrovascular (ACV) es una de las principales causas de muerte y discapacidad en el mundo (Yanez, Useche, Bayona, Porras, & Carrasquilla, 2020); es la primera causa de años de vida ajustados por discapacidad (AVAD), la segunda causa de años de vida potencial perdidos (AVPP) y la quinta causa de años de vida saludable perdidos (AVISA) en Latinoámerica (Feigin et al., 2019). La persona después de un ACV presenta problemas físicos, cognitivos y emocionales que perturban su capacidad de vivir de forma independiente y con frecuencia necesitan asistencia permanente para las actividades de la vida diaria (Khaku & Tadi, 2020; Rouanet & Silva, 2019). En la mayoría de los países de la región de América Latina y el Caribe, esta asistencia es proporcionada principalmente por los miembros de la familia, quienes se convierten en el principal cuidador (Ortega-Perez, Sanchez-Rubio, De las Salas, & Borja-Gonzalez, 2019). Cuidar en casa no es una actividad sencilla, el cuidador familiar (CF) adquiere este nuevo rol de forma inesperada, sin conocimientos, ni preparación para realizar actividades como alimentación, rehabilitación, higiene corporal y administración de medicamentos. Además, la carga de cuidado instrumental que trae este tipo de enfermedad es superior a las de otras enfermedades crónicas (D’Onofrio et al., 2015). La evidencia disponible en la literatura ha descrito las necesidades y dificultades que deben enfrentar los cuidadores familiares de personas con ACV durante la hospitalización de la persona y una vez estos son dados de alta. Entre estas se encuentran la necesidad de darle una mayor continuidad al cuidado pues el cuidador experimenta una falta de soporte y sentimientos de sentirse abandonado (Martinsen, Kirkevold, & Sveen, 2015), la necesidad de mayor acceso y soporte en la organización y establecimiento de una rutina Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 297 para el cuidado de la persona en el hogar y de soporte social y emocional (Hare, Rogers, Lester, McManus, & Mant, 2006). Otro aspecto importante es la falta de instrucción para acceder a servicios como la rehabilitación y fisioterapia pues se perciben muchas barreras para el acceso a estas (Cecil, Thompson, Parahoo, & McCaughan, 2013). Finalmente, los estudios describen la poca preparación que los cuidadores reciben en el alta hospitalaria de la persona con ACV lo que genera pánico y ansiedad (Cecil, Parahoo, Thompson, McCaughan, Power, & Campbell, 2011). El proceso de asumir el cuidado por primera vez de una persona con ACV se ha descrito de manera tangencial desde las necesidades que presentan los cuidadores en cada fase del proceso de enfermedad y estabilización de la condición de la persona enferma. Así, en un primer momento con la persona hospitalizado las necesidades principalmente giran en torno a la persona con ACV, la atención recibida y la información honesta. Luego en un segundo momento previo al alta las necesidades se expresan en información sobre la medicación, tratamiento y fisioterapia, los problemas físicos. Posterior al alta se identificó como necesario el reconocer cómo manejar los cambios comportamentales de la persona y las emergencias médicas, finalmente 3 meses después de la hospitalización las necesidades están enfocadas en la consecución de ayuda por parte de la red familiar y comunitaria (Tsai, Yip, Tai, & Lou, 2015). A la fecha la literatura ha reportado la experiencia de los cuidadores desde las necesidades, dificultades y desafíos (Pindus, Mullis, Lim, Wellwood, Rundell, Abd, et al., 2018), así como desde variables de resultado de la experiencia de cuidar como la sobrecarga del cuidado y la calidad de vida (Caminha, Denubila, & Marinho, 2018), pero la descripción del proceso de asumir por primera vez el cuidado de una persona con ACV documentando dicha experiencia desde el alta hospitalaria hasta el posterior cuidado en el hogar, no se ha realizado. Así pues, comprender esta experiencia como un continuo en el cuidador novato puede servir como punto de partida para mejorar la atención de otros cuidadores que se van a enfrentar a este proceso. En países de ingresos medios como Colombia, es necesario documentar la experiencia del cuidador de la persona con ACV pues las características del contexto, la cultura y el sistema de salud hacen que esta situación sea distinta a la documentada en cuidadores en otros países de ingresos altos (Rubiano, Carney, Chesnut, & Puyana, 2015). Por consiguiente, el objetivo de esta investigación fue comprender la experiencia de los CF que asumen por primera vez el cuidado de su familiar con ACV en Barranquilla-Colombia. Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 METODOLOGÍA Se desarrolló fundamentada 298 un constructivista estudio cualitativo siguiendo los con postulados abordaje de Kathy de teoría Charmaz (Charmaz, 2006). El interaccionismo simbólico como propuesta epistemológica de la teoría fundamentada permite reconocer la construcción y los significados atribuidos a la experiencia por parte de los participantes enmarcado en un proceso social que para este estudio fue asumir el cuidado de una persona, esta perspectiva además facilitó al investigador reconocer el sentido atribuido a las acciones desde la perspectiva de los participantes. En este sentido, en la interpretación de los datos se priorizaron los significados que los participantes atribuyeron a su experiencia (Corbin & Strauss, 2014). El estudio se desarrolló en la ciudad de Barranquilla–Colombia durante el primer semestre del 2019 y tuvo aprobación por parte del comité de ética de la Universidad del Norte (Acta no. 184). Participantes: se incluyeron cuidadores familiares mayores de 18 años que cuidaran a personas que sufrieron un ACV que estuvieran en la fase pos-temprana (menos de 3 meses), que fueran el principal respondiente del familiar, así como que fuera la primera vez que asumían el cuidado de una persona enferma. Se excluyeron aquellos cuidadores que fueran contratados, con experiencias previas como cuidadores, aquellos cuidadores de familiares que su lugar de alta fueran centros de cuidado crónico o residencias y aquellos que no fueran el cuidador principal de la persona. Se utilizó un muestreo teórico en el que a medida que la información era recolectada y analizada, se incluían más participantes para expandir cada una de las categorías. Recolección de la información: se realizó una llamada telefónica a los posibles participantes, se presentaron los objetivos del estudio y se verificaron los criterios de inclusión. Con los CF que aceptaron participar de forma verbal, se programó una visita domiciliaria y se firmó el consentimiento informado escrito. Los investigadores estaban entrenados en la realización de entrevistas semiestructuradas. Las siguientes preguntas sirvieron de guía para realizar la entrevista a los participantes: 1) ¿cómo fue la experiencia desde el alta del hospital hasta su llegada al domicilio con el familiar?, 2) ¿qué sintió usted como cuidador cuando tuvo que asumir por primera vez el cuidado de su ser querido en el domicilio? Mientras transcurría la entrevista se hicieron otras preguntas para profundizar en las respuestas dadas y se hicieron notas de campo para complementar la información. Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 299 En total se realizaron 16 entrevistas durante los meses de abril a mayo de 2019, cada uno de los participantes fue entrevistado solo una vez. Las entrevistas tuvieron una duración de 45 a 90 minutos. Todas las entrevistas fueron transcritas textualmente, así mismo fueron encriptadas para conservar el anonimato de los participantes. Se transcribieron 16 diarios de campo que fueron incluidos en el análisis de la información. Análisis de la información Las entrevistas se transfirieron al programa Atlas-ti para su análisis. La información fue analizada usando el método analítico constructivista propuesto por Charmaz (2006), inicialmente dos de los investigadores, este análisis inicial fue discutido con el otro investigador. El análisis de las entrevistas se llevó a cabo a través de 4 fases no lineales entre ellas: 1) codificación inicial de la información que resultó en 320 códigos. 2) codificación enfocada que permitió la asociación sistemática entre los códigos que resultó en 12 subcategorías, los códigos se asociaron por su familiaridad. 3) codificación axial en la que se establecieron las categorías principales y las subcategorías, esto determinado por sus relaciones y propiedades, aquí surgieron 5 categorías. 4) codificación teórica en la que se especificaron las relaciones entre las categorías de forma integrativa lo que permitió el desarrollo de una categoría central que describiera de forma general el fenómeno de estudio. Este proceso de recogida de datos y codificación se hizo de forma simultánea, aspecto que favoreció la recolección de información faltante. El muestreo teórico finalizó cuando no surgieron nuevos conceptos. Como criterios de rigor, se tuvo en cuenta durante todo el proceso de análisis y codificación, la interpretación de los códigos dentro del contexto, así mismo todo el equipo de investigación validó y definió la categoría central. Por otra parte, se cumplieron los criterios de reflexividad por parte de los autores cuando estos expresaron sus preconcepciones sobre el fenómeno, así como cuando se realizaron notas de campo y en los momentos en los que se discutió entre todos los investigadores las ideas frente a los hallazgos del estudio. En la misma línea la credibilidad se logró cuando los hallazgos se devolvieron a los participantes para ser confirmados y al momento de incluir en los hallazgos de este estudio los extractos de entrevistas que validan el contenido propuesto en la categoría. La transferibilidad se logró al hacer una descripción clara del contexto de investigación y las características sociodemográficas de los participantes del estudio (Guba & Lincoln, 1994). Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 300 RESULTADOS Se incluyeron un total de 16 cuidadores familiares, con una frecuencia de edad entre los 51 a 70 años; la mayoría eran mujeres (68,7%), amas de casa (62,5%), con nivel educativo de secundaria (62,5%) y con una relación con el familiar de esposa o hija. Estos cuidadores llevaban en promedio 6 meses cuidando y pasaban en promedio 12 horas al día dedicados al familiar. Las demás características sociodemográficas de los cuidadores se presentan en la tabla 1. Con respecto a las personas cuidadas en su mayoría eran hombres que presentaron ACV hemorrágico (n=11) e isquémico (n=5), con puntajes de la escala de Rankin modificada entre 4-5. Finalmente, se identificaron cinco categorias con doce subcategorías: 1) asumiendo el cuidado de mi familiar: una nueva experiencia, 2) cambiando el entono: haciendo más fácil la experiencia, 3) el apoyo de otros: sintiendo compañía en el cuidado, 4) sintiendo satisfacción: el resultado de un cuidado bien hecho, 5) viviendo el cuidado: entre el miedo, la fe y el amor. Dado que estas categorías plantean la vivencia de un proceso, se estructuró una categoría central que narra de forma longitudinal la experiencia que tienen los cuidadores desde el primer momento el cuidado de un familiar hasta que logran sentirse satisfechos. A continuacion se hace una descripcion de cada una de ellas. En la grafica 1 se muestra el esquema de la teoría. Asumiendo el cuidado de mi familiar: una nueva experiencia. Cuando la persona que ha sufrido un ACV regresa de su estancia hospitalaria al hogar, requiere del acompañamiento del CF para realizar algunas actividades de la vida diaria según la discapacidad o secuelas derivadas de la enfermedad. Ante esta situación, el CF debe asumir nuevas actividades en el hogar relacionadas con el cuidado como la alimentación, el baño y el confort de su ser querido, para las cuales no tiene entrenamiento ni se encuentra preparado. Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 301 Tabla 1. Características sociodemográficas de los participantes. Características Socio-demográficas de los CF N=16 (%) Género Femenino 11 (68,75%) Edad 18-30 3 (18,75%) 31- 50 5 (31,25%) 51-70 8 (50%) Grado de Secundaria 10 (62,5%) escolaridad Técnico 4 (25%) Universitario 2 (12,5%) Casado 11 (68,75%) Soltero 5 (31,25%) Católica 13 (81,25%) Evangélica 3 (18,75%) Si 2 (12,5%) No 13 (81,25%) Estrato 1 3 (18,75%) socioeconómico 2 13 (81,25%) Ocupación Hogar 10 (62,5%) Empleado – Trabajo independiente 6 (37,5%) Estado civil Religión Único cuidador Fuente: autores, 2020. La complejidad de estas actividades se relaciona con el grado de dependencia para el autocuidado de la persona con ACV y de las habilidades que vaya desarrollando el CF a través del tiempo. Estas actividades se adicionan a otras responsabilidades innatas en el cuidador según el rol convencional que ocupaba en el hogar. Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 302 Grafico 1. Representación gráfica de la teoría Fuentes: autores, 2020. Al respecto, un cuidador narra cómo fue asumir estas nuevas actividades y por tanto una nueva experiencia: “Y fue un proceso bien duro, después de que llegamos para acá la tuvimos que llevar de la mano, yo la bañaba…(E5YPm). Así mismo, otro de los participantes comentó como fue el proceso de actividades como el baño del familiar en el hogar: “yo me levanto, con el encima y lo llevo para el patio y allá lo baño, lo bañamos y todo eso”. (E2FTh) Ante estas nuevas experiencias el CF reconoce que no tiene los conocimientos y el entrenamiento para cuidar y asumir la dependencia, llevándolo a buscar información sobre cómo hacerlo de forma adecuada. Esta información la obtiene de personas conocidas que han vivido situaciones con familiares con un ACV y que por parte del personal de la salud. Algunos de los cuidadores informaron que desde el hospital Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 303 recibieron información parcial por parte del personal de enfermería que les ayudó a manejar a su familiar en el hogar. Por otra parte, un elemento fundamental en esta nueva experiencia es el proceso de comunicación entre el cuidador y el familiar. Cuando las secuelas del familiar están relacionadas con la pérdida de funciones cognitivas como el habla, se hace difícil la expresión de sus necesidades y llevan a que el cuidador desarrolle estrategias para mejorar la comunicación por medio de elementos o instrumentos que permitan a la persona enferma informar al cuidador cuando requiere algo. Por ejemplo, uno de los participantes diseñó una campana con materiales caseros para que su padre pudiera llamarlos cuando necesitara algo de ellos: “si él está allá y nosotras aquí en la sala, y si necesita algo ¿Cómo va a avisar? Entonces yo busque dos vasos de aluminio y le metí unas bolitas de cristal para que avisara si necesitaba algo” (E4Van) Cambiando el entorno: haciendo más fácil la experiencia Como parte del proceso de adaptación a la nueva experiencia de cuidar a una persona con ACV, el cuidador familiar realiza modificaciones a diversos elementos de su entorno físico y social con el fin de asumir de mejor manera el nuevo rol. Estas modificaciones en el cuidador pueden variar según el grado de dependencia con el que haya quedado la persona enferma, afectando las actividades cotidianas, tener menos tiempo libre y en ocasiones a abandonar su empleo: “…a mi ya no me quedaba tiempo de nada, entonces me tocó dejar el trabajo para cuidarlo, pero lo haría mil veces por mi papá” (E4VAn). Debido a las secuelas de la enfermedad, el cuidador realiza adecuaciones físicas y estructurales en algunos espacios de la casa con el objetivo de favorecer la comodidad y seguridad del familiar. Por ejemplo, ampliar la puerta del baño para que pueda atravesar una silla de ruedas sin dificultad, conseguir una cama hospitalaria para hacer más fácil el cambio de posiciones de su familiar y alquilar una casa nueva, son estrategias usadas para hacer más sencillo el cuidado: “como había que cargarlo para llevarlo al baño y no cabíamos todos por la puerta, tuvimos que tumbarla…después buscamos este apartamento más grande y nos vinimos para acá”. (E2FTh). Estas modificaciones ocurren en corto tiempo e impactan social y económicamente no solo al cuidador, sino a los demás miembros de la familia. El apoyo de otros: sintiendo compañía en el cuidado Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 304 Durante la experiencia de cuidar por primera vez en casa a una persona con secuelas de un ACV surgen situaciones en las cuales el cuidador requiere de ayuda. El apoyo recibido de la red primaria, es decir, amigos y familiares favorece el acompañamiento y el descargo o disminución de responsabilidades. Así lo narra una cuidadora: “mi sobrina ha sido un apoyo bastante grande, nos apoya bastante, nos ha ayudado mucho, todo ha sido más fácil gracias a su apoyo” (E5YPm). Cuando el cuidador siente que el apoyo que recibe no es suficiente manifiesta sus preocupaciones y necesidades a otros miembros de la familia en espera de recibir ayuda, así lo describió un participante: “…y yo le dije al esposo: contrata a alguien que me ayude porque yo sola no puedo, porque había días que me costaba levantarla para yo bañarla, para cambiarla…y siempre estaba yo sola” (E1DPh) El apoyo recibido por el personal de salud se hace necesario cuando se tienen inquietudes frente a cómo realizar un procedimiento como el baño en cama, la curación de una herida ocasionada por el largo tiempo en cama o la realización de terapias de rehabilitación física. Una de las cuidadoras narra que gracias a las enfermeras aprendió como bañar al familiar en la cama: “Para bañarlo una de las enfermeras me explicó: cuando lo vayas a bañar en la mañana lo bañas así y así”. (E6LYm). Así mismo, algunos de los cuidadores explicaron que las enfermeras los alertaron de cara a la realidad que ellos iban a enfrentar una vez tuvieran el familiar en el hogar, esto despertó el interés por prepararse y aprender del cuidado mientras el familiar se encontraba en el hospital. En algunas ocasiones, de acuerdo con el grado de dependencia o secuelas secundarias al ACV, es necesario el uso de elementos para el cuidado del familiar como absorbentes, cremas y protectores de piel que son costosos y deben ser asumidos directamente por el cuidador; sin embargo, cuando este cambia su estilo de vida, los ingresos económicos varían y se requiere el apoyo económico de otros para poder asumir dichos costos. Por ejemplo, una cuidadora recibió ayuda de su hija para comprar estos elementos: “como me quede sin trabajo la hija mía le dejo de todo lo que el necesitaba, las cremas, el jabón, la toalla, todo eso se lo compró ella, A él no le ha faltado nada, si le hace falta algo uno busca quien le ayude y se le compra” (E2FTh) Sintiendo satisfacción: el resultado de un cuidado bien hecho Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 305 Después de aprender a cuidar en casa y de tener una red de apoyo, las acciones de cuidado se hacen gratificantes para el cuidador y empiezan a tener un significado y sentido diferente. El cuidador resignifica su experiencia y siente satisfacción con los progresos que tiene su familiar, aquí se experimenta gusto y agradecimiento con la situación. Una cuidadora muestra cómo el progreso de su familiar le genera alegría: “Me siento feliz, él va mejorando, hemos logrado mucho…pero va a haber siempre unas secuelas, pero ya puede escribir y contar hasta 5” (E4VAe). Experimentar esta satisfacción hace que el cuidador se sienta seguro y confiado de los cuidados que realiza, y lo anima a enseñar a otros familiares cómo cuidar, además de convertirse en un apoyo para otros cuidadores que están pasando por lo mismo. Viviendo el cuidado: entre el miedo, la fe y el amor Durante toda esta nueva experiencia, el cuidador experimenta muchos sentimientos y emociones. Principalmente se evidenció que, dependiendo del estado de salud del familiar, los apoyos disponibles, la preparación del cuidador y el seguimiento por parte de los profesionales de la salud, el acto de cuidar puede ser percibido como una experiencia positiva o negativa. Algunos cuidadores refieren sentir emociones como miedo, ansiedad, tristeza y soledad, como es el caso una cuidadora quien expresó sus sentimientos de miedo frente a la condición del familiar: “A veces uno siente tristeza porque no va a volver a caminar…y es ese miedo de que se pare y se vaya a caer o que convulsione de nuevo” (E6LYm). En contraste, el cuidador experimenta otras emociones como la satisfacción, la alegría y el afrontamiento efectivo, las cuales surgen cuando la condición de salud del familiar mejora: “se siente una alegría muy grande el ver que ya dice algunas palabras…la hemos cuidado bien.” (E5YPm) Un elemento que acompaña el desarrollo de las emociones en el cuidador de personas con ACV es la espiritualidad, la cual se convierte en una forma de afrontamiento del nuevo rol y la condición de salud del familiar. Por medio de la espiritualidad el cuidador encuentra fortaleza y fe para continuar con sus labores, esperando siempre la mejora de la condición familiar. Varios de los cuidadores manifestaron que Dios es su fuente de apoyo y que gracias a Él todo es posible. Finalmente, el proceso de asumir el cuidado de una persona con ACV es percibido por el cuidador como una experiencia donde se aprende a reconocer el valor que tiene cuidar a otra persona. Para los participantes asumir el cuidado es considerado como un Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 306 acto de amor, así como se presenta en la siguiente nota: “Una experiencia amorosa, se estima más, se aprende, uno conoce lo que es un ser humano cuando está enfermo, una persona que no se puede valer y no se abandona” (E4VAe). Cuidando en el hogar: desde la falta de conocimiento a la satisfacción Esta categoría constituye el concepto central que describe la vivencia del cuidador familiar cuando asume por primera vez el cuidado de un familiar con ACV. El proceso inicia cuando el familiar regresa a casa y el cuidador descubre las implicaciones del rol que debe asumir para garantizar el bienestar y asumir la dependencia del cuidado de su ser querido. Ante esta situación, el cuidador identifica que no tiene la suficiente información, ni el entrenamiento adecuado sobre cómo cuidar, aquí empieza a buscar apoyo e instrucción de otras personas que han vivido situaciones similares de cuidado y del personal de salud. Con el paso de los días y de la experiencia acumulada, el cuidador va aprendiendo la forma correcta de satisfacer las necesidades de cuidado del familiar, lo cual se evidencia con un progreso en la condición del familiar y en su rehabilitación. Durante todo el proceso, el cuidador experimenta situaciones asociadas al cuidado, que generan emociones como ansiedad, angustia, tristeza y frustración, pero una vez superadas estas situaciones, se experimenta satisfacción al sentir que la dedicación para mejorar el bienestar y recuperación de su ser querido ha tenido buenos resultados. Durante todo el proceso de cuidado, la espiritualidad tiene un papel relevante en el proceso de adaptación del cuidador, debido a que en ella encuentra un soporte emocional importante que le permite comprender el por qué está viviendo la situación, haciendo una resignificación de la experiencia y la importancia de su rol para alcanzar. DISCUSIÓN En los hallazgos descritos se encontró que el impacto de asumir la experiencia de cuidado en el hogar es proporcional al grado de discapacidad y dependencia del familiar, para el caso de este estudio, se relacionó con la asistencia en actividades de la vida diaria. Estos hallazgos son soportados por Dixe et al. (2019) quienes encontraron que en el caso de personas con dependencia funcional existe una necesidad del 53,8% en suplir actividades de higiene y comodidad como alimentación, movilidad y comunicación. Así mismo, ese estudio encontró que asumir el cuidado de la persona asociado a su dependencia genera cansancio en los cuidadores. Otras investigaciones han documentado Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 307 que la fatiga del cuidador y el número de horas dedicadas al cuidado diario, tienen estrecha relación con una disminución de la calidad de vida de los cuidadores (Okoye et al., 2019). Los cuidadores de este estudio pasan más de 12 horas dedicados al cuidado diario de la persona con ACV, lo que se convierte en un factor que puede impactar negativamente su calidad de vida. Nueve de los cuidadores que participaron en este estudio dejaron sus empleos para dedicarse tiempo completo a la atención de sus familiares. Asociado con este tema, un estudio demostró que aquellos cuidadores que dejaron sus empleos experimentaron sensaciones como aislamiento social y problemas en las relaciones personales con otros familiares, generando en consecuencia un alto riesgo de depresión (Caro, Costa, & Da Cruz, 2018). Con relación al entorno donde se realizan las actividades de cuidado, es importante resaltar que el hogar es un espacio ideal para la recuperación y rehabilitación de los familiares; sin embargo, la estructura física de estos espacios no la adecuada. Por esta razón, los cuidadores se ven en la necesidad de modificar algunos espacios. En un estudio desarrollado por Kim, Zhao, Kim, & Ahn (2019) se encontró que las modificaciones al hogar generan consecuencias positivas como un mejor rendimiento en el cuidado y en las actividades de la vida diaria tanto para las personas con ACV como para los cuidadores, aportando a mejorar su calidad de vida. Las necesidades de apoyo fueron uno de los principales hallazgos. Los participantes del estudio recibieron apoyo para la satisfacción de sus necesidades por parte de su red de apoyo. De acuerdo con Rossinot, Marquestaut, & de Stampa (2019) los cuidadores sufren un agotamiento multifacético, tanto a nivel físico como mental que requiere de un apoyo por parte de su grupo social, lo que ayuda a disminuir emociones como angustia y soledad, que podrían hacer más compleja la situación de asumir el cuidado. El mismo estudio muestra que un aspecto importante para el cuidador es la posibilidad de expresar sus emociones y pensamientos a otras personas, por lo cual el soporte social de amigos o familiares se constituye como un recurso valioso para el bienestar del cuidador. A lo largo de toda la experiencia, el cuidador y su familia aprenden a cuidar de acuerdo con las situaciones que se vayan dando con el familiar. En este estudio, los participantes refirieron que con el tiempo fueron desarrollando habilidades para bañar y cambiar de posición a sus familiares, lo cual permitió que cada vez fuera más sencillo hacerlo. En un estudio realizado se evidenció que el cambio del hospital al hogar tiene un Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 308 impacto significativo en los roles y responsabilidades de los cuidadores, por lo cual es necesario realizar entrenamiento que permita el desarrollo de habilidades, tanto en aspectos técnicos como emocionales, con el fin de garantizar un afrontamiento adecuado (Cheng, Chair, & Chau, 2018). Entre las principales preocupaciones que experimenta el cuidador se encuentra la angustia ante lo desconocido, lo cual le genera disconfort. Lo anterior se evidenció en el estudio realizado por McCurley et al. (2019) quienes identificaron que la angustia emocional se encuentra relacionada con la aparición repentina del ACV, la incertidumbre sobre el futuro, las preocupaciones sobre las secuelas y cómo será la vida después del ACV. Las personas con ACV con dificultades de comunicación presentan problemas para expresar sus necesidades. Esta situación produce impotencia en los cuidadores al no comprender lo que quieren expresar sus familiares, esto lleva a diseñar estrategias para mejorar la comunicación. Wray, Clarke, & Forster (2019) manifiestan que los supervivientes de ACV con dificultades del habla y sus familiares emprendieron un trabajo significativo (práctico, relacional, emocional) para manejar su condición. La adaptación se facilita mediante el "hacer", es decir, probando qué actividades se podían gestionar de forma independiente y cuáles requerían apoyo adicional, demostrando ingenio y creatividad en la elaboración de estrategias para hacer frente a sus dificultades de comunicación. A pesar de la labor realizada, muchos expresaron una falta de confianza en su capacidad y sentimientos de impotencia. Finalmente, cuando el cuidador logra asumir el cuidado en el hogar y observa la evolución y recuperación de su ser querido, siente satisfacción. Esto lo motiva a continuar con su rol y vencer otras adversidades, a su vez, en este estudio se observó que cuando se genera satisfacción por el cuidado se fortalecen los lazos familiares y se produce un ambiente de tranquilidad para todos. En contraste, Lynch, Shuster, & Lobo (2018) informan que pocos cuidadores de personas con ACV alcanzan satisfacción debido al alto número de tareas de cuidado que debe realizar y al lento avance en su recuperación. Esto puede explicarse teniendo en cuenta que si bien los cuidadores de este estudio estaban dedicados gran parte del día al cuidado de la persona, no eran los únicos cuidadores pues se contaba con el apoyo de otros familiares para el cuidado. Futuros estudios deben Cultura de los Cuidados. 1º Cuatrimestre 2022. Año XXVI. nº 62 309 identificar si existe una asociación entre variables como el tiempo de dedicación al cuidado, el soporte social recibido con la satisfacción con el cuidado. Este estudio reconoce dentro de sus limitaciones que se realizó en cuidadores con niveles socioeconómicos bajos y con dificultades para acceder a recursos del sistema de salud, con lo cual los resultados descritos aquí representan las vivencias de un grupo poblacional de bajos ingresos. CONCLUSIONES El cuidador familiar y su familiar con ACV emprenden una importante labor de adaptación y gestión de la vida después de un ACV, desarrollan estrategias personalmente significativas y específicas del contexto para ajustarse, manejar la vida y suavizar la difícil transición al hogar; sin embargo, algunos cuidadores expresan sentimientos de impotencia y abandono al momento del egreso del hospital. La comprensión de las experiencias de cuidado de los cuidadores familiares de personas con ACV es fundamental para diseñar intervenciones de enfermería desde el plan de egreso que atiendan las necesidades específicas de esta población. Este estudio tiene varias implicaciones prácticas. La primera de ellas es la tarea urgente que tienen los profesionales de enfermería de reconocer al cuidador y disminuir la sensación de abandono que estos tienen durante el proceso de alta hospitalaria y el posterior paso al hogar. La segunda es la necesidad de implementar acciones como el reconocimiento del cuidador, su vinculación y preparación para el cuidado, la entrega de información oportuna y específica sobre el ACV y sobre los recursos disponibles, el fomento de las relaciones a largo plazo con los profesionales de la salud y un seguimiento constante después del alta, ya que estas acciones pueden facilitar la experiencia de convertirse en un cuidador de una persona con ACV. 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https://openalex.org/W2895003671	https://www.frontiersin.org/articles/10.3389/fpsyg.2018.01846/pdf	English	null	Keeping Emotions in Mind: The Influence of Working Memory Capacity on Parent-Reported Symptoms of Emotional Lability in a Sample of Children With and Without ADHD	Frontiers in psychology	2,018	cc-by	8,821	ORIGINAL RESEARCH published: 02 October 2018 doi: 10.3389/fpsyg.2018.01846 Citation: Jensen DA, Høvik MF, Monsen NJN, Eggen TH, Eichele H, Adolfsdottir S, Plessen KJ and Sørensen L (2018) Keeping Emotions in Mind: The Inﬂuence of Working Memory Capacity on Parent-Reported Symptoms of Emotional Lability in a Sample of Children With and Without ADHD. Front. Psychol. 9:1846. doi: 10.3389/fpsyg.2018.01846 Daniel André Jensen1,2,3, Marie Farstad Høvik4,5, Nadja Joseﬁne Nyhammer Monsen5, Thale Hegdahl Eggen6, Heike Eichele1, Steinunn Adolfsdottir1,5, Kerstin Jessica Plessen2,7,8 and Lin Sørensen1,2 1 The Department of Biological and Medical Psychology, University of Bergen, Bergen, Norway, 2 K.G. Jebsen Centre for Research on Neuropsychiatric Disorders, Bergen, Norway, 3 Betanien District Psychiatric Center (DPS), Bergen, Norway, 4 Department of Clinical Medicine, University of Bergen, Bergen, Norway, 5 Division of Psychiatry, Haukeland University Hospital, Bergen, Norway, 6 Division Mental Health Services, Akershus University Hospital, Lørenskog, Norway, 7 Child and Adolescent Mental Health Center, Copenhagen, Denmark, 8 Division of Child and Adolescent Psychiatry, Department of Psychiatry, Lausanne University Hospital, Lausanne, Switzerland Keywords: working memory, attention-deﬁcit/hyperactivity disorder, emotional lability, emotion regulation, letter– number sequencing Edited by: Edited by: Livio Provenzi, Eugenio Medea (IRCCS), Italy Emotional lability (EL) often co-occurs with attention-deﬁcit/hyperactivity disorder (ADHD) in children. However, difﬁculties of regulating intense emotions in ADHD are still poorly understood. We investigated the potential role of working memory (WM) as a protective factor against EL in children with ADHD by building on models describing the close relationship between WM and regulation of emotions. The parents of 41 children with ADHD and 34 typically developing children (TDC) ﬁlled out the emotional control scale (ECS) from the Behavior Rating Inventory of Executive Functioning and the child behavior checklist (CBCL). The children themselves completed the backward conditions of the digit span (DS) and spatial span (SS) tasks as well as the letter–umber sequencing (LNS) task. The results of a stepwise regression analysis conﬁrmed the negative relationship between parent reported EL measured using the ECS and scores on the LNS, when controlling for symptoms of ADHD and oppositional deﬁant disorder (ODD). WM thus seems to be important for the ability of the children to express emotions in an adaptive and ﬂexible way. We therefore suggest that a poorer WM capacity, which is often found in children with ADHD, may be a predictor of high levels of EL. Reviewed by: Vrinda Kalia, Miami University, United States Joanne Sara Johnston, University of Reading, United Kingdom Reviewed by: Vrinda Kalia, Miami University, United States Joanne Sara Johnston, University of Reading, United Kingdom Correspondence: Daniel André Jensen Daniel.A.Jensen@uib.no Specialty section: This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology Received: 06 April 2018 Accepted: 10 September 2018 Published: 02 October 2018 Keeping Emotions in Mind: The Inﬂuence of Working Memory Capacity on Parent-Reported Symptoms of Emotional Lability in a Sample of Children With and Without ADHD Daniel André Jensen1,2,3*, Marie Farstad Høvik4,5, Nadja Joseﬁne Nyhammer Monsen5, Thale Hegdahl Eggen6, Heike Eichele1, Steinunn Adolfsdottir1,5, Kerstin Jessica Plessen2,7,8 and Lin Sørensen1,2 INTRODUCTION page 18 of Gioia et al., 2000). The ability to monitor and modulate incoming information (WM capacity) is believed to be very important for the adaptive perception, experience, and expression of emotions (i.e., level of EL) (e.g., Gross, 2002; Sheppes and Gross, 2011; Sheppes et al., 2014; Smith and Lane, 2015). The high prevalence of EL diﬃculties in children and adults with ADHD (Skirrow et al., 2009) has been noted over time (Wender, 1972), and has been included as an associated feature to ADHD in the ﬁfth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5; American Psychiatric Association, 2013). Previously, eﬀorts to explain this association have predominantly focused on poorer inhibitory control (Barkley, 1997) and high levels of oppositional deﬁant disorder (ODD; e.g., Sobanski et al., 2010) as predictors of EL. ( g ) p Only one prior study (Banaschewski et al., 2012) has, to the best of our knowledge, investigated how inhibitory control and WM relate to EL in ADHD. They found no signiﬁcant association between these functions and parent-reported levels of EL after controlling for ADHD symptoms. However, the WM task applied, the digit span (DS), is probably not as sensitive as other measures of verbal WM in assessing the capacity to modulate incoming information (i.e., simple reversal of a single stimulus category may not be suﬃciently cognitively demanding; Shelton et al., 2009; Kasper et al., 2012). We therefore wanted to investigate a possible link between verbal WM and parent- reported EL by including a WM task that is assumed to place a higher load on the modulation of incoming information than the digit span, namely the letter–number sequencing (LNS) task (e.g., a “complex” task; Shelton et al., 2009). The LNS requires the participant both to remember (store, i.e., the phonological loop) and to sequence the digits and letters that are presented according to numerical and alphabetical order (integrating stored information and modulating it according to knowledge of the alphabet, i.e., the episodic buﬀer). Thus, introducing a greater processing demand and reliance of the central executive than simple reversal. It is important to note that WM, together with inhibitory control and cognitive ﬂexibility, are suggested to comprise the subfunctions of cognitive control (Miyake et al., 2000). WM is thus shown to load on inhibitory control, however, not on cognitive ﬂexibility (see Miyake and Friedman, 2012). INTRODUCTION Following Baddeley (1986) model, the central executive acts as an inhibitory control component. However, there is ample evidence of the importance of WM – and not inhibitory control alone – in several emotion regulation strategies (Smith and Lane, 2015), including cognitive reappraisal (McRae et al., 2012). This may be because these processes involve multiple components of WM. In addition to inhibitory control (i.e., central executive), the information is modulated by holding it in temporary storage (i.e., the phonological loop and the visuospatial sketchpad) at the same time as the information is integrated with existing knowledge and experience (i.e., the episodic buﬀer) (Baddeley, 2000, 2002). 2014; Smith and Lane, 2015). Baddeley’s (Baddeley, 1986, 2000, 2002) WM model can be used to understand the role of WM in emotional experiences and the modulation of these experiences. He describes WM as a hierarchical system comprising a central executive that regulates and controls the storing (the phonological loop and the visuospatial sketchpad) and integration (the episodic buﬀer) of information from multiple modalities. The central executive is, as such, essential for monitoring and modulating incoming information by regulating the allocation of attention in accordance with goal-oriented behavior. A higher WM capacity can help a child to modulate an emotional reaction by taking into perspective the situational expectancies (e.g., such as downregulating the emotional impact of a situation; Knudsen, 2007). Previous studies have shown that the contribution of the capacity to modulate the meaning and importance of emotional experiences is important in pursuing goal-oriented behavior (Gross, 2002; Sheppes and Gross, 2011; Bridgett et al., 2013; Sheppes et al., 2014; Smith and Lane, 2015). Gross (2002), Sheppes and Gross (2011), McRae et al. (2012), and Sheppes et al. (2014) have focused on the role of verbal WM in the experience, expression and regulation of emotions in typically developing adults and concluded that the ability to cognitively reappraise experiences eliciting negative emotions is related to better performance on verbal WM tasks. Typically, participants have been exposed to emotional stimuli with varying valence, such as emotion eliciting images, with the instruction to actively reduce the emotional impact of the stimuli by constructing alternative interpretations. One study ﬁnding supporting evidence in typically developing, young adults showed that the distribution of pre-made reappraisals, assumed to decrease the cognitive cost of reappraisal, increased the ability to down-regulate the intensity of negative emotions and thus facilitated the reappraisal process (Sheppes et al., 2014). INTRODUCTION Attention-deﬁcit/hyperactivity disorder (ADHD) is a frequent neurodevelopmental disorder present in around 5% of children (Polanczyk and Jensen, 2008; Willcutt, 2012). Problems of self-regulation associated with the disorder include diﬃculties in both cognitive (Willcutt et al., 2005) and emotional (Shaw et al., 2014) control functions (see Nigg, 2017). Typical diﬃculties October 2018 \| Volume 9 \| Article 1846 Frontiers in Psychology \| www.frontiersin.org 1 Inﬂuence of WM-capacity on EL Jensen et al. include a reduced performance on working memory (WM) tasks, which measure the capacity to monitor and modulate incoming information (see the meta-analyses of Martinussen et al., 2005; Kasper et al., 2012). At the same time, parents of children with ADHD tend to report that their children have problems controlling their emotional expressions (Skirrow et al., 2009). This has been described as emotional lability (EL; e.g., Sobanski et al., 2010), which includes frequent expressions of high intensity (negative) emotions (Skirrow et al., 2009; Shaw et al., 2014). Such diﬃculties can be assessed with parent reports on the emotional control scale (ECS) of the Behavior Rating Inventory of Executive Function that measures the ability to modulate emotional responses, with high scores indicating a high level of EL or explosiveness (BRIEF; cf. page 18 of Gioia et al., 2000). The ability to monitor and modulate incoming information (WM capacity) is believed to be very important for the adaptive perception, experience, and expression of emotions (i.e., level of EL) (e.g., Gross, 2002; Sheppes and Gross, 2011; Sheppes et al., 2014; Smith and Lane, 2015). of executive functioning, and emotional diﬃculties in children with ADHD (e.g., Nigg et al., 2004; Sheppes et al., 2015). include a reduced performance on working memory (WM) tasks, which measure the capacity to monitor and modulate incoming information (see the meta-analyses of Martinussen et al., 2005; Kasper et al., 2012). At the same time, parents of children with ADHD tend to report that their children have problems controlling their emotional expressions (Skirrow et al., 2009). This has been described as emotional lability (EL; e.g., Sobanski et al., 2010), which includes frequent expressions of high intensity (negative) emotions (Skirrow et al., 2009; Shaw et al., 2014). Such diﬃculties can be assessed with parent reports on the emotional control scale (ECS) of the Behavior Rating Inventory of Executive Function that measures the ability to modulate emotional responses, with high scores indicating a high level of EL or explosiveness (BRIEF; cf. Frontiers in Psychology \| www.frontiersin.org INTRODUCTION Similarly, research investigating the role of cognitive control in emotional experience from a developmental perspective (i.e., based on the model of Posner and Rothbart, 2000), have also implicated the importance of verbal WM capacity (Bridgett et al., 2013). This is in line with work suggesting a relationship between reduced WM capacity, as part Therefore, based on the theories of Gross (Gross, 2002; Sheppes and Gross, 2011; Sheppes et al., 2014) and Posner and Rothbart (Posner and Rothbart, 2000; Rueda et al., 2005; Rothbart et al., 2011; Bridgett et al., 2013) as well as a recent review (Smith and Lane, 2015), we expected an inverse relationship between verbal WM and parent reported diﬃculties related to EL. To examine this hypothesis we used three WM tasks – the DS and SS which can be described as “simple reversal” verbal and visuospatial span tasks, respectively, and the LNS October 2018 \| Volume 9 \| Article 1846 Frontiers in Psychology \| www.frontiersin.org 2 Inﬂuence of WM-capacity on EL Jensen et al. which can be described as a complex verbal WM task – and only expected verbal WM capacity to associate with EL, and then only with the WM task with the highest load on modulation of incoming information (i.e., the LNS; Shelton et al., 2009). We also wanted to explore whether this association was independent of parent reported symptoms of ADHD and ODD, and diagnostic status. As the reviewed studies (e.g., Gross, 2002; Sheppes and Gross, 2011; McRae et al., 2012; Sheppes et al., 2014) indicate an inverse relationship between EL and WM capacity in healthy samples, this inverse relationship may not distinguish between the ADHD group and the typically developing children (TDC). However, we expected higher levels of parent reported EL symptoms and a poorer WM capacity in the ADHD group than among the TDC. Furthermore, due to the noted association between WM and inhibition, as well as prior theories emphasizing the importance of diﬃculties related to inhibition, we also conducted supplementary analyses to investigate whether inhibition would be a signiﬁcant contributor to the current results (see Supplementary Materials). materials (Leckman et al., 1982). Comorbidities aﬀected several of the participating children. Among the children with ADHD, ODD was the most common comorbidity (n = 17) with three of these children also fulﬁlling the criteria for a conduct disorder. Working Memory Working memory was assessed with the backward conditions of the DS and the spatial span (SS) tasks, as well as the LNS task (Kaplan et al., 2004; Wechsler, 2003). In the backward conditions of the DS and the SS, children are instructed to recall and reproduce a list, or touch blocks, in the opposite order of that presented by the examiner (i.e., for the DS the examiner may read the sequence 2-7-1 and the child is to respond by reversing this sequence into 1-7-2), whereas the LNS requires the children to recall, rearrange, and reproduce a sequence of letters and numbers presented aloud by the examiner by ﬁrst repeating the numbers in ascending order and then the letters in alphabetical order (i.e., the sequence E-1-F is to be rearranged into 1-E-F; Kaplan et al., 2004). The DS and SS have been described as “simple” span tasks (i.e., even though the tasks include reversal of stimuli this may not be suﬃciently demanding to categorize such tasks as encompassing a high load on the central executive component of WM), whereas the LNS is the clinical measure which is most closely associated with laboratory measures of WM (i.e., additional processing of the stored information is required to correctly sort numbers by size and letters by alphabet placement; Shelton et al., 2009; Kasper et al., 2012). Participants The current study included 75 children between 8 and 12 years old, and consisted of 41 children with an ADHD diagnosis and a control group of 34 TDC. There were no group diﬀerences in sex or age distributions between the two groups (Table 1).All participating children had a full-scale intelligence quotient (FSIQ) above 75, however, the children with an ADHD diagnosis had lower FSIQ than the TDC. The study was carried out with the approval of the Regional Ethical Committee for Western Norway (REK-Vest), and written informed consent in accordance with the Declaration of Helsinki was obtained from all parents. Children with a suspected ADHD diagnosis were referred from outpatient child and adolescent psychiatric clinics serving the municipality of Bergen, Norway. A control group of TDC was recruited from schools in geographical areas overlapping with the areas served by the above mentioned outpatient clinics. INTRODUCTION Furthermore, 15 of the children with ADHD also fulﬁlled the criteria for an anxiety disorder and three the criteria for a tic disorder. One of the TDC fulﬁlled the criteria for a speciﬁc phobia. FSIQ was assessed using the Wechsler Intelligence Scale for Children – Fourth Edition (WISC-IV; Wechsler, 2003). The general ability index (GAI) score was also included as a measure of intellectual level in the current study, because WM scores are included in the calculation of the FSIQ scores (Table 1). Frontiers in Psychology \| www.frontiersin.org Emotional Lability (EL) Exclusion criteria for both groups were an existing ADHD diagnosis and prior use of psychostimulant medicine due to the wish to study cognitive functions that had not been modulated by treatment eﬀects (Eichele et al., 2016; Plessen et al., 2016; Sørensen et al., 2017). Further exclusion criteria were, suspicion of an autism spectrum disorder, or a prior head injury with loss of consciousness. The diagnosis of ADHD was given following the algorithm of the “Schedule for Aﬀective Disorders and Schizophrenia for School-Age Children – Present and Lifetime Version” (K-SADS-PL; Kaufman et al., 1997). Clinical professionals interviewed the children and their parents using the K-SADS-PL, and a board consisting of a child psychiatrist and a clinical psychologist ﬁnally conﬁrmed the diagnostic evaluations. Only children with a primary diagnosis of ADHD were included in the clinical group (n = 41), 26 children fulﬁlled the diagnostic criteria for the combined subtype, 12 had the predominantly inattentive subtype, and three the hyperactive/impulsive subtype on the basis of a best estimate diagnosis reviewing all available Emotional lability was measured with parent information on the emotional control subscale from the Behavior Rating Inventory of Executive Functioning, which “addresses the manifestation of executive functions within the emotional realm and measures a child’s ability to modulate emotional responses. Poor emotional control can be expressed as EL or emotional explosiveness” (Gioia et al., 2000, p. 18). This subscale asks the parents how they experience their children typically acting when they are upset, angry, or sad. Each item is evaluated according to a Likert-scale with three response alternatives: “often” (score 3), “sometimes” (score 2), or “never” (score 1). Internal reliability, as estimated by Cronbach’s alpha, is high (0.92; Gioia et al., 2000), also in a Norwegian sample in a comparable age group (Ranging from 0.80–0.98 for all subscales; Sørensen et al., 2011), and in the current sample (0.94 for the ECS). In the linear statistical analyses, we used the raw scores to secure a higher variability in scores (i.e., standardized scores are centralized around the mean). October 2018 \| Volume 9 \| Article 1846 Frontiers in Psychology \| www.frontiersin.org 3 Inﬂuence of WM-capacity on EL Jensen et al. TABLE 1 \| Descriptive characteristics of the sample. Emotional Lability (EL) TDC ADHD Between-group effects M SD M SD F/chi square Df p Post hoc Age (years) 9.47 1.08 9.65 1.25 0.43 1/72 ns FSIQ 105.76 11.07 91.15 7.33 4.71 1/72 <0.001 TDC > ADHD GAI 111.94 12.47 95.60 8.85 2.85 1/72 <0.001 TDC > ADHD ADHD 1.00 1.33 9.34 2.47 310.16 1/72 <0.001 TDC < ADHD ODD 0.76 1.28 4.49 2.95 46.88 1/72 <0.001 TDC < ADHD ECS 12.68 3.21 19.75 5.63 41.99 1/72 <0.001 TDC < ADHD DS 6.74 1.69 6.03 1.33 4.08 1/72 <0.05 TDC > ADHD SS 7.41 2.00 5.80 1.42 16.32 1/72 =0.001 TDC > ADHD LNS 15.85 4.05 12.55 3.62 13.72 1/72 =0.001 TDC > ADHD Boys/Girls 20/14 29/11 1.54 1 ns ODD-diagnosis (number/total) 0/34 16/40 17.35 1 <0.001 Pearson X2 FSIQ, full scale IQ; GAI, general ability index; ADHD, scores on the attention deﬁcit/hyperactivity problems scale of the CBCL; ODD, scores on the oppositional deﬁant problems scale of the CBCL; DS, score on the digit span backward condition; SS, score on the spatial span backward condition; LNS; score on the letter–number sequencing task; TDC, typically developing children; ODD-diagnosis, oppositional deﬁant disorder-diagnosis. TABLE 1 \| Descriptive characteristics of the sample. FSIQ, full scale IQ; GAI, general ability index; ADHD, scores on the attention deﬁcit/hyperactivity problems scale of the CBCL; ODD, scores on the oppositional deﬁant problems scale of the CBCL; DS, score on the digit span backward condition; SS, score on the spatial span backward condition; LNS; score on the letter–number sequencing task; TDC, typically developing children; ODD-diagnosis, oppositional deﬁant disorder-diagnosis. Preliminary Results Preliminary correlational analyses of the relationship between age and the variables of EL and the WM scores (LNS, SS, and DS), showed that age correlated signiﬁcantly with the WM scores of SS and LNS (Table 2). Age did, however, not correlate with the DS scores. Gender appeared to only be signiﬁcantly correlated with the DS scores and not with the other WM scores (LNS and SS). The parent-reported symptoms of EL, ODD and ADHD were not signiﬁcantly correlated with either age or gender (See Table 2). All three WM scores of LNS, SS, and DS correlated signiﬁcantly with each other. Dimensional Symptom Scales of ODD and ADHD we repeated the primary stepwise regression with the Stop- Signal Task score as an independent variable together with WM scores, symptoms of ADHD and ODD, age, and gender (see Supplementary Materials). We used the parent form of the child behavior checklist (CBCL), part of the Achenbach System of Empirically Based Assessment (ASEBA; Achenbach and Rescorla, 2001), to investigate the predictive validity of symptoms of ADHD and ODD on EL. The subscales of interest in the current study were the oppositional deﬁant problems scale (ODD symptom scale) and the attention- deﬁcit/hyperactivity problems scale (ADHD symptom scale). The CBCL is a highly validated and reliable measures in this age group (Achenbach and Rescorla, 2001), and also for use with clinical populations, including children and youth with ADHD and comorbidities (Achenbach and Rescorla, 2001; Krol et al., 2006; Biederman et al., 2008). Missing data for one child each on ADHD symptoms, ODD symptoms, and GAI were replaced with the series mean. Furthermore, an inspection of the studentized residuals showed that one participant belonging to the group of children with ADHD was an outlier (Aguinis et al., 2013). This child’s data were therefore omitted from the analyses. Frontiers in Psychology \| www.frontiersin.org Statistical Analyses All statistical analyses were conducted using IBM SPSS, version 25. Bivariate correlation analyses were conducted among all variables of interest. To test our main hypothesis, we conducted a linear stepwise regression analysis that included EL scores from the ECS as the dependent variable, and age, gender, symptoms of ODD and ADHD, GAI scores, and WM scores of DS, SS, and LNS scores as the independent variables. FSIQ was not included as it has been argued that controlling for it as a covariate is likely to distort ﬁndings (Dennis et al., 2009). The Relationship Between WM Capacity and Parent-Reported Emotional Lability EL, score on the emotional control scale of the BRIEF; ADHD, score on the attention deﬁcit/hyperactivity problems scale of the CBCL; ODD, score on the oppositional deﬁant problems scale of the CBCL; DS, score on the digit span backward condition; SS, score on the spatial span backward condition; LNS, score on the letter–number sequencing task. ∗∗Correlation is signiﬁcant at the 0.01 level (1-tailed); ∗correlation is signiﬁcant at the 0.05 level (1-tailed). esults from the forward stepwise regression model showing the prediction of EL based on symptoms of ODD and LNS-scores. TABLE 3 \| Results from the forward stepwise regression model showing the prediction of EL based on symptoms of ODD and LNS-scores. Model summary ECS Adjusted R2 1R df p B SE B β p Model 1 ODD 0.70 0.71 1/72 <0.001 1.65 0.13 0.84 <0.001 Model 2 ODD 0.73 0.03 1/71 <0.01 1.56 0.12 0.80 <0.001 LNS −0.26 0.09 −0.18 <0.01 ECS, score on the emotional control scale of the BRIEF; ODD, score on the oppositional deﬁant problems scale of the CBCL; LNS, Score on the letter–number sequencing task. FIGURE 1 \| A graphical representation of the moderation analysis (A), and a scatterplot showing the distribution of scores on the Letter–Number Sequencing (LNS) and Emotional Control Scale (ECS) as well as the equations describing the relationships between these for the group with attention-deﬁcit/hyperactivity disorder (ADHD), the No ADHD as well as the whole sample (B). Note: IV, independent variable; M, moderator; DV, dependent variable. Letter Number Sequencing = Centered scores on the Letter Number Sequencing task (i.e., individual scores minus the sample mean). FIGURE 1 \| A graphical representation of the moderation analysis (A), and a scatterplot showing the distribution of scores on the Letter–Number Sequencing (LNS) and Emotional Control Scale (ECS) as well as the equations describing the relationships between these for the group with attention-deﬁcit/hyperactivity disorder (ADHD), the No ADHD as well as the whole sample (B). Note: IV, independent variable; M, moderator; DV, dependent variable. Letter Number Sequencing = Centered scores on the Letter Number Sequencing task (i.e., individual scores minus the sample mean). The Relationship Between WM Capacity and Parent-Reported Emotional Lability FIGURE 1 \| A graphical representation of the moderation analysis (A), and a scatterplot showing the distribution of scores on the Letter–Number Sequencing (LNS) and Emotional Control Scale (ECS) as well as the equations describing the relationships between these for the group with attention-deﬁcit/hyperactivity disorder (ADHD), the No ADHD as well as the whole sample (B). Note: IV, independent variable; M, moderator; DV, dependent variable. Letter Number Sequencing = Centered scores on the Letter Number Sequencing task (i.e., individual scores minus the sample mean). As expected, higher LNS scores were related to lower EL symptoms after controlling for parent-reported ADHD and ODD symptoms. The follow-up moderation analysis supported that this relationship was independent of diagnostic status, although the levels of both WM capacity and EL diﬀered between the groups (i.e., children with ADHD had lower WM scores and higher EL scores than TDC, but the relationships between these scores did not signiﬁcantly diﬀer between groups). The results of the moderation analysis, investigating the interaction between ADHD diagnostic status and the LNS scores on the EL scores from the ECS, showed that there were no signiﬁcant interaction between the LNS scores and diagnostic status (see Figure 1). The Relationship Between WM Capacity and Parent-Reported Emotional Lability The Relationship Between WM Capacity and Parent-Reported Emotional Lability All three WM scores of LNS, SS, and DS were inversely correlated with the parent-reported EL scores on the ECS. The forward linear stepwise regression model including the EL scores from the ECS as the dependent variable and the independent variables of age, gender, ADHD symptoms, GAI scores, and the WM scores of LNS, DS, and SS, showed that only symptoms of ODD and the LNS scores signiﬁcantly predicted the parent-reported scores of EL on the ECS, and not age, gender, GAI, symptoms of ADHD, scores on the DS or scores on the SS (see Table 3). The stepwise regression analysis was followed by a moderation analysis as described by Kraemer et al. (2002) and Hayes (2012) building on the model of Baron and Kenny (1986). In our study this comprised a regression approach including the independent variable of the LNS scores (IV), a moderator variable of diagnostic status of ADHD versus TDC (M), and an interaction variable of the LNS scores by moderator variable of diagnostic status (IV × M) with the EL scores from the ECS as the dependent variable. To investigate the potential inﬂuence of inhibition on the relationship between WM-scores and EL scores from the ECS October 2018 \| Volume 9 \| Article 1846 Frontiers in Psychology \| www.frontiersin.org 4 Inﬂuence of WM-capacity on EL Jensen et al. TABLE 2 \| Correlations among the examined variables. 1 2 3 4 5 6 7 8 (1) EL – −0.37∗∗ −0.31∗∗ −0.21∗ 0.84∗∗ 0.70∗∗ 0.05 0.05 (2) LNS – 0.53∗∗ 0.49∗∗ −0.24∗ −0.40∗∗ 0.33∗∗ 0.03 (3) SS – 0.36∗∗ −0.24∗ −0.47∗∗ 0.36∗∗ −0.05 (4) DS – −0.09 −0.19 0.14 −0.21∗ (5) ODD – 0.72∗∗ 0.15 0.08 (6) ADHD – 0.09 0.10 (7) Age – 0.18 (8) Gender – EL, score on the emotional control scale of the BRIEF; ADHD, score on the attention deﬁcit/hyperactivity problems scale of the CBCL; ODD, score on the oppositional deﬁant problems scale of the CBCL; DS, score on the digit span backward condition; SS, score on the spatial span backward condition; LNS, score on the letter–number sequencing task. ∗∗Correlation is signiﬁcant at the 0.01 level (1-tailed); ∗correlation is signiﬁcant at the 0.05 level (1-tailed). TABLE 2 \| Correlations among the examined variables. DISCUSSION The current ﬁndings are in line with previous studies in healthy samples (e.g., McRae et al., 2012; Sheppes et al., 2014) in that a lower WM capacity seems to be related to an increased In line with our hypotheses, we found an inverse relationship between children’s verbal WM scores and parent reported EL. October 2018 \| Volume 9 \| Article 1846 Frontiers in Psychology \| www.frontiersin.org 5 Inﬂuence of WM-capacity on EL Jensen et al. probability of experiencing and expressing emotions in a way which is described as problematic by the children’s parents. Overall the current ﬁndings are therefore also in line with the model proposed by Gross and colleagues (Gross, 2002; Sheppes and Gross, 2011) and the previous ﬁndings that verbal WM is involved in expressing one’s emotions in an adaptive and goal- oriented way (Smith and Lane, 2015). The current ﬁndings can also be linked to similar ﬁndings from temperamental research showing an overlap between WM and eﬃcient control of one’s emotions (Bridgett et al., 2013). This may suggest that a higher WM capacity acts as a protective factor against developing clinically signiﬁcant diﬃculties in expressing one’s emotions (i.e., diﬃculties in controlling strong emotional outbursts – EL). development in the ability to answer the task requirements of the DS, with one period ending around the age of 8–9 years, and the second commencing around 12–13 years of age. The period between these, spanning the age range of our participants, seems to be characterized by small developmental changes, and this may therefore be the explanation for the lack of association found between age and DS scores. With regard to gender eﬀects on WM capacity in ADHD, previous studies show mixed results depending on the percentage of females included (Kasper et al., 2012). In studies with a more balanced gender distribution, as in the current study, smaller between-group eﬀect sizes appear related to the WM capacity. p y It is also worth mentioning some contrasts and similarities between the current ﬁndings and the ﬁndings of Banaschewski et al. (2012), as they found no association between WM and EL in children with ADHD. As stated in the introduction, we believe this may be due to the measure applied. The DS was the only measure of WM included in the study of Banaschewski et al. (2012), and our results support their conclusion that this measure is not closely associated with EL. DISCUSSION Future studies may therefore want to investigate whether this holds true in other populations with elevated levels of EL, such as in children with anxiety disorders (Maire et al., 2017) and in adults with bipolar disorders (Phillips et al., 2003), borderline personality disorder (Schoenleber et al., 2016) and post-traumatic stress disorder (Schoenleber et al., 2018). Interestingly, in the current study, neither age nor gender aﬀected the relationship between WM capacity as measured with the LNS and the level of parent-reported EL. This indicates that a poorer WM capacity seems to relate to higher levels of EL in general, independent of diagnosis, age and gender of the child. However, both an ADHD diagnosis and age showed an expected association with the performance on the WM tasks, with the exception that age did not correlate with the performance on the DS task. A diﬀerential eﬀect of gender on the WM task scores also appeared, with boys scoring lower on the DS task compared to the girls, whereas such a diﬀerence did not appear on the SS and LNS. Previously, small age related improvements have been reported on the DS task in samples with similar age ranges as included in the current study (see Brocki and Bohlin, 2004; Lensing and Elsner, 2018). These ﬁndings seem to indicate two distinct periods of marked The current ﬁndings highlight some interesting possible directions for future work. If the current results can be replicated in a larger sample we believe that this would also merit an investigation of whether measures of verbal WM could also be used to direct the implementation of clinical interventions aimed at reducing the impact of EL as an associated feature of ADHD, and at reducing the risk for comorbid diﬃculties related to EL (i.e., ODD; Sobanski et al., 2010). One potential intervention in this regard could be emotional WM training, which consists of a dual n-back task presenting a combination of auditory and visual stimuli where a majority of the stimuli have a negative emotional valence (Schweizer et al., 2011). Such training has been shown to have an eﬀect on a frontoparietal network assumed to underlie both WM and aﬀective control (Schweizer et al., 2013). Furthermore, results show that the eﬀects of such training generalizes to traditional measures of emotion regulation (Schweizer et al., 2013). DISCUSSION However, we believe that the current results support the assumption that WM is in fact meaningfully associated with EL, and that WM as measured using the LNS speciﬁcally, seems to be particularly important. Another distinction between the two studies is the inclusion of a measure of ODD in the current work. Given that ﬁndings show that EL in ADHD seems to be more closely associated to ODD than to ADHD (Sobanski et al., 2010), we believe that the signiﬁcance of the current ﬁndings even when controlling for symptoms of ODD further supports the notion that WM capacity may be an important protective factor against the development of EL. It is, however, worth noting the possibility that the close association between the LNS and EL may not be due to it being a speciﬁc measure of verbal WM, but of general WM capacity due to its’ higher demands on the modulation of information (Kasper et al., 2012). The ﬁndings may also be seen in connection with research on ADHD. There is a known relationship between ADHD and lower WM capacity (Martinussen et al., 2005). This lower WM capacity may be one of the factors contributing to the high prevalence of clinically signiﬁcant levels of EL in this group (Skirrow et al., 2009; Sobanski et al., 2010). This is also in line with the suggestion that there may be a connection between diﬃculties in cognitive and emotional control (Nigg et al., 2004), although the current results cannot give any indication on the causal relationship between these diﬃculties. Important to note, though, is that the inverse relationship found between a lower WM capacity and higher levels of parent-reported EL did not appear to be restricted to children with ADHD. Rather this was shown to be a dimensional relationship true for the whole sample. The results, therefore, indicate that the ﬁndings regarding a relationship between WM and emotion regulation from studies on typically developing adult populations reviewed in this article are also applicable to children, both with and without ADHD. However, the children with ADHD showed poorer WM capacity on the LNS (and the SS) and a higher frequency of parent- reported EL symptoms than the TDC, indicating that the inverse relationship between EL symptoms and WM capacity may be more signiﬁcant for their everyday functioning than for the group of TDC. Frontiers in Psychology \| www.frontiersin.org FUNDING This work was supported by grants from the Research Council of Norway under grant 190544/H110, the Western Norway Health Authority (MoodNet, the Network for Anxiety Disorders, and a postdoc grant) under grants 911435, 911607, 911827, and 911460 (MoodNet and the Network for Anxiety Disorders) under grants 911435, 911607, and 911827, and the National Norwegian ADHD network. All grants were given to KP, with the exception of the postdoc grant awarded to LS. DISCUSSION Another potential area of investigation is whether a screening of verbal WM can help inform the pharmacological treatment of ADHD. Building on the study by Cubillo et al. (2013) showing a diﬀerential eﬀect of methylphenidate and atomoxetine, in combination with ﬁndings indicating an anatomical overlap between WM and October 2018 \| Volume 9 \| Article 1846 Frontiers in Psychology \| www.frontiersin.org 6 Inﬂuence of WM-capacity on EL Jensen et al. common-method variance; Richardson et al., 2009). Ideally, the investigation should be replicated with the inclusion of observer measures of emotional reactions to reduce the impact of this limitation. self-regulation (e.g., Bridgett et al., 2015), it seems plausible to hypothesize that atomoxetine might be particularly beneﬁcial for the subgroup of children with ADHD who also have a low WM capacity. This is due to diﬀerential eﬀects showing that atomoxetine has a pronounced activating eﬀect on the dorsolateral prefrontal cortex, a region which has been shown to be involved in both WM and executive attention (Bridgett et al., 2015). Strengths and Limitations The current study had several important strengths and limitations. It employed neuropsychological measures which are often used in clinical practice (i.e., subtests from the WISC-IV and WISC-IV-Integrated) in combination with well validated and widely accessible questionnaires, thereby obtaining results which are available in, and transferable to, day-to-day clinical practice and may be replicated in many clinical settings. We also regard the use of dimensional analyses as a strength, as these allowed us to investigate the hypothesized pattern of results in both the children with ADHD and the TDC. This is in line with our expectations as the hypothesis was, to a large degree, based on studies of typically developing individuals. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2018.01846/full#supplementary-material CONCLUSION The current study found support for the hypothesis that WM is a protective factor against elevated levels of EL in children, thus supporting previous ﬁndings showing the importance of high (verbal) WM capacity in the adaptive display of emotions. The results, if replicated, may represent an approach to understanding the functional heterogeneity associated with ADHD. ACKNOWLEDGMENTS We would like to thank the children and parents who participated in this study. We also thank Anne Øfsthus and Randi Hopsdal for their technical assistance, Paul M. Kauserud, Marco A. Hirnstein, and Helene B. Halleland for their contributions in the preparations for this article, and Kristine Sirevåg for comments on the manuscript. AUTHOR CONTRIBUTIONS KP and LS conceived and designed the study. MH, NM, HE, SA, KP, and LS acquired the data. DJ and LS analyzed and interpreted the data. DJ, MH, TE, KP, and LS wrote the manuscript. All authors contributed to manuscript revision, read and approved the submitted version. yp y p g The main limitations of this study are the limited sample size and the cross-sectional nature of our data. Due to these limitations all of our participants with high levels of EL belonged to the diagnostic group, thus limiting the generalizability of our conclusions. Furthermore, the use of cross-sectional data does not allow for investigation of the developmental ordering of the children’s diﬃculties, which would be highly relevant with regards to the model proposed by Nigg et al. (2004). A closer examination of whether the results reported here are mainly due to the use of a measure of verbal WM or a WM task with high demands on the modulation of information is also necessary to improve our understanding of the relationship between WM and EL. At the current time it could equally well be argued that a complex visuospatial WM task would be equally as predictive of parent reported EL scores, and a direct comparison of two complex WM tasks where one is assumed to be reliant on the verbal and one on the visuospatial component of WM would, therefore, help to clarify this issue. The results would also have been strengthened if the investigation had included a measure of task switching, as this executive function may associate with level of EL (e.g., Dickstein et al., 2007). There is also the issue of a signiﬁcant diﬀerence in FSIQ between the two groups. Although this is common in studies of ADHD, and related to the known diﬀerence in WM capacity as well as likely to be related to test- taking behavior (Dennis et al., 2009), the ﬁndings should ideally be investigated in a sample with matched FSIQ scores. Lastly, the use of the same informant report when collecting information about symptoms of ADHD and ODD as well as EL may have reduced the statistical power of WM in the analyses (i.e., due to Aguinis, H., Gottfredson, R. K., and Joo, H. (2013). Best-practice recommendations for deﬁning, identifying, and handling outliers. Organ. Res. Methods 16, 270– 301. doi: 10.1177/1094428112470848 Assessment. Burlington, VT: Research Center for Children, Youth, & Families. Assessment. Burlington, VT: Research Center for Children, Youth, & Families. Aguinis, H., Gottfredson, R. K., and Joo, H. (2013). Best-practice recommendations for deﬁning, identifying, and handling outliers. Organ. Res. Methods 16, 270– 301. doi: 10.1177/1094428112470848 REFERENCES C., Kaiser, R., and Faraone, S. V. (2008). CBCL clinical scales discriminate adhd youth with structured-interview derived diagnosis of oppositional deﬁant disorder (ODD). J. Atten. 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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. Copyright © 2018 Jensen, Høvik, Monsen, Eggen, Eichele, Adolfsdottir, Plessen and Sørensen. 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. Sheppes, G., Suri, G., and Gross, J. J. (2015). Emotion regulation and psychopathology. Annu. Rev. Clin. 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https://openalex.org/W2769912530	https://acp.copernicus.org/articles/18/6867/2018/acp-18-6867-2018.pdf	English	null	Decrease in tropospheric O&lt;sub&gt;3&lt;/sub&gt; levels in the Northern Hemisphere observed by IASI	Atmospheric chemistry and physics	2,018	cc-by	15,979	Correspondence: Catherine Wespes (cwespes@ulb.ac.be) Correspondence: Catherine Wespes (cwespes@ulb.ac.be) Received: 29 September 2017 – Discussion started: 23 November 2017 Revised: 30 March 2018 – Accepted: 9 April 2018 – Published: 16 May 2018 Received: 29 September 2017 – Discussion started: 23 November 2017 Revised: 30 March 2018 – Accepted: 9 April 2018 – Published: 16 May 2018 and covariance) that we found to be the strongest at northern midlatitudes in summer. and covariance) that we found to be the strongest at northern midlatitudes in summer. Abstract. In this study, we describe the recent changes in the tropospheric ozone (O3) columns measured by the Infrared Atmospheric Sounding Interferometer (IASI), onboard the Metop satellite, during the ﬁrst 9 years of operation (Jan- uary 2008 to May 2017). Using appropriate multivariate re- gression methods, we differentiate signiﬁcant linear trends from other sources of O3 variations captured by IASI. The geographical patterns of the adjusted O3 trends are provided and discussed on the global scale. Given the large contri- bution of the natural variability in comparison with that of the trend (25–85 % vs. 15–50 %, respectively) to the total O3 variations, we estimate that additional years of IASI mea- surements are generally required to detect the estimated O3 trends with high precision. Globally, additional 6 months to 6 years of measurements, depending on the regions and the seasons, are needed to detect a trend of \|5\| DU decade−1. An exception is interestingly found during summer at mid- and high latitudes of the Northern Hemisphere (NH; ∼40 to ∼75◦N), where the large absolute ﬁtted trend values (∼\|0.5\| DU yr−1 on average) combined with the small model residuals (∼10 %) allow for detection of a band-like pat- tern of signiﬁcant negative trends. Despite no consensus in terms of tropospheric O3 trends having been reached from the available independent datasets (UV or IR satellites, O3 sondes, aircrafts, ground-based measurements, etc.) for the reasons that are discussed in the text, this ﬁnding is consis- tent with the reported decrease in O3 precursor emissions in recent years, especially in Europe and USA. The inﬂuence of continental pollution on that latitudinal band is further in- vestigated and supported by the analysis of the O3–CO rela- tionship (in terms of correlation coefﬁcient, regression slope Atmos. Chem. Phys., 18, 6867–6885, 2018 https://doi.org/10.5194/acp-18-6867-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction O3 plays a key role throughout the whole troposphere where it is produced by the photochemical oxidation of carbon monoxide (CO), non-methane volatile organic compounds (NMVOCs) and methane (CH4) in the presence of nitrogen oxides (NOx) (e.g., Logan et al., 1981). O3 sources in the troposphere consist of the in situ photochemical production from anthropogenic and natural precursors and the down- wards transport of stratospheric O3. Being a strong pollutant, a major reactive species and an important greenhouse gas in the upper troposphere, O3 is of greatest interest regarding air quality, atmospheric chemistry and radiative forcing studies. Thanks to its long lifetime (several weeks) relative to trans- port timescales in the free troposphere (Fusco and Logan, 2003), O3 also contributes to large-scale transport of pollu- tion far from source regions with further impacts on global air quality (e.g., Stohl et al., 2002; Parrish et al., 2012) and climate. Monitoring and understanding the time evolution of tropospheric O3 at a global scale is, therefore, crucial to ap- prehend future climate changes. Nevertheless, a series of lim- itations make O3 trends particularly challenging to retrieve and to interpret. While the O3 precursors anthropogenic emissions have increased and shifted equatorward in the developing coun- tries (Zhang et al., 2016) since the 1980s, extensive cam- paigns and routine in situ and remote measurements at spe- Decrease in tropospheric O3 levels in the Northern Hemisphere observed by IASI Catherine Wespes1, Daniel Hurtmans1, Cathy Clerbaux1,2, Anne Boynard2, and Pierre-François Coheur1 1Spectroscopie de l’Atmosphère, Service de Chimie Quantique et Photophysique, Faculté des Sciences, Université Libre de Bruxelles (ULB), Brussels, Belgium 2LATMOS/IPSL, UPMC Univ. Paris 06 Sorbonne Universités, UVSQ, CNRS, Paris, France 2 IASI O3 measurements and multivariate regression The IASI instrument is a nadir-viewing Fourier transform spectrometer that records the thermal infrared emission of the Earth–atmosphere system between 645 and 2760 cm−1 from the polar Sun-synchronous orbiting meteorological Metop series of satellites. Metop-A and -B were successively launched in October 2006 and September 2012. The third and last launch, of Metop-C, is planned for 2018, which will en- sure homogeneous long-term IASI measurements. The mea- surements are taken every 50 km along the track of the satel- lite at nadir and over a swath of 2200 km across track, with a ﬁeld of view of four simultaneous footprints of 12 km at nadir, which provides global coverage of the Earth twice a day (at 09:30 and 21:30 mean local solar time). The instru- ment has good spectral resolution and low radiometric noise, which allows for retrieval of numerous gas-phase species in the troposphere (e.g., Clerbaux et al., 2009, and references therein; Hilton et al., 2012; Clarisse et al., 2011). In this paper, we use the FORLI O3 proﬁles (Fast Opti- mal Retrievals on Layers for IASI processing chain set up at ULB; v20151001) retrieved from the IASI-A (aboard Metop- A) daytime measurements (deﬁned with a solar zenith an- gle to the sun of < 80◦), which are characterized by good spectral ﬁt (determined here by quality ﬂags on biased or sloped residuals, suspect averaging kernels, maximum num- ber of iteration exceeded, etc.) and which correspond to clear or almost-clear scenes (a ﬁlter based on a fractional cloud cover below 13 % has been applied; see Clerbaux et al., 2009; Hurtmans et al., 2012). These proﬁles are characterized by good vertical sensitivity in the troposphere and the strato- sphere (e.g., Wespes et al., 2017). The FORLI algorithm re- lies on a fast radiative transfer and retrieval methodology based on the optimal estimation method (Rodgers, 2000) and is fully described in Hurtmans et al. (2012). The FORLI O3 proﬁles, which are retrieved at 40 constant vertical layers from surface up to 40 km and an additional 40–60 km one, have already undergone thorough characterization and vali- dation exercises (e.g., Anton et al., 2011; Dufour et al., 2012; Gazeaux et al., 2013; Hurtmans et al., 2012; Parrington et al., 2012; Pommier et al., 2012; Scannell et al., 2012; Oet- jen et al., 2014; Boynard et al., 2016, 2018; Wespes et al., 2016; Keppens et al., 2018). C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6868 variance are examined for characterizing the origin of the air masses in regions of positive and negative trends. ciﬁc urban and rural sites have provided long-term but sparse datasets of tropospheric O3 (e.g., Cooper et al., 2014 and references therein). Ultraviolet and visible (UV/VIS) atmo- spheric sounders onboard satellites provide tropospheric O3 measurements with much wider coverage, but they result ei- ther from indirect methods (e.g., Fishman et al., 2005) or from direct retrievals which are limited by coarse vertical resolution (Liu et al., 2010). All these datasets also suffer from a lack of homogeneity in terms of measurement meth- ods (instrument and algorithm) and spatio-temporal sam- plings (e.g., Doughty et al., 2011; Heue et al., 2016; Lev- entidou et al., 2017). Those limitations, in addition to the large natural interannual variability (IAV) and decadal varia- tions in tropospheric O3 levels (due to large-scale dynamical modes of O3 variations and to changes in stratospheric O3, in stratosphere–troposphere exchanges, in precursor emissions and in their geographical patterns), introduce strong biases in trends determined from independent studies and datasets (e.g., Zbinden et al., 2006; Thouret et al., 2006; Logan et al., 2012; Parrish et al., 2012 and references therein). As a con- sequence, determining accurate trends requires a long period of high density and homogeneous measurements (e.g., Payne et al., 2017). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 3 Regression performance and sensitivity to trend In this section, we ﬁrst verify the performance of the MLR models (annual and seasonal; in terms of residual errors and variation explained by the model) to globally reproduce the time evolution of O3 records over the entire studied period (January 2008–May 2017). Based on this, we then investi- gate the statistical relevance of a trend study from IASI mea- surements in the troposphere by examining the sensitivity of the pair IASI-MLR to the retrieved LT term. For the purpose of this work, we focus on a tropospheric column ranging from ground to 300 hPa (MLT) that includes the altitude of maximum sensitivity of IASI in the tropo- sphere (usually between 4 and 8 km altitude), which lim- its the inﬂuences of the stratospheric O3 as much as possi- ble and which was shown in Wespes et al. (2017) to exhibit independent deseasonalized anomalies/dynamical processes from those in the stratospheric layers. The stratospheric con- tribution into the tropospheric O3 columns have been previ- ously estimated in Wespes et al. (2016) as ranging between 30 and 65 % depending on the region and the season with the smallest contribution and the largest sensitivity in the north- ern midlatitudes in spring–summer, where the O3 variations, thus, mainly originate from the troposphere. We use almost the same MLR model (in its annual or its seasonal formula- tion) as the one developed in the companion paper (see Eqs. 1 and 2; Sect. 2.2 in Wespes et al., 2017), which includes a se- ries of geophysical variables in addition to a linear trend (LT) term. In order to take account of the observed jump properly, we modiﬁed the previously used MLR model so that the con- stant term is split into two components covering the periods before and after the September 2010 jump, separately. The MLR which is performed using an iterative stepwise back- ward elimination approach to retain the most relevant ex- planatory variables (called “proxies”) at the end of the iter- ations (e.g. Mäder et al., 2007) is applied to the daily IASI O3 time series. The main proxies selected to account for the natural variations in O3 are namely the QBO (quasi-biennial oscillation), the NAO (North Atlantic Oscillation) and the ENSO (El Niño–Southern Oscillation; see Table 1 in Wespes et al., 2017 for the exhaustive list of the proxies used). 3 Regression performance and sensitivity to trend Their associated standard errors are estimated from the covariance matrix of the regression coefﬁcients and are corrected to take into account the uncertainty due to the autocorrelation of the noise residual (see Eq. 3 in Wespes et al., 2016). The com- mon rule that the regression coefﬁcients are signiﬁcant if they are greater in magnitude than 2 times their standard errors is applied (95 % conﬁdence limits deﬁned by 2σ level). The MLR model was found to give a good representation of the IASI O3 records in the troposphere over 2008–2016, allow- ing us to identify/quantify the main O3 drivers with marked regional differences in the regression coefﬁcients. Time lags of 2 and 4 months for ENSO are also included hereafter in the MLR model to account for a large but delayed impact of ENSO on mid- and high latitudes O3 variations far from the p Figure 1 presents the seasonal distributions of tropospheric O3 measured by IASI averaged over January 2008–May 2017 (Fig. 1a), along with the RMSE of the seasonal regres- sion ﬁt (in DU; Fig. 1b) and the contribution of the ﬁtted sea- sonal model into the IASI O3 time series (in percent; Fig. 1c), calculated as σ O3Fitted_model(t) σ(O3(t)) , where σ is the standard devia- tion relative to the regression models and to the IASI O3 time series. These two statistical parameters help to evaluate how well the ﬁtted model explains the variability in the IASI O3 observations. The seasonal patterns of O3 measurements are close to those reported in Wespes et al. (2017) for a shorter period (see Sects. 2.1 and 3.1 in Wespes et al., 2017 for a de- tailed description of the distributions) and they clearly show, for instance, high O3 values over the highly populated ar- eas of Asia in summer. The distributions from Fig. 1 show that the model reproduces between 35 and 90 % of the daily O3 variation captured by IASI and that the residual errors vary between 0.01 and 5 DU (i.e., the RMSEs relative to the IASI O3 time series are of ∼15 % on global average and vary between 10 % in the NH in summer and 30 % in speciﬁc tropical regions). C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6869 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere equatorial Paciﬁc, where the ENSO signal originates (We- spes et al., 2017). nard et al. (2018) from comparison with O3 sondes. This drift (∼2.8 DU decade−1 in the NH) is shown in Boynard et al. (2018) to result from a discontinuity (called “jump” by the author) in September 2010 in the IASI O3 time se- ries, for reasons that are unclear at present. Furthermore, the drift strongly decreases (< \|1\| DU decade−1 on average) af- ter the jump and it becomes even non-signiﬁcant for most of the stations (signiﬁcant positive drift is also found for some stations) over the periods before or after the jump, separately. 3 Regression performance and sensitivity to trend On an annual basis (data not shown), the model explains a large fraction of the variation in the IASI O3 dataset (from ∼45 to ∼85 %) and the RMSEs are lower than 4.5 DU everywhere (∼3 DU on the global average). The relative RMSE is less than 1 % in almost all situations indi- cating the absence of bias. Figure 1 presents the seasonal distributions of tropospheric O3 measured by IASI averaged over January 2008–May 2017 (Fig. 1a), along with the RMSE of the seasonal regres- sion ﬁt (in DU; Fig. 1b) and the contribution of the ﬁtted sea- sonal model into the IASI O3 time series (in percent; Fig. 1c), l l t d σ O3Fitted_model(t) h i th t d d d i The seasonal distributions of the contribution to the total variation in the MLT from the adjusted harmonic terms and explanatory variables, which account for the “natural” vari- ability, and from the LT term are shown in Fig. 2a and b, respectively. The grey areas in the LT panels refer to the LT terms rejected by the stepwise backward elimination process. The crosses indicate that the trend estimate in the grid cell is non-signiﬁcant at the 95 % conﬁdence limits (2σ level) when accounting for the autocorrelation in the noise residual at the end of the elimination procedure. While the large inﬂuences of the seasonal variations and of the main drivers – namely ENSO, NAO and QBO – on the IASI O3 records have been clearly attested in Wespes et al. (2017), we demonstrate in Fig. 2 that the LT also contributes considerably to the O3 variations detected by IASI in the troposphere. The LT con- 2 IASI O3 measurements and multivariate regression They demonstrated a good de- gree of accuracy, precision and vertical sensitivity, and no in- strumental drift, for capturing large-scale dynamical modes of O3 variability in the troposphere independently from the layers above (Wespes et al., 2017), with the possibility of further differentiating long-term O3 changes in the tropo- sphere (Wespes et al., 2016). Note, however, that a drift in the NH middle–low troposphere (MLT) O3 over the whole IASI dataset is reported in Keppens et al. (2018) and Boy- Such long-term datasets are now becoming obtainable with the new generation of nadir-looking and polar-orbiting instruments measuring in the thermal infrared region. In par- ticular, about one decade of O3 proﬁle measurements, with good sensitivity in the troposphere independent from the lay- ers above, is now available from the IASI (Infrared Atmo- spheric Sounding Interferometer) sounder aboard the Euro- pean Metop platforms, allowing the monitoring of regional and global variations in tropospheric O3 levels (e.g., Dufour et al., 2012; Saﬁeddine et al., 2014; Wespes et al., 2016). , ; , ; p , ) In this study, we examine the tropospheric O3 changes behind the natural IAV as measured by IASI over January 2008–May 2017. To that end, we use the approach described in Wespes et al. (2017), which relies on a multi-linear regres- sion (MLR) procedure, for accurately differentiating trends from other sources of O3 variations, the latter being robustly identiﬁed and quantiﬁed in the companion study. In Sect. 2, we brieﬂy review the IASI mission and the tropospheric O3 product, and we shortly describe the multivariate models (an- nual or seasonal) that we use for ﬁtting the daily O3 time se- ries. In Sect. 3, after verifying the performance of the MLR models over the available IASI dataset, we evaluate the fea- sibility of capturing and retrieving signiﬁcant trend param- eters, apart from natural O3 dependencies, by performing trend sensitivity studies. In Sect. 4, we present and discuss the global distributions of the O3 trends estimated from IASI in the troposphere. The focus is given in summer over and downwind anthropogenic polluted areas of the NH where the possibility of inferring signiﬁcant trends from the ﬁrst ∼9 years of available IASI measurements is demonstrated. Finally, the O3–CO correlations, enhancement ratios and co- www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 www.atmos-chem-phys.net/18/6867/2018/ www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6870 Figure 1. Seasonal distribution of O3 tropospheric columns (in DU, integrated from ground to 300 hPa) measured by IASI and averaged over January 2008–May 2017 (a), of the RMSE of the regression ﬁts (in DU, b) and of the fraction of the variation in IASI data explained by the regression model, calculated as 100 × σ O3Fitted_ model(t) /σ (O3(t)) (in percent, c). Data are averaged over a 2.5◦× 2.5◦grid box. Figure 1. Seasonal distribution of O3 tropospheric columns (in DU, integrated from ground to 300 hPa) measured by IASI and averaged over January 2008–May 2017 (a), of the RMSE of the regression ﬁts (in DU, b) and of the fraction of the variation in IASI data explained by the regression model, calculated as 100 × σ O3Fitted_ model(t) /σ (O3(t)) (in percent, c). Data are averaged over a 2.5◦× 2.5◦grid box. tribution generally ranges from 15 to 50 %, with the largest values (∼30 to ∼50 %) being observed at mid- and high lati- tudes in the SH (30–70◦S) and in the NH (45–70◦N) in sum- mer. In the SH, they are associated with the smallest tropo- spheric O3 columns (Fig. 1a) and the smallest natural con- tributions (< 25 %; Fig. 2a), while in the NH summer, they interestingly correspond to large MLT O3 columns, large nat- ural contributions (∼50 to ∼60 %) and the smallest RMSE (< 12 % or < 3 DU). From the annual regression model, the natural variation and the trend respectively contribute 30– 85 % and up to 40 % to the total variation in the MLT. from the adjustment with and without the LT term included in the MLR model (light blue and orange lines, respec- tively). The differences between the ﬁtted models with and without LT are shown in Fig. 3c (pink lines). They match fairly well the adjusted trend over the IASI period (Fig. 3c, grey lines; the trend and the RMSE values are also indi- cated) and the adjustment without LT leads to larger residuals (e.g., RMSE_JJA_w/o_LT = 3.37 DU vs. RMSE_JJA_with_LT = 3.21 DU in summer). www.atmos-chem-phys.net/18/6867/2018/ This result demonstrates the possibility of capturing trend information from ∼9 years of IASI-MLR with only some compensation effects from the other explana- tory variables, contrary to what was observed when consid- ering a shorter period of measurements or less-frequent sam- pling (i.e., monthly dataset; e.g., Wespes et al., 2016). It is also worth mentioning that the O3 change calculated over the whole IASI dataset in summer is larger than the RMSE of the model residuals (increase of 6.50 ± 2.35 DU vs. RMSE of 3.21 DU), underlying the statistical relevance of trend es- timates. p In Fig. 3, we further investigate the robustness of the estimated trends by performing sensitivity tests in regions of signiﬁcant trend contributions (e.g., in the NH midlat- itudes in summer; see Fig. 2). The ∼9-year time series of IASI O3 daily averages (dark blue) along with the re- sults from the seasonal regression model with and without the LT term included in the model (light blue and orange lines, respectively) are represented in Fig. 3a for one spe- ciﬁc location (highlighted by a blue circle in the JJA panel in Fig. 4). Figure 3b provides the deseasonalized IASI (dark blue line) and ﬁtted time series (calculated by subtracting the adjusted seasonal cycle from the time series) resulting The robustness of the adjusted trend is veriﬁed at the global scale in Fig. 4 which represents the seasonal dis- tributions of the relative differences in the RMSE with and without the LT included in the MLR model, calcu- www.atmos-chem-phys.net/18/6867/2018/ www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 6871 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere Figure 2. Seasonal distributions of the contribution from the seasonal and explanatory variables into the IASI O3 variations estimated as " 100 × σ 4;m P n=1;j=2 an;bn;xj cos(nωt);sin(nωt);Xnorm,j(t) ! /σ (O3(t)) # (in percent, a) and of the contribution from the linear trend calculated as 100 × σ xj=1 · trend /σ (O3(t)) (in percent, b). The grey areas and crosses refer to the non-signiﬁcant grid cells in the 95 % conﬁdence limits (2σ level). Note that the scales are different. Figure 2. Seasonal distributions of the contribution from the seasonal and explanatory variables into the IASI O3 variations estimated as " 100 × σ 4;m P n=1;j=2 an;bn;xj cos(nωt);sin(nωt);Xnorm,j(t) ! 4 O3 trend over 2008–2017 lated as (RMSE_w/o_LT −RMSE_with_LT) / RMSE_with_LT × 100 (in percent). An increase in the RMSE when excluding LT from the MLR is observed almost everywhere in regions of signiﬁcant trend contributions (Fig. 2), especially in mid- and high latitudes of the SH and of the NH in summer, where it reaches 10 %. This result indicates that adjusting LT im- proves the performance of the model and, hence, that a trend signal is well captured by IASI at a regional scale in the tro- posphere. From the annual model, the increase in the RMSE only reaches 5 % at mid- and high latitudes of the SH (data not shown). In regions of weak or non-signiﬁcant trend con- tribution (see crosses in Fig. 2), no improvement is logically found. www.atmos-chem-phys.net/18/6867/2018/ /σ (O3(t)) # (in percent, a) and of the contribution from the linear trend calculated as 100 × σ xj=1 · trend /σ (O3(t)) (in percent, b). The grey areas and crosses refer to the non-signiﬁcant grid cells in the 95 % conﬁdence limits (2σ level). Note that the scales are different. 4.1 Annual and seasonal trends The annual and the seasonal distributions of the ﬁtted LT terms which are retained in the annual and the seasonal MLR models by the stepwise elimination procedure are respec- tively represented in Fig. 5a and b (in DU yr−1). Generally, the mid- and high latitudes of both hemispheres and, more particularly, the NH midlatitudes in summer reveal signif- icant negative trends, while the tropics are mainly charac- terized by non-signiﬁcant or weak signiﬁcant trends. Even if trends in emissions have already been able to qualita- tively explain measured tropospheric O3 trends over spe- ciﬁc regions, the magnitude and the trend estimates consider- www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6872 Figure 3. (a) Example of daily time series of IASI O3 measurements (dark blue) and of the ﬁtted seasonal regression models with (light blue) and without (orange) the linear term in the troposphere. Daily time series (b) of the deseasonalized O3 (observations and regression models) and (c) of the difference of the ﬁtted models with and without the linear trend term as well as the adjusted annual trend (pink and grey lines, respectively; c; given in DU). The RMSE (annual and for the JJA period in DU) and the trend values (annual and for the JJA period in DU yr−1) are also indicated. Figure 3. (a) Example of daily time series of IASI O3 measurements (dark blue) and of the ﬁtted seasonal regression models with (light blue) and without (orange) the linear term in the troposphere. Daily time series (b) of the deseasonalized O3 (observations and regression models) and (c) of the difference of the ﬁtted models with and without the linear trend term as well as the adjusted annual trend (pink and grey lines, respectively; c; given in DU). The RMSE (annual and for the JJA period in DU) and the trend values (annual and for the JJA period in DU yr−1) are also indicated. Figure 4. Seasonal distribution of the differences between the RMSE of the regression ﬁts with and without the linear trend term [(RMSE_w/o_LT −RMSE_with_LT) / RMSE_with_LT × 100] (in percent). The blue circle in the JJA panel refers to the case presented in Fig. 3. Figure 4. 4.1 Annual and seasonal trends Seasonal distribution of the differences between the RMSE of the regression ﬁts with and without the linear trend term [(RMSE_w/o_LT −RMSE_with_LT) / RMSE_with_LT × 100] (in percent). The blue circle in the JJA panel refers to the case presented in Fig. 3. Figure 4. Seasonal distribution of the differences between the RMSE of the regression ﬁts with and without the linear trend term [(RMSE_w/o_LT −RMSE_with_LT) / RMSE_with_LT × 100] (in percent). The blue circle in the JJA panel refers to the case presented in Fig. 3. (e.g., Jonson et al., 2006; Cooper et al., 2010; Logan et al., 2012; Wilson et al., 2012; Hess and Zbinden, 2013; and references therein). As a result, comparing/reconciling the adjusted trends with independent measurements, even on a qualitative basis, remains difﬁcult. Nevertheless, several of the statistically signiﬁcant features observed in Fig. 5 show, ably vary between independent measurement datasets (e.g., Cooper et al., 2014; the TOAR-Climate report coordinated by the International Global Atmospheric Chemistry Project and available soon at http://www.igacproject.org/activities/ TOAR, last access: 8 May 2018, of Gaudel et al., 2018, and references therein) for the reasons discussed in Sect. 1, and they are not reproduced/explained by model simulations www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 6873 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere Figure 5. (a) Annual and (b) seasonal distributions of the adjusted trends in DU yr−1 from the multi-linear regression models. The grey areas and crosses refer to the non-signiﬁcant grid cells in the 95 % conﬁdence limits (2σ level). Figure 5. (a) Annual and (b) seasonal distributions of the adjusted trends in DU yr−1 from the multi-linear regression models. The grey areas and crosses refer to the non-signiﬁcant grid cells in the 95 % conﬁdence limits (2σ level). interestingly, qualitative consistency with respect to recent published ﬁndings: to the tropospheric O3 trends over that region (Liu et al., 2016, 2017). Nevertheless, this ﬁnding should be miti- gated by the fact that the trend value in the SH tropics is of the same magnitude as the RMSE of the regression residuals (∼2 to ∼4.5 DU; see Fig. 1). 4.1 Annual and seasonal trends – The SH tropical region extending from the Amazon to tropical eastern Indian Ocean seems to indicate a general increase with, for example, a DJF trend of ∼0.23 ± 0.18 DU yr−1 (i.e., 2.09 ± 1.70 DU over the IASI measurement period), despite the large IAV in the MLT which characterizes the tropics and which likely explains the high frequency of non-signiﬁcant trends. Enhanced O3 levels over that region have already been analyzed for previous periods (e.g., Logan, 1985; Lo- gan and Kirchhoff, 1986; Fishman et al., 1991; Moxim and Levy, 2000; Thompson and Wallace, 2000; Thomp- son et al., 2007; Sauvage et al., 2006, 2007; Heue et al., 2016; Ebojie et al., 2016; Archibald et al., 2018; Leven- tidou et al., 2017). For instance, the large O3 enhance- ment of ∼0.36 ± 0.25 DU yr−1 (i.e., 3.3 ± 2.3 DU over the whole IASI period) stretching from southern Africa to Australia over the northeast of Madagascar during the austral winter–spring likely originates from large IAV in the subtropical jet-related stratosphere–troposphere ex- changes, which have been found to primarily contribute – The SH tropical region extending from the Amazon to tropical eastern Indian Ocean seems to indicate a general increase with, for example, a DJF trend of ∼0.23 ± 0.18 DU yr−1 (i.e., 2.09 ± 1.70 DU over the IASI measurement period), despite the large IAV in the MLT which characterizes the tropics and which likely explains the high frequency of non-signiﬁcant trends. Enhanced O3 levels over that region have already been analyzed for previous periods (e.g., Logan, 1985; Lo- gan and Kirchhoff, 1986; Fishman et al., 1991; Moxim and Levy, 2000; Thompson and Wallace, 2000; Thomp- son et al., 2007; Sauvage et al., 2006, 2007; Heue et al., 2016; Ebojie et al., 2016; Archibald et al., 2018; Leven- tidou et al., 2017). For instance, the large O3 enhance- ment of ∼0.36 ± 0.25 DU yr−1 (i.e., 3.3 ± 2.3 DU over the whole IASI period) stretching from southern Africa to Australia over the northeast of Madagascar during the austral winter–spring likely originates from large IAV in the subtropical jet-related stratosphere–troposphere ex- changes, which have been found to primarily contribute Conversely, the tropical Paciﬁc region exhibits signif- icant negative trends that are similar to those reported from UV sounders in Ebojie et al. (2016) and in Leven- tidou et al. (2017) over previous periods, while Heue et al. C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere This ﬁnding is line with pre- vious studies which point out a possible leveling off of tropospheric O3 in summer due to the decline of anthro- pogenic O3 precursor emissions observed since 2010- 2011 in North America, in western Europe and also in some regions of China (e.g., Cooper et al., 2010, 2012; Logan et al., 2012; Parrish et al., 2012; Olt- mans et al., 2013; Simon et al., 2015; Ebojie et al., 2016; Archibald et al., 2018; Miyazaki et al., 2017). The present study even goes a step further by sug- gesting a possible decrease in the tropospheric O3 lev- els. Archibald et al. (2018) recently reported a net de- crease of ∼5 % in the global anthropogenic NOx emis- sions in the 30–90◦N latitude band, which is con- sistent with the annual signiﬁcant negative trend of −0.27 ± 0.15 DU yr−1 for O3 estimated from IASI in that band. We should also note that, even if these lati- tudes are characterized by the lowest stratospheric con- tribution (∼30 to ∼45 %; see Supplement in Wespes et al., 2016), it might partly mask/attenuate the variability in the tropospheric O3 levels. – The mid- and high latitudes of the SH show clear patterns of negative trends, throughout the year and in a more pronounced manner during winter–spring, with larger amplitudes than those of the RMSE values (∼−0.33 ± 0.14 DU yr−1 on average in the 35–65◦S band; i.e., a trend amplitude of ∼\|3.1\| ± 1.3 DU over the studied period vs. an RMSE value of ∼2.5 DU). These signiﬁcant negative trends in the SH are hard to explain, but considering the stratospheric contribu- tion into the tropospheric columns (natural and artiﬁ- cial due to the limited IASI vertical sensitivity) in the mid- and high latitudes of the SH (∼40 to ∼60 %; see Supplement in Wespes et al., 2016) and the nega- tive signiﬁcant trends previously reported from IASI in the UTLS/low stratosphere in the 30–50◦S band, they could be in line with those derived by Zeng et al. (2017) in the UTLS for a clean rural site of the SH (Lauder, New Zealand), which mainly originate from increasing tropopause height and O3-depleting substances. Signif- icant negative change in tropospheric O3 over these re- gions were also reported in Ebojie et al. (2016). 4.2 Expected year for trend detection In this section, we further verify that it is indeed possi- ble to infer, from the studied IASI period, the signiﬁcant negative trend derived in the 40–75◦N band in summer (∼\|0.5\| DU yr−1 on average; see Sect. 4.1) by determining the expected year from which such a trend amplitude would be detectable at a global scale. This is achieved by estimat- ing the minimum duration (with probability 0.90) of the IASI O3 measurements that would be required to detect a trend of a speciﬁed magnitude, and its 95 % conﬁdence level, fol- lowing the formalism developed in Tiao et al. (1990) and in Weatherhead et al. (1998): N∗≈ " 3.3 · σε τyr · s 1 + φ 1 −φ # 2 3 , (1) CLN∗= h N∗· e−B;N∗· e+B, i (2) (1) (2) – In the NH, a band-like pattern of negative trends is observed in the 40–75◦N band covering Europe and North America, especially during summer. Averaged annual trend of −0.31 ± 0.17 DU yr−1 and summer trend of −0.47 ± 0.22 DU yr−1 (i.e., −2.87 ± 1.57 and −4.36 ± 2.02 DU, respectively, from January 2008 to May 2017) are estimated in that latitudinal band. These trend values are signiﬁcantly larger than the RMSEs of the MLR model (< 3.5 DU in JJA; see Sect. 3, Fig. 1). Interestingly, both the annual and summer trends are ampliﬁed relative to the ones calculated in the midlatitudes of the NH over the 2008–2013 pe- riod of IASI measurements (−0.19 ± 0.05 DU yr−1 and −0.30 ± 0.10 DU yr−1 for the annual and the summer trends, respectively, calculated in the 30–50◦N band; where N∗is the number of the required years, σε is the stan- dard deviation of the autoregressive noise residual εt, τyr is the magnitude of the trend per year and φ is the lag-1 autocor- relation of the noise. The magnitudes of the variation and the autocorrelation in the noise residuals are taken into account for reaching a better trend estimate precision. Given that large variance (σ 2 ε ) and large positive autocorrelation φ of the noise induce small signal-to-noise ratio and long trend-like segments in the dataset, respectively, these two parameters increase the number of years that would be required for de- tecting a speciﬁed trend. C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6874 but also from a substantial change in the stratospheric contribution (Verstraeten et al., 2015), persist through 2008–2017 despite the recent decrease in O3 precursor emissions recorded in China after 2011 (e.g., Duncan et al., 2016; Krotkov et al., 2016; Miyazaki et al., 2017; van der A et al., 2017). This would indicate that this de- crease is probably too recent/weak to recover the 2008 O3 levels over the entire region. Signiﬁcant positive trends over Southeast Asia have also been reported from UV sounders over previous periods (e.g., Ebojie et al., 2016). Note, however, that this ﬁnding has to be taken carefully given the large model residuals (RMSE of ∼2 to ∼4 DU; see Sect. 3, Fig. 1) over that region. Finally, the large uncertainty in trend estimates over Southeast Asia might reﬂect the large IAV in the biomass-burning emissions and lightning NOx sources, in addition to the recent changes in emissions. but also from a substantial change in the stratospheric contribution (Verstraeten et al., 2015), persist through 2008–2017 despite the recent decrease in O3 precursor emissions recorded in China after 2011 (e.g., Duncan et al., 2016; Krotkov et al., 2016; Miyazaki et al., 2017; van der A et al., 2017). This would indicate that this de- crease is probably too recent/weak to recover the 2008 O3 levels over the entire region. Signiﬁcant positive trends over Southeast Asia have also been reported from UV sounders over previous periods (e.g., Ebojie et al., 2016). Note, however, that this ﬁnding has to be taken carefully given the large model residuals (RMSE of ∼2 to ∼4 DU; see Sect. 3, Fig. 1) over that region. Finally, the large uncertainty in trend estimates over Southeast Asia might reﬂect the large IAV in the biomass-burning emissions and lightning NOx sources, in addition to the recent changes in emissions. see Wespes et al., 2016). 4.1 Annual and seasonal trends (2016) mainly report a signiﬁcant positive trend over that region. – The trends over Southeast Asia are mostly non- signiﬁcant and vary by season. In spring–summer, some grid cells in India, in mainland China and eastern down- wind China exhibit signiﬁcant positive trends reach- ing ∼0.45 DU yr−1 (i.e., ∼4.2 DU over the IASI mea- surement period). This tends to indicate that the tropo- spheric O3 increases, which have been shown to mainly result from a strong positive trend in the Asian emis- sions over the past decades (e.g., Zhao et al., 2013; Cooper et al., 2014; Zhang et al., 2016; Cohen et al., 2017; Tarasick et al., 2018; and references therein) – The trends over Southeast Asia are mostly non- signiﬁcant and vary by season. In spring–summer, some grid cells in India, in mainland China and eastern down- wind China exhibit signiﬁcant positive trends reach- ing ∼0.45 DU yr−1 (i.e., ∼4.2 DU over the IASI mea- surement period). This tends to indicate that the tropo- spheric O3 increases, which have been shown to mainly result from a strong positive trend in the Asian emis- sions over the past decades (e.g., Zhao et al., 2013; Cooper et al., 2014; Zhang et al., 2016; Cohen et al., 2017; Tarasick et al., 2018; and references therein) www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere In summary, these large-scale indirect ENSO- related variations in tropospheric O3 and the lightning NOx impact on O3, which are not accounted for in the MLR by speciﬁc representative proxies, are misrepresented in the re- gression models. They induce large noise residuals, i.e., large σε, and hence extend the time period needed to detect a trend of any given magnitude. the uncertainty in φ (the uncertainty in σε being negligible given that only a few years of data are needed to estimate it; see Weatherhead et al., 1998). As a result, based on the available IASI-A and proxies datasets and assuming that the MLR model used in this study is accurate, we estimate, in Fig. 6a and b, the expected year when an O3 trend ampli- tude of \|5\| DU decade−1 (i.e., τyr = \|0.5\| DU yr−1 which cor- responds to the averaged absolute value of the ﬁtted nega- tive trends in the NH summer; see Fig. 5b) is detectable, and its associated maximal conﬁdence limit, respectively. The re- sults in Fig. 6 clearly demonstrate the possibility of infer- ring, from the available IASI dataset, such signiﬁcant trends in the mid- and high latitudes of the NH in summer and fall (trend detectable from ∼2016 to 2017 with an uncertainty of ∼6 to ∼9 months; see Fig. 6b). Conversely, the tropi- cal regions and the NH in winter–spring would require ad- ditional ∼6 months to ∼6 years of measurements to detect an amplitude of \|0.5\| DU yr−1 (trend signiﬁcant only from ∼2017 to 2023 or after depending on the location and the season). Note also that the strongest negative trends (up to −0.85 DU yr−1, i.e., τyr = \|0.85\| DU yr−1; see Fig. 5b) ob- served in speciﬁc regions of the NH midlatitudes would only require ∼6 years of IASI measurements for being detected. The mid- and high latitudes of the SH would require the shortest period of IASI measurement for detecting a speci- ﬁed trend, with only ∼7 years ± ∼6 months of IASI mea- surements to detect a \|0.5\| DU yr−1 trend (trend detectable from ∼2015). That band-like pattern in the SH corresponds to the region with the weakest IAV and contribution from large-scale dynamical modes of variability in the IASI MLT columns (see Sect. 3, Figs. 1 and 2), which translates into small σ 2 ε and φ. C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere Note however that an additional few months of IASI data are required to conﬁrm the smaller negative trend of ∼−0.35 DU yr−1 measured on average in the SH (see Fig. 5; a period ∼9 years ± ∼6 months being necessary to detect a trend amplitude of \|3.5\| DU decade−1). Given that large σε means large noise residual in the IASI data, the re- gions of short (long) required measurement period coincide, as expected, well with the small (high) RMSE values of the regression residuals (see Sect. 3, Fig. 1). Figure 6, ﬁnally, suggests that a long duration is also re- quired, especially in summer, above and east of China to quantify the anthropogenic impact on the local changes in the MLT: additional 3 ± 1.5 or 5 ± 1.5 years for a given \|5\| or \|3.5\| DU decade−1 trend are respectively calculated. This result could be explained by large perturbations in the MLT columns induced by recent decreases after decades of almost constant increases in surface emissions in China (e.g., Cohen et al., 2017; Miyazaki et al., 2017). 4.3 Multi-linear vs. single linear model Even if MLR models have already been used for extracting trends in stratospheric and total O3 columns (e.g., Mäder et al., 2007; Frossard et al., 2013; Rieder et al., 2013; Knibbe et al., 2014), single linear regression (SLR) models, which do not differentiate the natural (chemical and dynamical) factors describing the O3 variability, are still commonly used (e.g., Cooper et al., 2014; the TOAR-Climate report of Gaudel et al., 2018; and references therein). They are, however, sus- pected to contribute to trend biases retrieved between inde- pendent measurements. In addition to the time-varying in- strumental biases, trend biases can also be related to dif- ferences in the spatial and the temporal samplings (e.g., Doughty et al., 2011; Saunois et al., 2012; Lin et al., 2015), in the measurement period, in the upper boundary of the O3 columns, in the algorithm and in the vertical sensitivity of the measurements. The latter artiﬁcially alters the character- istics of the sounded layer by contaminations from the above and below layers leading to a mixing of the trend and also of the natural characteristics originating from these different layers (e.g., troposphere and stratosphere). The differences in the studied period, in the tropopause deﬁnition and in the The regions of the longest measurement periods required in the tropics for the \|5\| DU decade−1 trend detection (up to ∼16 years of IASI data) correspond to known patterns of widespread high O3: (a) above intense biomass burning in Amazonia and eastwards across the tropical Atlantic (Lo- gan and Kirchhoff, 1986; Fishman et al., 1991; Moxim and Levy, 2000; Thompson and Wallace, 2000; Thompson et al., 2007; Sauvage et al., 2007), (b) eastwards of Africa across the southern Indian Ocean which is subject to large varia- tions in the stratospheric inﬂuences during the winter–spring austral period (JJA–SON; Liu et al., 2016, 2017), (c) east- wards of Africa across the northern Indian Ocean to India likely due to large lightning NOx emissions above central Africa during the wet season associated with the northeast- ward jet conducting a so-called “O3 river” (Tocquer et al., C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 2015) and (d) above regions of positive ENSO “chemical” ef- fect in equatorial Asia/Australia and eastwards above north- ern and southern tropical regions (Wespes et al., 2016) ex- plained by reduced rainfall and biomass ﬁres during El Niño conditions (e.g., Worden et al., 2013). In fact, most of these patterns (a, b and d) are closely connected with strong El Niño events which extend the duration of the dry season and cause severe droughts, producing intense biomass-burning emissions, for instance, over South America (e.g., Chen et al., 2011; Lewis et al., 2011) and South Asia/Australia (e.g., Oman et al., 2013; Valks et al., 2014; Ziemke et al., 2015), and which alter the tropospheric circulation by increasing the transport of stratospheric O3 into the troposphere (e.g., Voulgarakis et al., 2011b; Neu et al., 2014) and the transport of biomass-burning air masses to the Indian Ocean (Zhang et al., 2012). In summary, these large-scale indirect ENSO- related variations in tropospheric O3 and the lightning NOx impact on O3, which are not accounted for in the MLR by speciﬁc representative proxies, are misrepresented in the re- gression models. They induce large noise residuals, i.e., large σε, and hence extend the time period needed to detect a trend of any given magnitude. 2015) and (d) above regions of positive ENSO “chemical” ef- fect in equatorial Asia/Australia and eastwards above north- ern and southern tropical regions (Wespes et al., 2016) ex- plained by reduced rainfall and biomass ﬁres during El Niño conditions (e.g., Worden et al., 2013). In fact, most of these patterns (a, b and d) are closely connected with strong El Niño events which extend the duration of the dry season and cause severe droughts, producing intense biomass-burning emissions, for instance, over South America (e.g., Chen et al., 2011; Lewis et al., 2011) and South Asia/Australia (e.g., Oman et al., 2013; Valks et al., 2014; Ziemke et al., 2015), and which alter the tropospheric circulation by increasing the transport of stratospheric O3 into the troposphere (e.g., Voulgarakis et al., 2011b; Neu et al., 2014) and the transport of biomass-burning air masses to the Indian Ocean (Zhang et al., 2012). 4.2 Expected year for trend detection CLN∗is the 95 % conﬁdence limit, which is not symmetric around N∗and depends on B, an es- timated uncertainty factor calculated as 4 3 √ D q 1+φ 1−φ , with D the number of days in the IASI datasets, which accounts for where N∗is the number of the required years, σε is the stan- dard deviation of the autoregressive noise residual εt, τyr is the magnitude of the trend per year and φ is the lag-1 autocor- relation of the noise. The magnitudes of the variation and the autocorrelation in the noise residuals are taken into account for reaching a better trend estimate precision. Given that large variance (σ 2 ε ) and large positive autocorrelation φ of the noise induce small signal-to-noise ratio and long trend-like segments in the dataset, respectively, these two parameters increase the number of years that would be required for de- tecting a speciﬁed trend. CLN∗is the 95 % conﬁdence limit, which is not symmetric around N∗and depends on B, an es- timated uncertainty factor calculated as 4 3 √ D q 1+φ 1−φ , with D the number of days in the IASI datasets, which accounts for www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 6875 Atmos. Chem. Phys., 18, 6867–6885, 2018 www.atmos-chem-phys.net/18/6867/2018/ The ﬁtted signal of the ENSO proxy from the MLR model (calculated as xjXnorm,j following Wespes et al., 2017) is also represented (Fig. 8c). That example clearly shows that the ENSO inﬂuence is considerably compensated for by the adjustment of the linear trend in the SLR (annual trend of −0.48 ± 0.06 DU yr−1 from SLR vs. −0.23 ± 0.16 DU yr−1 from MLR for that example). Finally, differences between the SLR and the MLR models are also observed in the region with strong positive NAO inﬂuence over the Icelandic/Arctic region during MAM (see Wespes et al., 2016, for a descrip- tion of the NAO-related O3 changes). Conversely, the sub- tropical SH exhibits similar seasonal patterns of negative trends from both the SLR and the MLR. This results from the weak natural IAV and contributions in tropospheric O3 above that region (see Sect. 3, Figs. 1 and 2), which, hence, limits the compensation effects. spatio-temporal sampling might also imply differences in the natural inﬂuence on the measured O3 variations. While the impact of the natural contribution is taken into account in the MLR model, it might introduce an additional bias in the trend determined from SLR, making further challenging to compare trends estimated from a series of inhomogeneous independent measurements. Substantial efforts in homogenizing independent tropo- spheric O3 column (TOC) datasets have been made in the TOAR-Climate assessment report (Gaudel et al., 2018), but large SLR trend biases remain between the TOAR datasets, in particular, between the satellite datasets where the lack of homogeneity in terms of tropopause calculation (same tropopause deﬁnition but different temperature proﬁles are used), of instrument vertical sensitivities and of spatial sam- pling have been speciﬁcally raised as possible causes for the trend divergence. If reconciling the trend biases between the datasets by ap- plying the vertical sensitivity of each measurement type to a common platform, as proposed in the TOAR-Climate assess- ment report is beyond the scope of this study and if, at this stage, there is no consensus in determining tropospheric O3 trends, the improvement in using a MLR instead of a SLR model for determining more accurate/realistic trends is ex- plored here by comparing the seasonal distributions of the trends estimated from MLR (see Fig. 5b in Sect. 4.1.) and from SLR (presented in Fig. 7). www.atmos-chem-phys.net/18/6867/2018/ www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6876 e 6. (a) Estimated year of tropospheric IASI O3 trend detection (with a probability of 90 %) for a given trend of \|5\| D ng at the beginning of the studied period (20080101) and (b) associated maximal conﬁdence limits from the annual (top pa nal (bottom panels) regression models. os. Chem. Phys., 18, 6867–6885, 2018 www.atmos-chem-phys.net/18/6 Figure 6. (a) Estimated year of tropospheric IASI O3 trend detection (with a probability of 90 %) for a given trend of \|5\| DU decade−1 starting at the beginning of the studied period (20080101) and (b) associated maximal conﬁdence limits from the annual (top panel) and the seasonal (bottom panels) regression models. www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6877 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere Figure 7. Seasonal distributions of the ﬁtted linear term trends (given in DU yr−1) derived from a single linear regression model. The crosses refer to the non-signiﬁcant grid cells in the 95 % conﬁdence limits (2σ level). Figure 7. Seasonal distributions of the ﬁtted linear term trends (given in DU yr−1) derived from a single linear regression model. The crosses refer to the non-signiﬁcant grid cells in the 95 % conﬁdence limits (2σ level). LT term and other covariates. For instance, the regions with high signiﬁcant SLR trends (∼0.3 to ∼0.6 DU yr−1 over the tropical western and middle Paciﬁc) during the period extending from September to May match the regions with strong El Niño/Southern Oscillation inﬂuence (see Figs. 8 and 12 in Wespes et al., 2016). Conversely, the MLR model shows generally weak signiﬁcant negative or non-signiﬁcant trends in the Paciﬁc El Niño region during that period and it would even need additional ∼3 to ∼4 years of IASI mea- surements for detecting the ﬁtted SLR trends (see Sect. 4.2 above). The effect of ENSO in overestimating the ﬁtted SLR trend is further illustrated in Fig. 8 which represents the time series of O3 observed by IASI and adjusted by the annual MLR model (Fig. 8a) along with the deseasonalized times series (Fig. 8b) and the ﬁtted SLR and MLR trends (Fig. 8c). www.atmos-chem-phys.net/18/6867/2018/ Note that the constant term in the SLR model is split into two components (covering the periods before and after the September 2010 jump) to take account of the observed jump (see Sect. 2). The highest dif- ferences in the ﬁtted trends derived from the two methods are found in the tropics and in some regions of the midlati- tudes of the NH. They likely result from overlaps between the In summary, despite the fact that considering a long period of measurements is usually recommended in SLR studies to www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6878 Figure 8. Daily time series of (a) O3 measured by IASI (dark blue) and adjusted by the multivariate annual regression model (light blue), (b) the deseasonalized O3 and (c) the ﬁtted signal of ENSO proxy (one of the main retained proxies in the multivariate regression model) calculated as xjXnorm,j (given in DU) along with the adjusted trends derived from the single and the multivariate linear regressions (SLR and MLR) over the equatorial central Paciﬁc (region of negative ENSO “dynamical” effect). The RMSE of the MLR ﬁt and the ﬁtted SLR and MLR trend values are also indicated. Figure 8. Daily time series of (a) O3 measured by IASI (dark blue) and adjusted by the multivariate annual regression model (light blue), (b) the deseasonalized O3 and (c) the ﬁtted signal of ENSO proxy (one of the main retained proxies in the multivariate regression model) calculated as xjXnorm,j (given in DU) along with the adjusted trends derived from the single and the multivariate linear regressions (SLR and MLR) over the equatorial central Paciﬁc (region of negative ENSO “dynamical” effect). The RMSE of the MLR ﬁt and the ﬁtted SLR and MLR trend values are also indicated. Bertschi and Jaffe, 2005). However, there are only a few stud- ies using global satellite data for this purpose (Zhang et al., 2006; Voulgarakis et al., 2011a; Kim et al., 2013) and the analysis of the O3–CO relationship for better understanding the origin of O3 trends in the troposphere has, to the best of our knowledge, never been explored. www.atmos-chem-phys.net/18/6867/2018/ overcome the dynamical cycles and, hence, to help in differ- entiating their inﬂuences from trends, we show that, consid- ering that some dynamics have irregular or no particular pe- riodicity (e.g., NAO, ENSO), it is not accurate enough. Fur- thermore, accurate satellite measurements of tropospheric O3 at a global scale are quite recent, limiting the period of avail- able and comparable datasets (e.g., Payne et al., 2017). As a consequence, we support here that using a reliable multivari- ate regression model based on geophysical parameters and adapted for each speciﬁc sounded layer is a robust method for differentiating a “true” trend from any other sources of variability and, hence, that it should help in resolving trend differences between independent datasets. Figure 9a and b show the seasonal patterns of the O3–CO correlations (referred as RO3−CO) and of the dO3 / dCO re- gression slopes calculated in the troposphere (from the sur- face to 300 hPa) over the studied IASI period (January 2008– May 2017). The dO3 / dCO regression slopes, which rep- resent the so-called O3–CO enhancement ratio, are used to evaluate the photochemical O3 production in continental out- ﬂow regions. The RO3−CO and the dO3 / dCO distributions are similar and clearly show regional and seasonal differ- ences in the strength of the O3–CO relationships. The pat- terns of positive and negative correlations allows differen- tiating the outﬂow regions characterized by photochemical O3 production from precursors (including CO) or CO de- struction (both identiﬁed by positive RO3−CO) from the re- gions characterized by O3 loss (chemical destruction or sur- face deposition) or by strong stratospheric contaminations (both identiﬁed by negative RO3−CO). Negative RO3−CO and dO3 / dCO are measured at high latitudes of both hemi- spheres all over the year, but more speciﬁcally at high lat- 4.4 Continental inﬂuence In this section, we use the capabilities of IASI to simultane- ously measure O3 and CO in order (1) to differentiate tro- pospheric and stratospheric air masses, (2) to identify the regions inﬂuenced by the continental export/intercontinental transport of O3 pollution and (3) to evaluate that continen- tal inﬂuence on tropospheric O3 trends as observed by IASI. Similar tracer correlations between CO and O3 have already been used to give insights into the photochemical O3 en- hancement in air pollution transport (e.g., Parrish et al., 1993; www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6879 Figure 9. Global distributions of (a) the correlation coefﬁcients (RO3−CO), (b) the regression slope (dO3 / dCO in mol cm−2/mol cm−2) and (c) the covariances (COVO3−CO in 1033 mol cm−2 × mol cm−2) of daily median IASI tropospheric O3 and CO over January 2008–May 2017. Data are averaged over a 2.5◦× 2.5◦grid box. Crosses in RO3−CO (a) refer to the non-signiﬁcant grid cells in the 95 % conﬁdence intervals (2σ level). Figure 9. Global distributions of (a) the correlation coefﬁcients (RO3−CO), (b) the regression slope (dO3 / dCO in mol cm−2/mol cm−2) and (c) the covariances (COVO3−CO in 1033 mol cm−2 × mol cm−2) of daily median IASI tropospheric O3 and CO over January 2008–May 2017. Data are averaged over a 2.5◦× 2.5◦grid box. Crosses in RO3−CO (a) refer to the non-signiﬁcant grid cells in the 95 % conﬁdence intervals (2σ level). itudes of the SH in summer–fall (with RO3−CO < −0.25 on averages in DJF and MAM). Given that high latitudes ex- perience more O3 destruction than the low latitudes due to a lack of sunlight, the negative correlations for the high latitude regions might also reﬂect air masses originating from/characterizing the stratosphere due to natural intrusion or to artiﬁcial mixing with the troposphere introduced by the limited vertical sensitivity of IASI in the highest lati- tudes (stratospheric contribution varying between ∼40 and ∼65 %; see Supplement in Wespes et al., 2016). These pro- cesses are likely at the origin of the band-like pattern of negative trends in the SH discussed in Sects. 3 and 4.1. 4.4 Continental inﬂuence Negative RO3−CO and dO3 / dCO are also found above the Caribbean, the Arabic Peninsula and the northern Indian Ocean in JJA/SON and the South Atlantic in DJF. They are in line with Kim et al. (2013) and they likely reﬂect the in- ﬂuence of lightning NOx which produce O3 but also OH ox- idizing CO (e.g., Sauvage et al., 2007; Labrador et al., 2004). and over the southern Africa/Amazonia/South Atlantic re- gion. These O3–CO correlation patterns from IASI are fully consistent with those measured by TES (Zhang et al., 2006, 2008; Voulgarakis et al., 2011a) and by OMI/AIRS (Kim et al., 2013), which have been interpreted with global chemical transport models (CTMs) as originating from Asian pollu- tion inﬂuences and combustion sources including biomass burning, respectively. The high positive RO3−CO found in JJA at midlatitudes of the NH are detected to a lesser ex- tent in DJF reﬂecting the decreasing photochemistry. It is also worth pointing out the clear hemispheric differences in Fig. 9 in the RO3−CO and dO3 / dCO values at mid- and high latitudes and, in particular, the shift of positive RO3−CO and dO3 / dCO towards higher latitudes of the NH during sum- mer (e.g., RO3−CO = 0.24 in summer vs. 0.038 in spring in the 35–55◦N band). As a consequence, these results sug- gest that the band-like pattern of negative trends measured by IASI in summer might substantially reﬂect the continen- tal pollution inﬂuence and, hence, that it could result from the decline of anthropogenic O3 precursor emissions. Nev- ertheless, interpreting O3–CO correlations in the free tropo- sphere, especially in photochemically aged pollution plumes far from the emission sources towards the highest latitudes, remains complicated by the mixing of the continental com- Strong positive correlations are identiﬁed in both RO3−CO and dO3 / dCO patterns over the tropical regions and for midlatitudes of both hemispheres during the peak of photochemistry in summer. Maxima (RO3−CO > 0.8 and dO3 / dCO > 0.5) are detected in continental pollution out- ﬂow regions in the NH, especially downwind Southeast Asia www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere The distributions have allowed us to identify, in particular, strong positive O3–CO correlations, regression slopes and covariance in the NH midlatitudes and northward during summer, which suggest a continental pol- lution inﬂuence in the NH band-like pattern of high signif- icant negative trends recorded by IASI and, hence, a direct effect of the policy measures taken to reduce emissions of O3 precursor species. ) Finally, we also provide in Fig. 9c the seasonal patterns of O3–CO covariances (COVO3−CO). They conﬁrm the band- like pattern of the weak natural variation captured in the SH midlatitudes (see Sects. 3 and 4.1) and help identify- ing the region downwind eastern China, the northern mid- latitudes outﬂow region and the southern tropical region as the ones with the highest pollution variability, in addition to the strongest O3–CO correlations. To conclude, the particu- larly strong positive O3–CO relationship in terms of RO3−CO, dO3 / dCO and COVO3−CO measured over and downwind northeast India/eastern China in summer in comparison with the ones measured downwind eastern USA and over Eu- rope indicate that Southeast Asia experiences the most of the intense pollution episodes of the NH with the largest O3–CO variability (COVO3−CO > 40 × 1033 mol2 cm−4) and the largest O3 enhancement (dO3 / dCO > 0.5) over the last decade. The strong O3–CO relationship in that region is as- sociated with the signiﬁcant increase that is detected in the IASI O3 levels downwind east of Asia (see Sect. 4.1) despite the net decrease in O3 precursor emissions recorded in China after 2011 (e.g., Cohen et al., 2017; Miyazaki et al., 2017). C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6880 bustion outﬂow with stratospheric air masses, in addition to the background dynamic and photochemistry (e.g., Liang et al., 2007). found interesting signiﬁcant positive trends in the SH trop- ical region extending from the Amazon to the tropical east- ern Indian Ocean and over Southeast Asia in spring–summer, which should however be carefully considered given the high RMSEs of the regression residuals in these regions. The MLR analysis reveals a band-like pattern of high signiﬁcant negative trends in the NH mid- and high latitudes in summer (−0.47 ± 0.22 DU yr−1 on average in the 40–75◦N band). The statistical signiﬁcance of such trend estimates is fur- ther veriﬁed by estimating, based on the autocorrelation and on the variance of the noise residuals, the minimum number of years of IASI measurements that are required to detect a trend of a \| 5\| DU decade−1 magnitude. The results clearly demonstrate the possibility of determining such a trend am- plitude from the available IASI dataset and the used MLR model at northern mid- and high latitudes in summer, while much larger measurement periods are necessary elsewhere. In particular, the regions with the longest required period, in the tropics, highlight a series of known processes that are closely related to the El Niño dynamic, which underlies the lack of associated parameterizations in the MLR model. The importance of using reliable MLR models in understanding large-scale O3 variations and in determining trends is fur- ther explored by comparing the trends inferred from MLR and from SLR, the latter still being commonly used by the international community. The comparison has clearly high- lighted the advantage of MLR in attributing the trend-like segments in natural variations, such as ENSO, to the right processes and, hence, in avoiding misinterpretation of “ap- parent” trends in the measurement datasets. Nevertheless, it is worth noting that there could be a possible impact of the sampling (because of the cloud and quality ﬁlters applied) and of the jump in September 2010 that has been identiﬁed in the IASI dataset (see Sect. 2), in both MLR and SLR estima- tions. Finally, by exploiting the simultaneous and vertically resolved O3 and CO measurements from IASI, we have pro- vided and used the O3–CO correlations in the troposphere to help determine the origins/characteristics of patterns of negative or positive trends. www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere Climate assessment report of Gaudel et al., 2018). Currently, no consensus in terms of O3 trends in the troposphere is reached from the available measurements (UV or IR satel- lites, O3 sondes, aircrafts, ground-based measurements, etc.) for several reasons (time-varying instrumental biases, dif- ferences in the methodology used for calculating trends, in the measurement period, in the upper boundary of the O3 columns, in the retrieval algorithm, in the spatio-temporal sampling, in the vertical sensitivity of the instrument, etc.; see Sect. 4.3 and the TOAR-Climate report of Gaudel et al., 2018). However, determination, with IASI, of robust trends in tropospheric O3 at the global scale will be achievable in the near future by merging homogeneous O3 proﬁles from the three successive instruments onboard Metop-A (2006); -B (2012) and –C (2018) platforms and from the IASI next- generation instrument onboard the Metop Second Generation series of satellites (Clerbaux and Crevoisier, 2013; Crevoisier et al., 2014). A long record of tropospheric O3 measurements will be also provided by the Cross-track Infrared Sounder (CrIS) onboard the Joint Polar Satellite System series of satellites. References Aeris: The IASI O3 products processed with FORLI-O3 v20151001, available at: http://iasi.aeris-data.fr/O3/ (last access: 14 May 2018), 2017a. Aeris: The IASI CO products processed with FORLI-CO v20151001, available at: http://iasi.aeris-data.fr/CO/ (last access: 14 May 2018), 2017b. Anton, M., Loyola, D., Clerbaux, C., Lopez, M., Vilaplana, J., Banon, M., Hadji-Lazaro, J., Valks, P., Hao, N., Zimmer, W., Coheur, P., Hurtmans, D., and Alados-Arboledas, L.: Validation of the Metop-A total ozone data from GOME-2 and IASI using reference ground-based measurements at the Iberian peninsula, Remote Sens. Environ., 115, 1380–1386, 2011. Archibald, A., Elshorbany, Y., et al.: Tropospheric Ozone Assess- ment Report: Critical review of the present-day and near future tropospheric ozone budget, Elem. Sci. 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The IASI O3 and CO data respectively pro- cessed with FORLI-O3 and FORLI-CO v0151001 can be down- loaded from the Aeris portal (http://iasi.aeris-data.fr/O3/ and http: //iasi.aeris-data.fr/CO/, respectively; Aeris, 2017a, b). Boynard, A., Hurtmans, D., Garane, K., Goutail, F., Hadji-Lazaro, J., Koukouli, M. E., Wespes, C., Keppens, A., Pommereau, J.- P., Pazmino, A., Balis, D., Loyola, D., Valks, P., Coheur, P.-F., and Clerbaux, C.: Validation of the IASI FORLI/Eumetsat ozone products using satellite (GOME-2), ground-based (Brewer- Dobson, SAOZ) and ozonesonde measurements, Atmos. Meas. Tech. Discuss., https://doi.org/10.5194/amt-2017-461, in review, 2018. 5 Conclusions In this study, we have explored, for the ﬁrst time, the possi- bility of inferring signiﬁcant trends in tropospheric O3 from the ﬁrst ∼10 years (January 2008–May 2017) of IASI daily measurements at a global scale. To this end, MLR analyses have been performed by applying a multivariate regression model (annual and seasonal formulations), including a linear trend term in addition to chemical and dynamical proxies, on gridded mean tropospheric ozone time series. This work follows on the analysis of the main dynamical drivers of O3 variations measured by IASI, which was recently published in Wespes et al. (2017). We have ﬁrst veriﬁed the perfor- mance of the MLR models in explaining the variations in daily time series over the whole studied period. In particu- lar, we have shown that the model reproduces a large part of the O3 variations (> 70 %) with small residuals errors (RMSE of ∼10 %) at northern latitudes in summer. We have then performed O3 trend sensitivity tests to verify the possibility of capturing trend characteristics independently from natural variations. Despite the weak contribution of trends to the to- tal variation in the MLT O3 columns at a global scale, the re- sults demonstrate the possibility of differentiating signiﬁcant trends from the explanatory variables, especially in summer at mid- and high latitudes of the NH (∼40–75◦N) where the contribution and the sensitivity of trends are the largest (contribution of ∼30 to ∼50 % and a ∼10 % increase in the RMSE excluding the LT in the model). We then focused on the interpretation of the global trend estimates. We have Overall, this study supports the importance of us- ing (1) high density and long-term homogenized satellite records, such as those provided by IASI and (2) complex models with predictor functions that describe the O3 regres- sors dependencies for more accurate determination of trends in tropospheric O3 – as required by the scientiﬁc commu- nity, e.g., in the Intergovernmental Panel on Climate Change (IPCC, 2013) – and for further resolving trend biases be- tween independent datasets (Payne et al., 2017; the TOAR- www.atmos-chem-phys.net/18/6867/2018/ Atmos. Chem. Phys., 18, 6867–6885, 2018 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6881 C. Wespes et al.: Decrease in tropospheric O3 levels in the Northern Hemisphere 6882 ern mid-latitudes, as seen by IAGOS from 1995 to 2013, Atmos. Chem. Phys. 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https://openalex.org/W4392696400	https://www.researchsquare.com/article/rs-4034917/latest.pdf	English	null	Constructing an Ovarian Cancer Prognostic Model Based on Shared m6A Modifications and Cellular Senescence-Related Genes with Polycystic Ovary Syndrome	Research Square (Research Square)	2,024	cc-by	8,820	Constructing an Ovarian Cancer Prognostic Model Based on Shared m6A Modifications and Cellular Senescence-Related Genes with Polycystic Ovary Syndrome Yang Liu Department of Reproductive Medicine, The Second Affiliated Hospital of Kunming Medical University Yujie Gengxiao Department of Reproductive Medicine, The Second Affiliated Hospital of Kunming Medical University Yanzhi Wu Department of Reproductive Medicine, The Second Affiliated Hospital of Kunming Medical University Xi Hu Department of Reproductive Medicine, The Second Affiliated Hospital of Kunming Medical University Yingying Ma Department of Reproductive Medicine, The Second Affiliated Hospital of Kunming Medical University Chunyi Sun Department of gynecology, The Second Affiliated Hospital of Kunming Medical University Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Page 1/25 Abstract Background: Polycystic ovary syndrome (PCOS) and ovarian cancer (OV) are significant women's health concerns. This study aims to identify genes related to m6A modification and cellular senescence that are shared by PCOS and OV and to develop a prognostic model for OV outcomes. Methods: Transcriptomic datasets from GEO and TCGA were collected to identify differentially expressed genes (DEGs) associated with m6A modifications and cellular senescence in both PCOS and OV. We identified hub genes through WGCNA and assessed their prognostic significance. An OV prognostic model was developed and validated using multiple datasets, and correlations between risk scores and tumor immune microenvironment were evaluated. Drug sensitivity was predicted based on risk scores. qRT-PCR was performed to verify model gene alterations in OV cell lines. Results: We identified 73 DEGs that were common to both PCOS and OV, with eight genes associated with m6A modification and cellular senescence. WGCNA revealed 242 DEhub genes. A 19-gene prognostic model was established, exhibiting impressive efficacy with AUCs exceeding 0.7 for predicting OV patient survival. High-risk scores correlated with increased M0 macrophages and monocytes and decreased M1, CD8+ T cells, and T follicular helper cells. Drug sensitivity varied based on risk scores. Model gene alterations were verified in external OV datasets and OV cell lines. In external datasets, CXCR4 emerged as a key target gene of m6A modification. Conclusions: The derived 19-gene prognostic model offers potential avenues for risk assessment and personalized therapy in OV patients. Introduction In this study, we aimed to investigate the shared molecular mechanisms between PCOS and OV to uncover a potential link from PCOS to OV. We employed a comprehensive approach, including transcriptomic data analysis, co-expression network analysis, and the development of a prognostic mode for OV patients. Our methods involved the integration of multiple datasets, identification of differentially expressed genes (DEGs), and the exploration of gene modules associated with m6A modification and cellular senescence. Additionally, we constructed a prognostic risk signature and validated its effectiveness across different OV cohorts. Our results may identify novel biomarkers and therapeutic avenues for OV, ultimately improving patient care and outcomes. Introduction Polycystic Ovary Syndrome (PCOS) is an endocrine disorder that affects 5-20% of reproductive-age women worldwide[1]. Characterized by hormonal imbalances, irregular menstrual cycles, and ovarian cysts, PCOS has been intimately associated with a myriad of health issues[2-4]. Of these, one of the most concerning is the potential link between PCOS and ovarian cancer (OV)[5], a lethal gynecological malignancy that accounted for 4.2% of all cancer-related deaths worldwide in 2020[6]. The frequency of both disorders and their potential connection has led to increasing concerns about the progression from PCOS to OV[7, 8]. Unraveling this relationship is crucial, not only for understanding disease etiology but also for the development of timely interventions. Recent studies have demonstrated the significant involvement of N6-methyladenosine (m6A) RNA modification in OV. Elevated expression of the methyltransferase-like-3 (METTL3) protein in OV has been associated with tumor growth and invasion[9], along with other m6A-related proteins such as YTH domain family protein 1 (YTHDF1), YTHDF2, and obesity-associated protein (FTO)[10-12]. Interestingly, these genes have also been found to be upregulated in PCOS[13]. Furthermore, m6A-related osteoglycin has been identified as a risk marker linking PCOS to OV[14]. Abnormal m6A modification has been linked to cellular senescence[15-17], which is a process where cells lose their ability to divide and contribute to Page 2/25 age-related diseases, including ovarian cancer. It plays a multifaceted role in OV pathobiology, including the disease-promoting activity of normal peritoneal mesothelial cells, the effect of drugs on the senescence of normal cells, and spontaneous senescence in OV cells[18]. PCOS patients with irregular menstruation have been shown to share mRNA expression profiles similar to those observed in OV, including the alterations in cellular senescence-related genes[8]. However, the roles of the shared genes in OV development and prognosis remain largely unknown. In this study, we aimed to investigate the shared molecular mechanisms between PCOS and OV to uncover a potential link from PCOS to OV. We employed a comprehensive approach, including transcriptomic data analysis, co-expression network analysis, and the development of a prognostic model for OV patients. Our methods involved the integration of multiple datasets, identification of differentially expressed genes (DEGs), and the exploration of gene modules associated with m6A modification and cellular senescence. Additionally, we constructed a prognostic risk signature and validated its effectiveness across different OV cohorts. Our results may identify novel biomarkers and therapeutic avenues for OV, ultimately improving patient care and outcomes. Functional enrichment analysis Functional enrichment analysis of the DEhub genes was conducted using the R package "clusterProfiler." Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed. To enhance the robustness of the results, P-values were corrected using the Benjamini-Hochberg method. Enrichment results associated with the corrected P-values were reported. ssGSEA analysis of DEGs in PCOS and OV for m6A and cellular senescence The DEGs that consistently exhibited altered expression in both PCOS and OV were combined. Subsequently, these genes were intersected with m6A target genes and cellular senescence genes, resulting in the identification of m6A_CellAgeRGs. The m6A_CellAgeRG score was then computed using the "GSVA" package to evaluate m6A and cell aging-related metrics. Data retrieval and processing The schematic overview of the study is shown in Fig. S1. Transcriptomic data were obtained from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/geo/), including PCOS datasets GSE34526, GSE137684, GSE80432, GSE114419, and GSE102293, as well as OV datasets GSE18520, GSE27651, and GSE140082 (Table 1). The PCOS datasets were merged for DEG identification, comprising 28 PCOS granulocyte samples and 22 normal granulocyte samples. The GSE18520 OV dataset was used for the identification of OV-related DEGs, consisting of 53 OV cases and 10 normal controls. An external dataset, GSE27651, was combined with GSE18520 to explore model gene expression in OV. GSE18520 and the external dataset GSE140082 were used to validate the efficacy of the prognostic risk model, comprising 380 cancer tissue samples with prognosis data. GEO data processing involved matching probes to gene names, removing empty probes, and calculating the median expression value for genes with multiple corresponding probes. Batch-effect correction of the merged datasets was carried out using the "ComBat" function from the "sva" package. Transcriptomic data of The Cancer Genome Atlas (TCGA) OV dataset were retrieved from the UCSC Xena Browser (https://xenabrowser.net/) to develop a prognostic model for OV patients. FPKM values were converted into TPM using a formula: and were subsequently subjected to a log2(x + 1) transformation for subsequent analysis. m6A target genes in humans were obtained from m6A2Target m6A target genes in humans were obtained from m6A2Target (http://rm2target.canceromics.org/#/home). A total of 701 m6A target genes were selected as they were validated m6A targets reported in the literature for the human genome (hg38). A total of 866 genes Page 3/25 related to cellular senescence were sourced from the CellAge database (https://genomics.senescence.info/cells/). Weighted Gene Co-Expression Network Analysis (WGCNA) A WGCNA network was constructed using the "WGCNA" R package. In this analysis, the m6A_CellAgeRG score was used to identify gene modules associated with this score. For each cohort (PCOS and OV), disease samples from patients were selected. The top 50% of genes with the highest variance were used as input data. Outliers were removed, and the optimal soft threshold was determined based on the scale- free topology criterion. Subsequently, the weighted adjacency matrix and topological overlap matrix transformation were constructed. Modules containing more than 30 genes were identified using the hierarchical clustering tree method, with a merged similarity threshold set at 0.25. Each module was visually distinguished by a unique color. Furthermore, modules underwent additional scrutiny based on their correlation with the m6A_CellAgeRG score. Hub genes within these modules were identified using dual criteria: \|Module Membership (MM)\| > 0.6 and Gene Significance (GS) > 0.5. Those belonging to OV- related DEGs (DEhub) were extracted for subsequent analysis. Identification of DEGs Differential gene expression analysis between the disease and control groups was performed using the R package "limma." In the integrated PCOS dataset, DEGs were identified with a significance threshold of P < 0.05. In the OV dataset GSE18520, DEGs were determined based on the criteria of \|logFC\| > 1 and an adjusted P-value < 0.05. Construction of protein-protein interaction (PPI) network To explore the interactions between model genes and their functionally similar counterparts, a PPI was constructed using GeneMANIA (www.genemania.org) Construction of a prognostic risk signature for OV A prognostic risk signature for OV was established using TCGA OV cohort data. Prognosis-related DEhub genes were identified through univariate Cox analysis based on a significance threshold of P-value < 0.05. Subsequently, TCGA OV patients were randomly divided into training and test sets in a 7:3 ratio. LASSO Page 4/25 COX regression analysis was conducted within the training set using the "glmnet" R package. The optimal penalty parameter (λ) was determined, and nineteen genes with non-zero coefficients were selected as significant contributors to the model. To calculate the risk score, weight coefficients from the model were combined with gene expression levels using the following formula: COX regression analysis was conducted within the training set using the "glmnet" R package. The optimal penalty parameter (λ) was determined, and nineteen genes with non-zero coefficients were selected as significant contributors to the model. To calculate the risk score, weight coefficients from the model were combined with gene expression levels using the following formula: Immune response prediction The Tumor Immune Dysfunction and Exclusion (TIDE) tool (http://tide.dfci.harvard.edu) was used to predict the response of patients within the TCGA cohort to immunotherapy. The TIDE scores were compared among patients with different risk levels. Immune cell infiltration assessment The "CIBERSORT" algorithm was used to assess the infiltration levels of 22 different immune cell types within the TCGA OV cohort. To ensure reliable analysis, immune cell types with an abundance of 0 in more than half of the samples were removed. The remaining immune cells were evaluated for their infiltration patterns in patients with different risk scores. The R package "estimate" was utilized to evaluate the immune score, tumor purity, and stromal score in patients at varying risk levels. Construction and evaluation of a prognostic nomogram Univariate and multivariate Cox analyses were conducted to assess the prognostic value of the risk score and clinical variables, including age, stage, and grade. The R package "forestplot" was utilized to visualize the results of the Cox analyses in the form of forest plots. A nomogram was constructed using the R package “regplot”. The performance, which measures the agreement between predicted and observed outcomes, of the nomogram was evaluated using calibration curves generated by the calibrate function of the R package "rms". The clinical decision-making performance was assessed using decision curve analysis (DCA) curves generated by the R package "ggDCA". Identification of DEGs in PCOS and OV To identify shared DEGs between PCOS and OV, we analyzed gene expression data from public datasets. The synthesis of five PCOS datasets yielded data from 28 PCOS and 22 normal granulocyte samples. The TCGA OV dataset GSE18520 provided 53 cancer and 10 normal tissue samples. Our analysis revealed 1179 PCOS-associated DEGs (520 upregulated and 659 downregulated; Fig. 1A) and 2092 OV- related DEGs (1237 upregulated and 855 downregulated; Fig. 1B). Remarkably, there was an overlap of 28 upregulated and 45 downregulated DEGs between PCOS and OV (Fig. 1C). Statistical analysis Statistical analysis was carried out using R (v4.3.0). Survival analysis and visualization were performed using the R packages "survival" and "survminer". Heat maps were generated using the "pheatmap" package. Time-dependent Receiver Operating Characteristic analysis (ROC) was conducted using the "timeROC" package. Venn diagrams were generated using the "ggvenn" package. Other results were visualized using ggplot2 or plot functions. Correlation analysis was performed using the Pearson method. The significance of differences between two groups was assessed using the Wilcox test. A P- value of less than 0.05 was considered statistically significant. Drug Sensitivity Prediction Drug sensitivity was predicted in the training set of the TCGA OV cohort using the "calcPhenotype" function of the "oncoPredict" package, utilizing data from the Genomics of Drug Sensitivity in Cancer (GDSC2) and Cancer Therapeutics Response Portal (CTRP V2) databases. Page 5/25 Page 5/25 qRT-PCR qRT-PCR was conducted to determine mRNA expression of model genes in SKOV3 and A2780 OV cell lines compared to human ovarian surface epithelial cells (HOSEpiC). Total RNA was isolated using Trizol reagent (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions, followed by cDNA synthesis using Hifair® III 1st Strand cDNA Synthesis SuperMix (Yeasen Biotechnology, China). The cDNA amplification was performed using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology) following the manufacturer’s protocol. GAPDH was used as the internal reference, and relative expression was calculated using the 2-ΔΔCt method. The experiment was repeated three times. The primer sequences are summarized in Table S4. Association of DEGs with m6A modification and cellular senescence To identify DEGs associated with both m6A modification and cellular senescence (m6A_CellAgeRG), we aligned the DEGs from both PCOS and OV with known m6A target genes and cellular senescence genes. This intersection produced 8 significant m6A_CellAgeRGs, including LMNB1, SNAI2, NOTCH1, DDIT3, MEIS2, STAT5A, TACC3, and VCAN (Fig. 1D). A subsequent m6A_CellAgeRG score was derived using the ssGSEA algorithm, which revealed elevated scores in PCOS and OV patients compared to controls (Fig. 1E and 1F). This finding suggests that m6A modification and cellular senescence may play a pivotal role in the pathogenesis of both PCOS and OV. Functional enrichment and pathway analysis of DEhub genes To investigate the biological functions and pathways of the DEhub genes, we performed GO and KEGG enrichment analyses. For functional annotation, GO analysis revealed that the DEhub genes were significantly enriched in 2627 biological processes, including cell-substrate adhesion, positive regulation of angiogenesis, and positive regulation of vasculature development. They were also associated with 18 cellular components, such as the collagen-containing extracellular matrix, external side of the plasma membrane, and membrane raft, as well as 11 molecular functions, including extracellular matrix structural constituent, coreceptor activity, and cytokine binding (Fig. 3D, Table S1). Furthermore, through KEGG pathway enrichment analysis, we identified 11 significant pathways, including Adherens junction, Fc gamma R-mediated phagocytosis, and the AGE-RAGE signaling pathway in diabetic complications (Fig. 3E, Table S2). These results suggest that these pathways and processes may be potential therapeutic targets for OV. Identification of core modules and OV-related hub genes To identify core modules related to the m6A_CellAgeRG score, we conducted WGCNA on PCOS and TCGA OV datasets. After initial sample clustering, we detected and removed one outlier in the PCOS dataset (Fig. 2A). To establish an optimal scale-free topology and connectivity, we set the soft threshold β to 12 (Fig. 2B). Utilizing hierarchical clustering, we categorized the genes into 10 distinct modules (Fig. 2C). Notably, the blue module exhibited the highest correlation (R = 0.8) with the m6A_CellAgeRG score (Fig. 2D). Similarly, in the TCGA OV dataset, we identified and removed 13 outliers (Fig. 2E), selected a soft threshold β of 8 (Fig. 2F), and organized the genes into 19 modules (Fig. 2G), with the brown module showing the highest correlation (R = 0.74) with the m6A_CellAgeRG score (Fig. 2H). By applying the criteria GS > 0.5 and \|MM\| > 0.6, we identified 1246 hub genes within the blue module in the PCOS cohort (Fig. 3A) and 308 hub genes within the brown module in the OV cohort (Fig. 3B). Out of these hub genes, 242 were also among the 2092 DEGs associated with OV (termed as DEhub genes) (Fig. 3C). These data suggest the involvement of these hub genes in OV development and progression. Page 6/25 Page 6/25 Construction and validation of the prognostic nomogram To explore the clinical utility of the prognostic model, we identified independent prognostic clinical factors through univariate and multivariate Cox analyses. Both analyses unveiled significant associations between OS and the risk score (both HR = 2.7, P < 0.001; Fig. 5A and 5B). Subsequently, we constructed a nomogram by integrating the risk score with tumor stage, grade, and age, aiming to forecast 1-year, 2- year, and 3-year survival probabilities (Fig. 5A). The calibration plot showed good agreement between the observed and predicted survival rates spanning 1, 2, and 3 years (Fig. 4D-F). The DCA for these durations further underscored the clinical utility of this nomogram (Fig. 4G-I). These data suggest that the constructed nomogram provides a valuable tool for predicting survival outcomes in OV patients. Efficacy of prognostic risk score model across OV cohorts To explore the prognostic implications of the risk score, we performed Kaplan-Meier (KM) survival analysis on the TCGA OV cohort. The results showed diminished OS for high-risk patients in both the training and test sets (Fig. S2A and S2B). The ROC curves indicated AUC values of 0.72, 0.77, and 0.74 for 1-year, 2-year, and 3-year OS in the training set and 0.61, 0.67, and 0.64 in the test set (Fig. S2D and 2E). When evaluating the model's reliability on an external OV cohort, GSE140082, a significant decrease in OS for the high-risk group was observed (Fig. S2C). The corresponding ROC curves showed AUC values of 0.56, 0.63, and 0.64 for 1-year, 2-year, and 3-year survival (Fig. S2F). Upon analyzing the correlation between risk scores and OS among the TCGA OV patients, we categorized these patients based on their age, tumor stage, and grade. The data showed that high-risk patients generally had lower OS rates compared to their low-risk counterparts across the overall patient population (P < 0.0001; Fig. S2G) and when differentiated by age (P < 0.0001; Fig. S2H and S2I). The difference in OS was notably significant among patients with high-grade (G3+G4, P < 0.0001; Fig. S2J) and advanced-stage tumors (III+IV, P < 0.0001; Fig. S2K). However, for those diagnosed with early-stage (I+II, P = 0.25; Fig. S2L) or low-grade tumors (G1+G2, P = 0.46; Fig. S2M), there wasn't a significant difference in OS between the high and low- risk groups. The results suggest that the risk score is a reliable prognostic indicator for OS in OV, particularly among high-grade and advanced-stage tumors. Development and validation of an OV prognostic model To identify hub genes related to the prognosis of OV, we conducted a univariate Cox analysis to assess the correlation between the expression levels of 242 DEhub genes and patient prognosis in the TCGA OV cohort. Using a significance threshold of P < 0.05, we identified 31 DEhub genes significantly associated with patient prognosis (Table S3). To address potential issues such as variable collinearity and overfitting, we performed LASSO Cox analysis on these 31 DEhub genes in the training set (Fig. 4A), selecting the minimum λ value from the model (Fig. 4B). This process led us to identify 19 genes (APBB2 C5AR1, CCDC80, CFI, CXCR2, CXCR4, DOCK11, FNIP1, GBP5, KATNAL1, KCTD1, LILRA2, MRC1, P2RX1, P2RY14, PI3, PYGB, TMOD2, and ZBP1) with corresponding weight coefficients shown in Fig. 4C. Subsequently, these 19 genes underwent multivariate Cox analysis (Fig. 4D), and their functional interactions with closely related proteins were explored (Fig. 4E). Using the expression levels and regression coefficients of these 19 DEhub genes, we calculated risk scores for each TCGA sample. Page 7/25 Page 7/25 Patients were then divided into high-risk and low-risk groups based on the median risk score, and the risk score formula was as follows: risk score = (-0.102) × APBB2 + 0.016 × C5AR1 + 0.13 × CCDC80 + (-0.066) × CFI + 0.485 × CXCR2 + (-0.133) × CXCR4 + 0.073 × DOCK11 + 0.067 × FNIP1 + (-0.088) × GBP5 + 0.108 × KATNAL1 + (-0.041) × KCTD1 + 0.165 × LILRA2 + 0.05 × MRC1 + (-0.192) × P2RX1 + (-0.678) × P2RY14 + 0.089 × PI3 + 0.177 × PYGB + 0.073 × TMOD2 + (-0.13) × ZBP1. A heatmap depicted the expression of these 19 DEhub genes in each TCGA OV sample (Fig. 4F). Drug sensitivity analysis in different risk categories To investigate how the risk score reflects therapeutic responsiveness, we projected drug sensitivity in patients. Differences in drug sensitivity between risk groups were determined based on the prediction scores (Fig. 7A). The lower the prediction score, the higher the sensitivity of the corresponding patients to the drug. Three drugs, namely Sabutoclax_1849, AGI-6780_1634, and BMS-536924_1091, emerged as the most correlated with the risk scores (Fig. 7B). However, the prediction scores of Sabutoclax_1849 showed no difference between the two groups. Thus, we only display the Pearson correlation coefficient (r) and P- value between the risk score and the prediction scores of AGI-6780_1634 and BMS-536924_1091 (Fig. 7C and 7D). BMS-536924_1091 response was negatively correlated with the risk score (r = -0.22, p = 1.5e-05), while AGI-6780_1634 response was positively correlated with the risk score (r = 0.25, p = 1.4e-06). These patterns suggest that risk scores could potentially serve as informative indicators for therapeutic strategies in OV. Association of risk score with immune cell infiltration To explore the potential interplay between the risk score and the tumor's immune microenvironment, we used the CIBERSORT algorithm to quantify the infiltration of immune cells within the tumor and compared these measurements across risk groups. The results revealed significant differences in the Page 8/25 Page 8/25 Page 8/25 abundance of seven immune cell types between the risk groups (Fig. 6A and 6B). In particular, the high- risk group was characterized by elevated counts of M0 macrophages and monocytes, coupled with a subtle rise in M2 macrophages. Conversely, counts of M1, CD8+ T cells, and T follicular helper (Tfh) cells decreased significantly in this group. By employing the ESTIMATE algorithm, we found that the ESTIMATEScore and stromal score were noticeably higher in the high-risk group compared to the low-risk group (P = 0.014 and 0.00011, respectively; Fig. 6C and 6E), while tumor purity reduced significantly in the high-risk group (P = 0.014; Fig. 6D). However, no marked difference was observed in the immune score across the groups (Fig. 6F). Subsequent TIDE analyses to measure the potential for immunotherapy response revealed that interferon gamma (IFNG), CD8, microsatellite instability (MSI), and myeloid- derived suppressor cell (MDSC) levels decreased in the high-risk group, while T cell dysfunction, exclusion, and cancer-associated fibroblasts (CAF) increased (Fig. 6G). These observations suggest an intricate relationship between the risk score and the immune landscape of the tumor. Discussion In this study, shared DEGs were identified between PCOS and OV using multiple datasets. Eight DEGs were associated with both m6A modification and cellular senescence, suggesting their role in both conditions. A 19-gene prognostic model was developed and validated for OV, demonstrating its effectiveness in predicting patient outcomes and revealing associations with immune cell infiltration and drug sensitivity. Furthermore, qRT-PCR analysis confirmed notable upregulation or downregulation of the model genes in SKOV-3 and A2780 cell lines compared to HOSEpiC, consistent with the gene expression patterns observed in OV datasets. The developed 19-gene prognostic model for OV offers potential clinical significance by providing a valuable tool for predicting patient outcomes and guiding personalized therapeutic approaches. We identified eight significant m6A_CellAgeRGs, including LMNB1, SNAI2, NOTCH1, DDIT3, MEIS2, STAT5A, TACC3, and VCAN, which are common between PCOS and OV. This overlap suggests potential shared molecular mechanisms or pathways that might underlie the progression from PCOS to OV. LMNB1, from the lamin family, is pivotal for maintaining the structure and function of the cell nucleus[19]. Notably, its related protein, LMNB2, has been identified as a potential biomarker for ovarian cancer risk in women with PCOS[20]. Moreover, LMNB1 is one of the top hub genes in the PPI network of PCOS, playing a central role in the core network of PCOS-related genes[21]. This connection suggests that changes in the nuclear lamina could bridge PCOS and OV. The association between irregular menstrual cycles and an elevated risk of OV further strengthens the link between PCOS and OV[22]. SNAI2 exhibits differential expression in ovarian tissue from PCOS patients with irregular menstruation compared to those with regular cycles[8]. This implies that hormonal imbalances or irregular menstrual cycles, common in PCOS, might set the stage for cellular changes that predispose to OV. DDIT3's association with both PCOS and OV is also intriguing. While it correlates positively with tumor purity in OV, its role as a core ferroptosis-related gene in PCOS suggests that cellular stress and death pathways might be a bridge connecting these two conditions[23]. Moreover, the enrichment of DEhub genes in pathways like the AGE-RAGE signaling pathway in OV, offers another potential connection between PCOS and OV, given its role in chronic inflammation[24], a hallmark of PCOS[25-27]. In OV, the AGE-RAGE pathway is associated with tumor growth, angiogenesis, and metastasis[28, 29]. Model gene expression in external OV datasets and OV cell lines. Lastly, we examined the alterations in model gene expression in external OV datasets and OV cell lines. The expression patterns of these model genes in GSE18520 and GSE27651 datasets (Fig. 8A) are summarized in Table 2. Notably, our analysis revealed that CXCR4 emerged as the only m6A target gene, subject to regulation by m6A-associated genes FTO, METTL14, ELAVL1, and YTHDF2 (Fig. 8B). This finding suggests a complicated regulatory network governing CXCR4 in OV. Furthermore, in SKOV-3 and A2780 OC cell lines, qRT-PCR analysis revealed notable upregulation of C5AR1, CFI, CXCR2, CXCR4, KCTD1, PI3, PYGB, ZBP1, and LILRA2 (only in SKOV3 cells) compared to HOSEpiC. Conversely, CCDC80, FNIP1, GBP5 (only in SKOV3 cells), KATNAL1, MRC1, P2RX1, and TMOD2 (only in A2780 cells) showed significant downregulation (all P < 0.05). The other genes did not exhibit significant changes (all P > 0.05; Fig. 9). These findings are generally consistent with the gene expression alterations observed in OV datasets (Table 2). Page 9/25 Conclusion In conclusion, our study identified shared dDEGs between PCOS and OV, highlighting the potential involvement of m6A modification and cellular senescence in both conditions. We developed a prognostic model for OV based on 19 hub genes, offering promise for risk assessment in OV patients. This model demonstrated efficacy across multiple OV cohorts and revealed associations with the tumor immune microenvironment and drug sensitivity. Further research and clinical validation are needed to fully realize the clinical significance of these findings and their potential impact on OV patient care. Discussion These results highlight the need for personalized immunotherapeutic strategies that address the specific immune challenges faced by high-risk OV patients. The limitations of this study include the reliance on bioinformatics and data analysis, which requires further experimental validation to confirm the functional roles of identified genes and their clinical significance. Additionally, the study's findings are based on retrospective data, and prospective clinical studies are needed to validate the prognostic model's effectiveness in real-world clinical settings. Lastly, the study focuses on associations and does not establish causation, warranting future mechanistic investigations. Discussion These results suggest that the shared DEGs and signaling pathways might collectively drive the progression from PCOS to OV. In this study, we observed that the high-risk group exhibited increased levels of M0 macrophages and monocytes, accompanied by a slight elevation in M2 macrophages. Conversely, we observed a significant reduction in the counts of M1 macrophages, CD8+ T cells, and TFH cells within this group. These findings collectively suggest a distinct immunological profile in the high-risk individuals, potentially indicating an immune microenvironment conducive to OV progression. Elevated levels of M0 macrophages and monocytes indicate a potential pro-tumorigenic environment, as these cells can promote tumor growth and suppress anti-tumor immune responses[30]. Malignant OV cells can release M2-like cytokines, including IL-10, CCL2/3/4/5/7/8, CXCL12, VEGF, and PDGF, as part of their strategy to attract and recruit additional monocytes and M0 macrophages to the tumor site, subsequently inducing their transformation Page 10/25 Page 10/25 into the M2 phenotype[31]. The subtle increase in M2 macrophages further supports a tumor-promoting milieu, as M2 macrophages are known for their immunosuppressive functions in OV[32]. Conversely, the significant decrease in M1 macrophages, CD8+ T cells, and TFH cells suggests impaired anti-tumor immunity, as these cells play crucial roles in mounting effective immune responses against OV[33]. High- grade serous OV tumors with higher estimated proportions of TFH and M1 macrophages were significantly and independently associated with long-term survival[34]. This distorted immune profile in the high-risk group may contribute to disease progression and highlights the importance of therapeutic strategies aimed at restoring a balanced and robust anti-tumor immune response in OV patients at risk. TIDE analyses in the high-risk group indicated a decrease in key immune factors such as IFNG, CD8 T cells, MSI, and MDSC, coupled with an increase in indicators of T cell dysfunction, immune exclusion, and the presence of CAF. In OV immunotherapy, these findings suggest that the high-risk group may face challenges in responding effectively to immunotherapeutic interventions. The decrease in CD8 T cells, which are crucial for anti-tumor immune responses, and the rise in immunosuppressive MDSCs can compromise the immune system's ability to target and control OV[35, 36]. Additionally, elevated T cell dysfunction and immune exclusion markers indicate a potentially hostile tumor microenvironment that hinders immune cell infiltration and function in OV[37, 38]. The presence of CAFs, known for their role in promoting tumor growth and immune evasion, further complicates the therapeutic landscape[39, 40]. Authors' contributions YL and CYS carried out the studies, participated in collecting data, and drafted the manuscript. YJGX and YZW performed the statistical analysis and participated in its design. YYM and XH participated in acquisition, analysis, or interpretation of data and draft the manuscript. All authors read and approved the final manuscript. Abbreviations Polycystic ovary syndrome (PCOS); ovarian cancer (OV); differentially expressed genes (DEGs); ovarian cancer (OV); N6-methyladenosine (m6A); methyltransferase-like-3 (METTL3); YTH domain family protein Page 11/25 Page 11/25 1 (YTHDF1); Weighted Gene Co-Expression Network Analysis (WGCNA); Gene Ontology (GO); Genomics of Drug Sensitivity in Cancer (GDSC2); Cancer Therapeutics Response Portal (CTRP V2); Receiver Operating Characteristic analysis (ROC); Kaplan-Meier (KM); T follicular helper (Tfh); interferon gamma (IFNG); microsatellite instability (MSI); myeloid-derived suppressor cell (MDSC); cancer-associated fibroblasts (CAF) 1 (YTHDF1); Weighted Gene Co-Expression Network Analysis (WGCNA); Gene Ontology (GO); Genomics of Drug Sensitivity in Cancer (GDSC2); Cancer Therapeutics Response Portal (CTRP V2); Receiver Operating Characteristic analysis (ROC); Kaplan-Meier (KM); T follicular helper (Tfh); interferon gamma (IFNG); microsatellite instability (MSI); myeloid-derived suppressor cell (MDSC); cancer-associated fibroblasts (CAF) Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Funding The study was supported by the Famous Doctor Project of Xingdian Talent Support Program in Yunnan Province [XDYC-MY-2022-0057]; Kunming Medical UniversityYoung and Middle-aged Discipline Leaders and Reserve Candidates -"Riding the Wind* Talent Cultivation Programme [2023(108)]; External Cooperation Research Project of the Second Affiliated Hospital of Kunming Medical University - Mechanism of histone lactate regulation of METTL3 in PCOS affecting the proliferation of ovarian granulosus cells [2022dwhz03]. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Availability of data and materials All data generated or analysed during this study are included in this published article. Acknowledgements Not applicable. Page 12/25 Page 12/25 References 1. Kh MM, Boboev K. Modern aspects of etiology, diagnosis, and treatment of polycystic ovarian syndrome. European journal of modern medicine and practice. 2022;2:86-93. 2. 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Tables Table 1 Information of public datasets Page 15/25 ID Platform Sample type Sample size Data type Disease GSE34526 GPL570 Normal or PCOS granulocytes 10 mRNA array PCOS GSE137684 GPL17077 Normal or PCOS granulocytes 12 mRNA array PCOS GSE80432 GPL6244 Normal or PCOS granulocytes 16 mRNA array PCOS GSE114419 GPL17586 Normal or PCOS granulocytes 6 mRNA array PCOS GSE102293 GPL570 Normal or PCOS granulocytes 6 mRNA array PCOS GSE18520 GPL570 Normal or cancer tissue 63 mRNA array OV GSE27651 GPL570 Normal or cancer tissue 49 mRNA array OV GSE140082 GPL14951 Cancer tissue with prognosis data 380 mRNA array OV TCGA-OV Cancer tissue with prognosis data 373 RNASeq OV ID Table 2 Model gene expression in external OV datasets Table 2 Model gene expression in external OV datasets Page 16/25 Gene OV_GSE18520 OV_GSE27651 PCOS_DEGs APBB2 Up Down Down C5AR1 Down - Up CCDC80 Down Down - CFI Down - - CXCR2 Down - Up CXCR4 Up - - DOCK11 Down Down Up FNIP1 Down - - GBP5 Up - Up KATNAL1 Down Down - KCTD1 Up Up - LILRA2 Down Down Up MRC1 Down Down Up P2RX1 Up Down - P2RY14 Down Down - PI3 Up - - PYGB Up - - TMOD2 Down Down - ZBP1 Up - - Figures Figures Page 17/25 Figure 1 Identification of differentially expressed genes (DEGs) associated with both polycystic ovary syndrome (PCOS) and ovarian cancer (OV). Five PCOS datasets (GSE34526, GSE137684, GSE80432, GSE114419, and GSE102293) were merged, including 28 PCOS granulocyte samples and 22 normal granulocyte samples. The TCGA OV dataset GSE18520 contained 53 cancer tissue samples and 10 normal control tissue samples. (A, B) Volcano plots illustrate DEGs associated with PCOS (A) and OV (B), where red represents upregulated genes, blue represents downregulated genes, and gray represents non-significant changes. (C) Venn diagrams depict the intersection of upregulated (left) and downregulated (right) DEGs Figure 1 Identification of differentially expressed genes (DEGs) associated with both polycystic ovary syndrome (PCOS) and ovarian cancer (OV). Five PCOS datasets (GSE34526, GSE137684, GSE80432, GSE114419, and GSE102293) were merged, including 28 PCOS granulocyte samples and 22 normal granulocyte samples. The TCGA OV dataset GSE18520 contained 53 cancer tissue samples and 10 normal control tissue samples. (A, B) Volcano plots illustrate DEGs associated with PCOS (A) and OV (B), where red represents upregulated genes, blue represents downregulated genes, and gray represents non-significant changes. (C) Venn diagrams depict the intersection of upregulated (left) and downregulated (right) DEGs Identification of differentially expressed genes (DEGs) associated with both polycystic ovary syndrome (PCOS) and ovarian cancer (OV). Five PCOS datasets (GSE34526, GSE137684, GSE80432, GSE114419, and GSE102293) were merged, including 28 PCOS granulocyte samples and 22 normal granulocyte samples. The TCGA OV dataset GSE18520 contained 53 cancer tissue samples and 10 normal control tissue samples. (A, B) Volcano plots illustrate DEGs associated with PCOS (A) and OV (B), where red represents upregulated genes, blue represents downregulated genes, and gray represents non-significant changes. (C) Venn diagrams depict the intersection of upregulated (left) and downregulated (right) DEGs Page 18/25 Page 18/25 in both PCOS and OV cohorts. (D) Venn diagram depicting the intersection of PCOS and OV-related DEGs with m6A target genes and cellular senescence genes (m6A_CellAgeRG). (E and F) Comparison of the m6A_CellAgeRG scores in merged PCOS dataset (E) and TCGA OV dataset GSE18520 (F) between disease and control groups. with m6A target genes and cellular senescence genes (m6A_CellAgeRG). (E and F) C m6A_CellAgeRG scores in merged PCOS dataset (E) and TCGA OV dataset GSE185 disease and control groups. Pag Figure 2 Figure 2 Page 19/25 Identification of hub genes in PCOS and OV cohorts. Figures (A, E) Sample clustering with the removal of outliers in PCOS (A) and TCGA OV (E) cohorts. (B, F) Soft threshold β selection. (C, G) Co-expression module clustering. Different colors represent various co-expression modules. (D, H) Correlation between modules and m6A_CellAgeRG Score. Figure 3 Identification of hub genes. (A, B) Hub gene selection within the blue module of the PCOS cohort (A) and the brown module of the OV cohort (B). (C) Intersection of the hub genes with the DEGs associated with OV (D E) GO (D) and KEGG (E) enrichment analyses on intersected hub genes Figure 3 Identification of hub genes. (A, B) Hub gene selection within the blue module of the PCOS cohort (A) and the brown module of the OV cohort (B). (C) Intersection of the hub genes with the DEGs associated with OV. (D, E) GO (D) and KEGG (E) enrichment analyses on intersected hub genes. Page 20/25 Page 20/25 Figure 4 Identification of prognostic-related hub genes in OV. (A) Distribution of LASSO coefficients of 31 prognosis-related DEhub genes. Coefficients are plotted against log lambda (λ), depicting the trend of approaching zero as λ increases. (B) λ selection by 10-fold cross-validation. The vertical dot lines represent the minimum λ and optimal λ values for the model. (C) Bar plot showing the weight coeffici of the 19 selected candidate genes in the model. (D) Forest plot of the multivariate Cox regression analysis for the model genes. (E) Protein interaction network illustrating the functional interactions of model genes with proteins that have similar functions. (F) Heatmap displaying the expression pattern the model genes in each TCGA OV sample Figure 4 Figure 4 Identification of prognostic-related hub genes in OV. (A) Distribution of LASSO coefficients of 31 prognosis-related DEhub genes. Coefficients are plotted against log lambda (λ), depicting the trend of approaching zero as λ increases. (B) λ selection by 10-fold cross-validation. The vertical dot lines represent the minimum λ and optimal λ values for the model. (C) Bar plot showing the weight coefficients of the 19 selected candidate genes in the model. (D) Forest plot of the multivariate Cox regression analysis for the model genes. (E) Protein interaction network illustrating the functional interactions of the model genes with proteins that have similar functions. (F) Heatmap displaying the expression patterns of the model genes in each TCGA OV sample. Page 21/25 Page 21/25 Figure 5 Construction and evaluation of the nomogram. (A, B) Forest plots showing the results of univariate and multivariate Cox analyses for the risk score and clinical factors. (C) The nomogram constructed based on the risk score, age, stage, and grade. (D-F) Calibration plots of the nomogram for predicting 1-year, 2-year, and 3-year survival rates. (G-I) Clinical decision curve analysis (DCA) curves evaluating the predictive performance of the model for 1-year, 2-year, and 3-year survival rates. Figure 5 Construction and evaluation of the nomogram. (A, B) Forest plots showing the results of univariate and multivariate Cox analyses for the risk score and clinical factors. (C) The nomogram constructed based on the risk score, age, stage, and grade. (D-F) Calibration plots of the nomogram for predicting 1-year, 2-year, and 3-year survival rates. (G-I) Clinical decision curve analysis (DCA) curves evaluating the predictive performance of the model for 1-year, 2-year, and 3-year survival rates. Construction and evaluation of the nomogram. (A, B) Forest plots showing the results of univariate and multivariate Cox analyses for the risk score and clinical factors. (C) The nomogram constructed based on the risk score, age, stage, and grade. (D-F) Calibration plots of the nomogram for predicting 1-year, 2-year, and 3-year survival rates. (G-I) Clinical decision curve analysis (DCA) curves evaluating the predictive performance of the model for 1-year, 2-year, and 3-year survival rates. Page 22/25 Page 23/25 Figure 6 Correlation between risk score and immune cell infiltration. (A) Heatmap illustrating the differential abundance of immune cells in different risk groups. (B)Box plots showing the abundance of immune cells in different risk groups. Figure 4 (C-F) Box plots depicting the ESTIMATEScore, tumor purity, stromal score, and immune score in different risk groups. (G) Violin plot demonstrating the interferon gamma (IFNG) response, T cell dysfunction scores, T cell exclusion scores, CD8 levels, MSI expression signature, CAF score, and MDSC score in different risk groups. Figure 6 Figure 6 Figure 6 Correlation between risk score and immune cell infiltration. (A) Heatmap illustrating the differential abundance of immune cells in different risk groups. (B)Box plots showing the abundance of immune cells in different risk groups. (C-F) Box plots depicting the ESTIMATEScore, tumor purity, stromal score, and immune score in different risk groups. (G) Violin plot demonstrating the interferon gamma (IFNG) response, T cell dysfunction scores, T cell exclusion scores, CD8 levels, MSI expression signature, CAF score, and MDSC score in different risk groups. Page 23/25 Page 23/25 Figure 7 Predicting drug sensitivity in patients from different risk groups.(A) Comparison of drug prediction scores between different risk groups. (B) Correlation between drug sensitivity and risk score. (C, D) the Pearson correlation coefficient (r) and p-value (p) between the risk score and the prediction scores of BMS- 536924_1091 (C) and AGI-6780_1634 (D). Figure 7 Predicting drug sensitivity in patients from different risk groups.(A) Comparison of drug prediction scores between different risk groups. (B) Correlation between drug sensitivity and risk score. (C, D) the Pearson correlation coefficient (r) and p-value (p) between the risk score and the prediction scores of BMS- 536924_1091 (C) and AGI-6780_1634 (D). Page 24/25 Figure 8 Model gene expression in external OV datasets. (A) Box plot of model gene expression in GSE27651 dataset. (B) CXCR4 is regulated by FTO, METTL14, ELAVL1, and YTHDF2. Figure 8 Model gene expression in external OV datasets. (A) Box plot of model gene expression in GSE27651 dataset. (B) CXCR4 is regulated by FTO, METTL14, ELAVL1, and YTHDF2. Page 24/25 Page 24/25 Figure 9 Model gene expression in OV cell lines. qRT-PCR was conducted to determine mRNA levels of model genes in SKOV3 and A2780 cells compared to human ovarian surface epithelial cells (HOSEpiC). Data are expressed as mean ± standard deviation. P < 0.05, P < 0.01, **P < 0.0001, ns, non-significant; n = 3. Figure 9 Model gene expression in OV cell lines. qRT-PCR was conducted to determine mRNA levels of model genes in SKOV3 and A2780 cells compared to human ovarian surface epithelial cells (HOSEpiC). Data are expressed as mean ± standard deviation. P < 0.05, P < 0.01, **P < 0.0001, ns, non-significant; n = 3. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementarymaterial.docx TableS1.xlsx TableS2.csv TableS3.csv TableS4.docx Page 25/25
https://openalex.org/W4239479587	https://osf.io/tvm6q/download	English	null	A study on English communication skills at tertiary level	null	2,021	cc-by	6,456	Thu Huynh Ngoc Anh 1 * 3 Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). Thu Huynh Ngoc Anh 1 * 3 1 Ho Chi Minh City University off Food industry; huynhthu0810@gmail.com * Correspondence: huynhthu0810@gmail.com; Tel.: +84 982 898 737 Abstract: Communication skills are one of the most important soft skills in the 21st century. There is a sad fact that even young Vietnamese, although learning English as an official subject at school, almost always show a lack of confidence when communicating with foreigners. Many high school graduates or even university students, when meeting for- eigners, teachers or speaking in front of the class, still can not say anything more than simple questions such as greetings and introducing themselves. This study was conducted by the Ho Chi Minh City University of Food Industry (HUFI). With the participation of 120 students. Through the questionnaire. This study was for the purpose of indicating the errors that students make, as well as finding solutions that support their skills to learn and improve their ability to communicate. The results of this study showed the participa- tion of 120 students. Only 50 out of 120 students received good reviews in the research, 45 students still lack skills and confidence when communicating and received an average rating. The rest of the 120 study participants were rated as weak, lacking a lot of commu- nication skills. This condition needs to be interested in, as if not even when communi- cating with Vietnamese in English, you will lack confidence. Keywords: Communication; communication skills; tertiary lever (or tertiary education); 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Citation: Lastname, F.; Lastname, F.; Lastname, F. Title. Information 2021, 12, x. https://doi.org/10.3390/xxxxx Academic Editor: Firstname Last- name Received: date Accepted: date Published: date Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2021 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Citation: Lastname, F.; Lastname, F.; Lastname, F. Title. Information 2021, 12, x. https://doi.org/10.3390/xxxxx Academic Editor: Firstname Last- name Received: date Accepted: date Published: date Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2021 by the authors. 1. Introduction In general, communication might be described as the procedure of sending and re- ceiving data from people giving the knowledge to the person receiving the knowledge via verbal and nonverbal strategies. The most common way to communicate is verbal, which may be a two-way process with responses to the received data. The sharing of thoughts, views, and knowledge for a shared goal is usually mentioned as communication. Apart from verbal communication, information can also be communicated by the utilization of symbols or signage. Language and textiles are is the 2 elements that have been parallel throughout history and are inseparable. Language itself is the culture of a country and culture can be expressed through language. Therefore, to learn the language better, you can take the time to learn about culture through cultural subjects as well as news channels. In this way, when understanding culture, language learning as well as using language are more efficient and appropriate. In addition, the job of loving and respecting a culture is also a springboard to promoting students to learn the language of that culture well. Com- munication is the process of passing information and understanding from one person to another. Therefore, all communication influences at least two people: the one who sends the message and the one who receives it. Consistent with this definition, elements of com- munication include the individual providing the knowledge, the infomation gained, and the receiver’s feedback, with the replication of those processes leading to knowledge cre- ation. 39 40 41 42 43 44 45 Copyright: © 2021 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). Additionally, for self-study, students should make the most of outside help, espe- cially help and orientation from subject teachers. Instructors guide students to learn and 45 2 of 12 2 of 12 develop English skills from simple to journal. At the same time, provide sufficient infor- mation to students so that students can understand the content, objectives and require- ments of the training program. 1. Introduction 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 Speaking is one of the important parts of English skills that should be mastered by students besides reading and listening. Speaking and most of all, communication skills play an important role in language proficiency. Many researchers have been conducted discussing the various problems facing Vietnamese learners of English. But few reports mention how to learn and how to apply communication skills in practice. Currently, there are still problems that need to be solved in learning English communication skills among students. Including lack of vocabulary, lack of speaking skills and lack of non-verbal com- munication-body language. This survey will show the current situation as well as some specific solutions to help students acquire proficient English communication skills. 2. Literature review Since the 1970s, there have been many different conceptions of communication by different authors. Everyone looks at the problem from a different angle and gives different concepts: (Kôlôminxki, 1981) described: “Communication is the interaction of objects and in- formation between man and man''. (Tarone, 1997) has analyzied about communication is used by an individual to over- come the crisis which occurs when language structures are inadequate to convey the in- dividual’s thought”. (Tarone, 1980) also provides a broad explanation of communication as a “mutual attempt of two interlocutors to agree on a meaning in situations where req- uisite meaning structures do not seem to be shared” According to Nguyen Thac and Hoang Anh (1991), there are 4 signs of communica- tion: According to Nguyen Thac and Hoang Anh (1991), there are 4 signs of communica- tion: + Communication is a phenomenon specific to humans, only humans have real com- munication. + Communication is a phenomenon specific to humans, only humans have real com- munication. + Communication is a way of expressing relationships with one or more other people on the basis of economic and political relations in society. + Communication is carried out through the exchange of information and mutual understanding. + Communication is based on mutual understanding between peopl g p p (Cleland, Foster, & Moffat, 2005) They found that a positive communication environ- ment provides opportunities for students to find ways to communicate, and thus, have better communication skills to be better prepared for the work market after completing their degree, university students should find ways to attach. When the individual receiv- ing the message understands and practices the skills, communication improves. Commu- nication is easier if the receiver of the information can understand and practice the skills. Further, communication are getting to be more meaningful if physical, spiritual and social factors are taken into account during the communication process. As a college student preparing to start their chosen profession, they ought to participate in any activities that improve communication skills in a broad and comprehensive manner to develop and im- prove communication skills. There are many kinds of communication skills, but generally it involves oral and written skills, there are essentially three sorts of communication, which are interpersonal communication, management communication (communication during a group), and public communication (speech making) has proposed by (Rahman., 2005). 3.1 Participants The research was carried out between March and May 2021, and included 120 stu- dents (aged from 18 to 24 years old), from Ho Chi Minh City University of Food Industry (HUFI). According to random selection, the distribution according to gender is uneven, as the sample includes 49 men (40.83%), 70 women (58.33%), and 1 gender X (0.83%). They were asked to fill out a questionnaire. The data was collected and (was) analyzed. The questionnaire has eight questions, including four questions with a multiple choice format, and four questions with a one-to-five-number rating corresponding to the explanation of each number below: 1 = Worst/very unskilled. 2 = Not good/unskilled. 3 = Neutral/normal. 4 = Skilled. 4 = Skilled. 5 = Very skilled. 3. Materials and Methods The questionnaire was designed using the available communication skills literature. The frameworks of communication skills (oral skills, reading skills, listening and social skills) during this paper were decided to use the communication definitions mentioned previously. 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 2. Literature review (Cleland, Foster, & Moffat, 2005) They found that a positive communication environ- ment provides opportunities for students to find ways to communicate, and thus, have better communication skills to be better prepared for the work market after completing their degree, university students should find ways to attach. When the individual receiv- ing the message understands and practices the skills, communication improves. Commu- nication is easier if the receiver of the information can understand and practice the skills. Further, communication are getting to be more meaningful if physical, spiritual and social factors are taken into account during the communication process. As a college student preparing to start their chosen profession, they ought to participate in any activities that improve communication skills in a broad and comprehensive manner to develop and im- prove communication skills. There are many kinds of communication skills, but generally Communication generally involves four elements, which are the speaker, the re- ceiver, channel and feedback. Some researchers have defined communication as verbal communication, written language, non-verbal communication, listening and giving feed- back. Concurrently, (Rodiah & Idris, 2010) has said that communication as a non-verbal skill, giving feedback, presenting ideas verbally and in written form, doing presentations 3 of 12 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 and negotiating to achieve a goal and getting agreement in our globalized world, univer- sity students have to master communication skills in several cultural contexts. As a re- sponse, universities need to provide plenty more ways to help students communicate more effectively to beat the stress of a globalized world. As a result, the aim of this study would be to look into the stage of communication skills of many students. 4. Results The questionnaire has been analyzed and linked according to each section having an impact on the other. Below are four headings, along with the charts to be analyzed. 4.1 The correlation between study time and proficiency in essential English s 4.1 The correlation between study time and proficiency in essential English skills 4.2 Freshman and level of proficiency off essential English skills 4.1 The correlation between study time and proficiency in essential English skills Through the questionnaire, it is easy to see how time (school year) affects students' proficiency in using English skills. Learning a foreign language is a complex, time-con- suming process. That is, the development of the linguistic ability of learners is influenced by many influencing factors. The theory of second language acquisition covers many fac- tors that are likely to affect language learning and development. These factors may be related to the teaching and learning process in the curriculum for a school year. 4.2 Freshman and level of proficiency off essential English skills 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 4 of 12 0% 10% 20% 30% 40% 50% 60% SPEAKING LISTENING READING SPEAKING LISTENING READING VERY GOOD 0% 0% 10% GOOD 9% 7% 21% NEUTRAL 49% 56% 52% NOT GOOD 12% 34% 10% WORST 30% 3% 7% 0% 10% 20% 30% 40% 50% 60% SPEAKING LISTENING READING SPEAKING LISTENING READING VERY GOOD 0% 0% 10% GOOD 9% 7% 21% NEUTRAL 49% 56% 52% NOT GOOD 12% 34% 10% WORST 30% 3% 7% 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 Table 1. Freshman and level of proficiency of essential English skills. At HUFI, first year students will cultivate all four basic and essential skills in English: listening, speaking, reading, and writing. Most of the time studying is at school because students are not familiar with self-study at home, so most of them depend on lectures from books and knowledge passed from lecturers. Speaking is one of the most critical and fundamental abilities that must be practiced to communicate orally. The factors affecting speaking skills are lack of subject, improper listening skills, lack of proper vocabulary, anxiety, and being overwhelmed by the fast and good grades of learners in the class. One common issue seen among freshman is that they think that they have nothing to say about a specific theme. In reality, they may be bored or feel that the subject is unrelated to anything they know. On the off chance that this is often the case, they will have no inspiration to talk other than the truth that they ought to be partaking in it effectively. 4.3 Sophomore and level of proficiency off essential English skills 4.1 The correlation between study time and proficiency in essential English skills This is because they are still dependent on topics limited to textbooks and have limited vocabulary. Attached was their anxiety about being asked to speak in English. Listening and reading skills are similar. Class time is limited and students are unfamiliar with having to study at home (except doing homework). All things have formed the habit of relying on, waiting for teaching from their class. The above factors are also based on the questionnaire. It was concluded that fresh- man English-speaking skills were assessed as below average on a five-point scale. Listen- ing skills are assessed on average, mainly because the lack of vocabulary also affects hear- ing ability. Reading skills are assessed to be simpler than speaking because there are al- ready words in the text. Students will have no difficulty in looking up the meaning of words, so reading skills are assessed as fairly good. 5 of 12 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 0% 10% 20% 30% 40% 50% 60% 70% SPEAKING LISTENING READING SPEAKING LISTENING READING VERY GOOD 14% 7% 10% GOOD 15% 15% 30% NEUTRAL 54% 60% 55% NOT GOOD 7% 18% 5% WORST 10% 0% 0% 0% 10% 20% 30% 40% 50% 60% 70% SPEAKING LISTENING READING SPEAKING LISTENING READING VERY GOOD 14% 7% 10% GOOD 15% 15% 30% NEUTRAL 54% 60% 55% NOT GOOD 7% 18% 5% WORST 10% 0% 0% Table 2. Sophomore and level of proficiency of essential English skills In the sophomore year, students are introduced to their majors. Sophomores are thor- oughly instructed in theory as well as practical skills. From here on, the necessary skills will be gradually developed. Sophomore students have gradually adapted to self-study, but few spend a great deal of time on it. Moreover, the ratings system has also been improved. Less "Worst" ratings, instead fairly good higher than the rating in Table 1. In Table 2, it was observed that as- sessment of speaking and listening skills improved from worse to better, as well as slight changes in the ratings of reading skills. Students realized that studying at home was equally important to improving essen- tial skills in English during the second year of study, after a first year of study with a surprise. Table 3. Junior and level of proficiency of essential English skills 4.1 The correlation between study time and proficiency in essential English skills This is the year during which the knowledge delivered by the faculty become extremely practical, no longer contingent upon the topics provided. Likewise, the ability to listen and speak has greatly improved, since today's topics are diverse and the sophomores will not be worried about running out of things to talk about. Yet, the ability to speak relies on factors such as public speaking anxiety as well as poor performance in actual communication. 4.4 Junior and level of proficiency off essential English skills Table 3. Junior and level of proficiency of essential English skills 0% 10% 20% 30% 40% 50% 60% SPEAKING LISTENING READING SPEAKING LISTENING READING VERY GOOD 30% 6% 22% GOOD 23% 27% 36% NEUTRAL 47% 56% 31% NOT GOOD 0% 11% 11% WORST 0% 0% 0% 4.4 Junior and level of proficiency off essential English skills Table 3. Junior and level of proficiency of essential English skills 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 6 of 12 The third year of education saw juniors being exposed to English in different fields, such as hotels, tourism, accounting, finance, and banking. Learning English through a variety of careers greatly improved vocabulary and sentence building skills. Besides learning English by profession. A translation and interpretation subject is also taught to juniors, which is crucial to improving their English language skills. During that period, listening and speaking skills were assessed as good, while reading skills were ex- amined as fair. 4.5 Senior and level of proficiency off essential English skills 0% 10% 20% 30% 40% 50% 60% SPEAKING LISTENING READING SPEAKING LISTENING READING VERY GOOD 32% 14% 24% GOOD 35% 23% 20% NEUTRAL 33% 55% 49% NOT GOOD 0% 8% 7% WORST 0% 0 0% 4.5 Senior and level of proficiency off essential English skills 0% 10% 20% 30% 40% 50% 60% SPEAKING LISTENING READING SPEAKING LISTENING READING VERY GOOD 32% 14% 24% GOOD 35% 23% 20% NEUTRAL 33% 55% 49% NOT GOOD 0% 8% 7% WORST 0% 0 0% 4.5 Senior and level of proficiency off essential English skills Table 4. Senior and level of proficiency of essential English skills All seniors completed all academic requirements at the school over the past four years in order to meet the English proficiency requirements. 4.1 The correlation between study time and proficiency in essential English skills As seen in Table 4, English proficiency is ranked from good to excellent in the listening, speaking, and reading do- mains. Speaking level is dependent on the school year. Students' skills improve more quickly the longer they study. 4.6 Genders affect communicating in English A total of 120 individuals took part in the survey, including 49 men (40.83%), 70 women (58.33%), and one gender X (0.83%). These results reveal that English learning is unequally distributed between men and women. In terms of gender and language, there are three main differences: First, language differences between men and women occur due to differences in human structure, including the position of language-containing regions in the brain, as well as differences in sound structure. A second difference is the language used by men and women, or, more specifically, the ways in which women speak. Lan- guages have different words meant for different genders and some words that could not be used for another. Third, gender-based language differences can be seen in the dialects each gender speaks. The distinction is in the way the words are used; each gender uses words different from one another to describe the same issue. It's no secret that every gen- der receives or learns language differently. Learning styles differ among men and women, particularly when learning a second language (English). It has been observed that males possess a more powerful right hemisphere while females have a more balanced left hem- isphere. Because of that, men can better visualize, understand space, or use logic, whereas women excel at vocabulary exercises, or in tasks related to music, humanities, etc. There are significant differences between male and female learning styles. Males tend to be more motoric, tactile, and intuitive. Environments with a high level of dynamic in- formation appeal to them. Additionally, they are less likely to follow the rules, but show 7 of 12 7 of 12 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 better support for their partners than women. The chances of male students learning from listening are lower than those of female students. On the other hand, women tend to be hearing-oriented, and they abide by the rules of the class. Communication involves mak- ing decisions. This is one of the key components of communication. While many men show the opposite traits, women are more likely to demonstrate moderation. Therefore, this greatly hinders both genders' ability to communicate effectively in English when in- vited to have a conversation. 4.6 Genders affect communicating in English Male students often speak briefly, sometimes make opposi- tional remarks, and often give emotional feedback. Despite the fact that female students are usually obedient when speaking in full sentences, they focus on the speaker and offer appropriate feedback. Researchers have found that native speakers believe women communicate with tag questions, including polite words such as "please" or "thank you", as well as emphasizing words such as "very", "really," and so on. As opposed to women, men use short words like: "thanks" instead of "thank you", and "It is..." instead of "It is very...". By analyzing this study, it is possible to conclude that gender really does exist in language communication. In terms of communication, a person's ability to communicate has two dimensions, the first is the gender-based influence on style and method, and the second is how gender is expressed through communication. 4.7 Vocabulary size in communication. 4.7 Vocabulary size in communication. 4.7 Vocabulary size in communication. Using dictionary when communicating with English to foreigners Of course Never Using dictionary when communicating with English to foreigners Using dictionary when communicating with English to foreigners Of course Never The density of HUFI students using dictionaries in English communication is very high. A total of 70 candidates (54.30%) out of 120 participants stated that they all needed to use a dictionary while speaking. They also admitted that they were unaware of the context in which the word was used in terms of communication, and that they did not know how to reply in English. The students mentioned using dictionaries to look up words, match sentences, and then converse with native speakers. These results indicate a lack of vocabulary among students. Unlike the mother tongue, the second language (here English) requires constant updating in terms of the usage and meaning of words. Indirectly, vocabulary impacts a student's communication skills; a limited vocabu- lary makes communication more challenging for students. The English language has a number of different meanings, which is why words are used in an inflexible way when learning them. Over time, this negatively affects their communication skills, leading to decreased confidence in communication. It is common for students to learn a word's main meaning, but, in reality, each English word has a plethora of different meanings. Further- more, students often don't pay much attention to learning the correct pronunciation of 8 of 12 8 of 12 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 words, leading to students making lisper pronunciations that alter the meaning of the sentence. Consequently, the survey found that students generally have an average vocab- ulary size. It is therefore imperative to practice and to increase the amount of vocabulary every day to avoid being surprised when communicating. 4.8 Body language enhances English communication 4.8 Body language enhances English communication Body language is rarely used in communication, as shown by the chart. When stu- dents were asked how they usually express themselves, they said that they stand upright, cross their arms, or hold their hands together. 4.7 Vocabulary size in communication. These people do not even make simple gestures like nodding in support of the speaker or smiling upon receiving a compliment; rather, they simply say "thank you" to the speaker. Language is used to express someone's thoughts, intentions, or conditions, and can also be used to conceal or distract them. A language associated with consciousness should 5% 22% 34% 23% 16% Using body language when communicating Always Usually Often y g g g 5% 22% 34% 23% 16% Using body language when communicating Always Usually Often y g g g 5% 22% 34% 23% 16% Using body language when communicating Always Usually Often Using body language when communicating Body language is rarely used in communication, as shown by the chart. When stu- dents were asked how they usually express themselves, they said that they stand upright, cross their arms, or hold their hands together. These people do not even make simple gestures like nodding in support of the speaker or smiling upon receiving a compliment; rather, they simply say "thank you" to the speaker. Language is used to express someone's thoughts, intentions, or conditions, and can also be used to conceal or distract them. A language associated with consciousness should be controlled by consciousness. Furthermore, there is an unattached kind of language, called body language, that has little or no connection with the conscious mind. In the com- munication process, gestures, facial expressions, and limb movements are utilized by this language. An individual's speech is composed of three elements: language, non-verbal cues, and tone. In which language accounts for only 7% of the impact on the listener, 38% is due to tone, and 55% is due to nonverbal communication of body language. University of Cal- ifornia, Los Angeles psychology professor Albert Mehrabian is responsible for establish- ing this rule. This conclusion was reaffirmed by Dr. Ray Birdwhistell - Professor of Psy- chiatry at the University of Pennsylvania, that listeners receive 7% of words and content; 38% of the content words (voice: press, stop, high, low, loud, small...) and 55% are not related to words, such as posture, costumes, movements, swing, facial expressions… of the speaker. 5.3 Improve vocabulary size A third way to reinforce the word is by applying it immediately after learning it; repeating it repeatedly and doing some extra activities to reinforce the word. Almost an- ything that is repeated in a repetitive manner requires more time for memorization. Reg- ular testing will do more than help students gauge their level. Nothing is harmful about this, it is only beneficial. It is essential that students have access to high-quality vocabulary learning resources. Students can find English vocabulary learning websites that develop online vocabulary for mainstream units, as well as application software that is developed by reputable units. E.g.: 1. https://www.vocabulary.com 2. https://quizlet.com 3. https://dictionary.cambridge.org 4. https://www.oxfordlearnersdictionaries.com 5.1 Self-study It is essential to constantly practice and improve your English, classroom lectures are time-limited, and teachers can not solely focus on teaching an individual and ignore oth- ers. Self-study is the best method. You will not be able to learn anything by waiting for class and may not even comprehend what the instructor has said, so students should de- cide what time makes the most sense to study. Do students work or study more efficiently in the mornings or afternoons? Choose for the individual a period of time during which the individual's brain is most focused on language learning. The English language can be studied through online courses as well as apps in every individual's spare time. Students can study wherever they want, at any time. 5.2 Improve vocabulary size 5.2 Improve vocabulary size Language is based on vocabulary. It is the "backbone" of communication in English. Due to its high vocabulary size, it helps individuals acquire other skills. When grammar is absent, not much information can be communicated. Vocabulary is crucial, but without it, you can't communicate anything. There can be no doubt that it is the most accurate conclusion about the importance of vocabulary. The following are some tips that can help students overcome difficulties with vocabulary learning: + Make sure you learn slowly according to your level and develop gradually. Avoid studying at an intricate level. If an individual does not know their level, they can not choose the right study program. Thus, students will make a list of appropriate vocabulary to focus on according to their level. There is no need to learn all the words since you will have difficulty remembering them. In the learning process, it is important to remember not to force yourself to memorize or rote study. In order to become familiar with English words, it is necessary to practice them. It is a very effective way to learn English vocabu- lary that you need to remember. + Learn words through books, movies, and music, which also happens to be the most frequently heard advice. Start by presenting information on topics that are familiar, rather than overwhelming, specialized information. It is not necessary for individuals to look up every new word in a dictionary. Continuously searching will interrupt thinking, making the ability to receive information more limited. To determine what the idea is, you should highlight unknown words in context, or based on related words in a sentence. 5. Discussion In the fourth part of the paper (Results), the scientists raise a number of issues. Here, the survey suggests possible solutions. In particular, the following recommendations fo- cus on improving communication skills in English and learning English with greater pre- cision among university students in general, as well as HUFI students. There is no doubt that learning and using English as fluently as a native speaker can be very challenging. Practicing a second language daily and continuously helps promote knowledge that can 9 of 12 be readily applied in practice. All the solutions listed below are not the fastest solutions for students, but they can be applied immediately, and the effects will improve gradually. be readily applied in practice. All the solutions listed below are not the fastest solutions for students, but they can be applied immediately, and the effects will improve gradually. 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 6. Conclusions The purpose of this study is to investigate how university students' English commu- nication skills have improved over their years at HUFI. The research paper consists of eight survey questions that are selected and evaluated by the students themselves. The survey was analyzed and the above results were given (in the Results and Discuss sec- tion). The survey is analyzed into four parts as follows: First, the correlation between study time and proficiency in essential English skills. This section is based on the academic year along with the students' evaluation of their own work. Second, genders affect how we communicate in English. Based on gender, the analysis of factors related to the com- munication ability of the two different genders is given. Third, vocabulary size in com- munication. The analysis is based on the amount of vocabulary that students have, thereby analyzing students' ability to use a dictionary when practicing communication with native speakers. The last part - body language enhances English communication. Known as a communication aid, this section will analyze the gestures and actions of stu- dents when communicating face-to-face. Along with the analysis are the recommenda- tions that have been given. And the important part is have realistic expectations is im- portant for the learner. Set goals that are consistent with your abilities. Do not rush, do not be impatient, and be patient. Moreover, it takes at least two years for native speakers to speak their first words as children are only able to listen before they speak. This means you can not master a language in a matter of weeks or months. Additionally, students should not put pressure on teachers to completely improve their English. It's up to you to learn. The teacher is just a guide, giving methods to learn. Whether you go far or not depends on your feet, your thoughts. Since there are 40 students in a class, the teacher shares the same amount of knowledge with all of them. Practicing both speaking and lis- tening skills every day is key to improving communication skills, and in order to fluently communicate in English, students need to practice every day. The role of listening is to receive information from the speaker, and the role of speaking is to give back information. The ability to communicate in English at any time is gained by using English often, not just in the classroom. 5.4 Speaking slowly The fourth item is the speed of speech, also the most important issue students need to be aware of. It's common for students to say that they surf the internet often, which is 10 of 12 10 of 12 not good. Considering the personal starting point is that of a language learner, not a native speaker, the pronunciation and speaking speed will be different from that of a native speaker. Even though a student can answer without thinking, students should slow down their speech. Students may worry that the person they are speaking to is not patient enough, so they will try to speak quickly, but this is not a good idea. The reason is that people often want an honest answer, which is also a sign that you value their opinion. But silence is also disrespectful, as it can give the individual time to think by inserting words with different contexts such as "Well", "Basically" and "Honestly", or "Oh, yeah", "You know", and "I mean"... You need to insert a lot of words when searching for the right an- swer, so to do this, you need to learn more vocabulary. 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 6. Conclusions Never be afraid to make errors when communicating in English because native speakers sometimes make errors in their communication and also need to correct them. Students should not be too dependent on the dictionary to guess the mean- ings of words and sentences by referring to the content of the reading text, listening text and communication situations, because this fosters bad habits. A dictionary isn't always carried or used. Students will notice significant improvements in results by using these methods for about 2-3 months. It is hoped that if this research topic is undertaken again, based on the efforts of the students, it will produce a better result than the current one. 7. Patents Funding: This research received no external funding Conflicts of Interest: The authors declare no conflict of interest. Funding: This research received no external funding Conflicts of Interest: The authors declare no conflict of interest. 11 of 12 Appendix Survey questions investigating for English communicating skills. 1. Your gender: Male Female X 2. Year of learning: Freshman Sophomore Junior Senior 3. Level of proficiency in speaking skills in English ? 1 = Worst / very unskilled 2 = Not good / unskilled 3 = Neutral / normal 4 = Skilled 5 = Very skilled 4. Level of proficiency in listening skills in English ? 1 = Worst / very unskilled 2 = Not good / unskilled 3 = Neutral / normal 4 = Skilled 5 = Very skilled 5. Level of proficiency in reading skills in English ? 1 = Worst / very unskilled 2 = Not good / unskilled 3 = Neutral / normal. 4 = Skilled 5 = Very skilled 6. Level of availability when communicating with foreigners ? Unwilling Neutral Willing 7. Using dictionary when communicating with foreigners ? Yes No 8. Level of using body language when communicating with foreigners ? 1 = Never 2 = Sometimes 3 = Often 4 = Usually 5 = Always Appendix 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 4. Kôlôminxki. (1981). Những cơ sở của tâm lí học sư phạm. Nhà xuất bản Giáo dục. 5. Martínez, & Nosnik. (1998). Practical Organizational Communication. Mexico.: Editorial T 6. Rahman., M. H. (2005). Malaysia: Siti Rahaya Ariffin, Shahrir Abdullah & Abd. 9. Tarone. (1997). Conscious communication strategies in interlanguage. Washington DC. 8. Tarone. (1980). Communication strategies. Washington DC. 10. Thạc, N., & Anh, H. (1991). Vài thực nghiệm về KNGT sư phạm của sinh viên vốn có nhu cầu giao Nam: Tạp chí nghiên cứu Giáo dục. Appendix Survey questions investigating for English communicating skills. 1. Cleland, Foster, & Moffat. (2005). Undergraduate students’ attitudes to communication skills learning differ depending 4 on year of study and gender. 5 2. Chiavenato, & Idalberto. (2006). Introduction to the General Theory of Administration. Spain: McGraw-Hill 6 Interamericana. 7 3. Hernández, A., & Garay, O. (2005). Communication in the sport context. Spain: Wanceulen Editorial Deportiva, 428 S.L. 429 12 of 12 4. Kôlôminxki. (1981). Những cơ sở của tâm lí học sư phạm. Nhà xuất bản Giáo dục. 7. Rodiah, & Idris. (2010). Pemeriksaan Ciri-ciri Psikometrik dan Pembentukan Penanda Aras Pentadbiran Kemahiran Genarik. Malaysia. 8. Tarone. (1980). Communication strategies. Washington DC. 10. Thạc, N., & Anh, H. (1991). Vài thực nghiệm về KNGT sư phạm của sinh viên vốn có nhu cầu giao tiếp khác nhau. Việt 7 Nam: Tạp chí nghiên cứu Giáo dục. 8 11. Wikipedia contributors. (2021, May 6). https://en.wikipedia.org/wiki/Tertiary_education. Retrieved from 39 en.wikipedia.org: https://en.wikipedia.org/wiki/Tertiary_education 40
https://openalex.org/W4387099162	https://www.emerald.com/insight/content/doi/10.1108/CDI-08-2023-0278/full/pdf?title=are-we-talking-about-the-same-thing-the-case-for-stronger-connections-between-graduate-and-worker-employability-research	English	null	Are we talking about the same thing? The case for stronger connections between graduate and worker employability research	Career development international	2,023	cc-by	8,277	The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/1362-0436.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/1362-0436.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/1362-0436.htm CDI 29,1 Received 15 August 2023 Revised 28 August 2023 Accepted 28 August 2023 Note: This is an invited submission by the Editor-in-Chief, Dr Jennifer A. Harrison, to Car Development International g Funding details: This study was supported by Interne Fondsen KU Leuven / Internal Funds K Leuven (project C24 M/19/012). Career Development International Vol. 29 No. 1, 2024 pp. 80-92 Emerald Publishing Limited 1362-0436 DOI 10.1108/CDI-08-2023-0278 Abstract Purpose and approach – This article presents the case for creating stronger connections between research on graduate and worker employability. We offer a narrative review of commonalities and differences between these research streams and offer thoughts and suggestions for further integration and mutual learning. Findings – We outline some of the main theories and concepts in the graduate and worker employability domains. Furthermore, we analyze how these show considerable overlap, though they have barely connected with each other yet. We also formulate an agenda for future research that would spur stronger connections between the fields. Finally, we turn to our fellow authors, reviewers, and editors to encourage a more open approach to each other’s work that would enable more cross-fertilization of knowledge. Findings – We outline some of the main theories and concepts in the graduate and worker employability domains. Furthermore, we analyze how these show considerable overlap, though they have barely connected with each other yet. We also formulate an agenda for future research that would spur stronger connections between the fields. Finally, we turn to our fellow authors, reviewers, and editors to encourage a more open approach to each other’s work that would enable more cross-fertilization of knowledge. Implications – We hope our narrative review, critical analysis and future research suggestions will lead to more collaborations and mutual learning among employability researchers in the educational, career and psychology areas. a o e ope app oac to eac ot e s wo t at wou d e ab e o e c oss e t at o o ow edge. Implications – We hope our narrative review, critical analysis and future research suggestions will lead to more collaborations and mutual learning among employability researchers in the educational, career and psychology areas. Keywords Employability, Graduate employability, Worker employability, Careers, Education Paper type Viewpoint © Jos Akkermans, William E. Donald, Denise Jackson and Anneleen Forrier. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/ legalcode Career Development International Vol. 29 No. 1, 2024 pp. Disclosure statement: No potential competing interests were reported by the a Are we talking about the same thing? The case for stronger connections between graduate and worker employability research Jos Akkermans Department of Management and Organization, School of Business and Economics, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands 80 Received 15 August 2023 Revised 28 August 2023 Accepted 28 August 2023 William E. Donald Southampton Business School, University of Southampton, Southampton, UK and Organizational Behavior and HRM, Ronin Institute, Montclair, New Jersey, USA Denise Jackson School of Business and Law, Edith Cowan University, Joondalup, Australia, and Anneleen Forrier Faculty of Economics and Business, KU Leuven, Leuven, Belgium Denise Jackson School of Business and Law, Edith Cowan University, Joondalup, Australia, and Anneleen Forrier Faculty of Economics and Business, KU Leuven, Leuven, Belgium Employability research: a promising but divided scholarly area Connecting graduate and worker employability Research on employability has gained considerable momentum, most notably in career research (Akkermans and Kubash, 2017; Byington et al., 2019) and educational research (Healy et al., 2022; Tomlinson, 2012). Employability is typically defined as an individual’s employment potential in the internal and external labor market (Forrier and Sels, 2003). Within this research area, there is a particular emphasis on graduates’ immediate and long- term career outcomes in the higher education research field (Clarke, 2018; Donald et al., 2019), whereas career and psychology research primarily focuses on how employability can serve as a resource that facilitates career mobility and success (Forrier et al., 2018; Fugate et al., 2021). 81 Employability matters. Research on employability among workers – which we will refer to as worker employability in this article to compare it to the common term of graduate employability – has provided clear support for the relevance of studying employability. For example, in their review article, Fugate et al. (2021) concluded that employability has considerable benefits for employees, such as being a predictor of career success, serving as a resource to help deal with insecurity and challenges and enhancing work-related and general well-being. Despite the lack of a systematic overview of the findings of graduate employability research [1], research in this area also shows convincing evidence of its value for graduates, such as offering transferable and career management skills (Clarke, 2018; Tomlinson, 2012). Furthermore, research on school-to-work transitions – often separated from but closely related to (graduate) employability – has established that employability is a critical asset for successfully transitioning from education to work and laying the foundation for a sustainable career (De Vos et al., 2019, 2020). Although employability research is timely, it is also criticized for being “fuzzy” (Van Harten et al., 2022). Definitions and conceptualizations of employability still tend to differ considerably across and within the streams of worker and graduate employability (Forrier et al., 2018). To illustrate, research on worker employability has long been divided across three strands (Van Harten et al., 2022): (1) personal strengths that increase employment potential (such as competence-based employability, see Van der Heijde and Van der Heijden, 2006), (2) perceived employability (see, e.g. Vanhercke et al., 2014) and (3) job transitions (see, e.g. De Vos et al., 2021). Abstract 80-92 Emerald Publishing Limited 1362-0436 DOI 10.1108/CDI-08-2023-0278 y Note: This is an invited submission by the Editor-in-Chief, Dr Jennifer A. Harrison, to Career Development International Note: This is an invited submission by the Editor-in-Chief, Dr Jennifer A. Harrison, to Career Development International Employability research: a promising but divided scholarly area Although some empirical studies have tested the associations between these strands, such as Forrier et al.’ (2015) test of all three in a dynamic chain, the number is surprisingly low, and comparisons are difficult due to the large variety of measurement instruments used to assess employability (Van Harten et al., 2022). Similarly, graduate employability research has focused on different themes, such as individual capital and competencies and institutional policies and practices aimed at strengthening employability (Healy et al., 2022). Furthermore, R€omgens et al. (2020) argued that the availability of so many different competency-based frameworks of graduate employability has hindered scholarly progress. In addition to these problems and disconnects within the worker and graduate employability discourses, the connections between them are even rarer, despite calls for more integration (e.g. Akkermans and Kubash, 2017; Clarke, 2018) as the concept of employability is, at its core, highly similar whether we talk about graduates or workers. Some recent conceptual attempts have been made to connect the worker and graduate employability areas. For example, Healy et al. (2022) formulated several key pedagogical principles underlying a more integrative approach to how employability may be strengthened by reconciling ideas from the graduate and worker employability literature streams. He advocated using career development theories to increase our understanding and application of careers and employability learning. Similarly, R€omgens et al. (2020) integrated the workplace learning and higher education literature streams to formulate several central dimensions of graduate employability learning that narrow down the wide range of available concepts across the worker and graduate employability literature. Furthermore, Donald et al. CDI 29,1 (2020) combined career ecosystem and psychological contract theory to design a model that helps understand how graduates may enter their professional careers successfully and sustainably. As a final example, Akkermans et al. (2023) presented a model of initial employability development, emphasizing the signaling and social exchange processes that evolve between graduates and employers and that could enhance (or undermine) employability development. (2020) combined career ecosystem and psychological contract theory to design a model that helps understand how graduates may enter their professional careers successfully and sustainably. As a final example, Akkermans et al. (2023) presented a model of initial employability development, emphasizing the signaling and social exchange processes that evolve between graduates and employers and that could enhance (or undermine) employability development. Theorizing employability h k l bili li The worker employability literature has been criticized for being a-theoretical. Indeed, Forrier et al. (2018) observed that theories – if they are used at all – are primarily used as a post hoc means of explaining research findings instead of using theory systematically to formulate research hypotheses. Van Harten et al.’s (2022) literature review confirmed this, as they found that most relationships tested between employability strands were not-theory based. Despite the lack of systematic theorizing in worker employability research, we do observe several theories that have been proposed and used most often. First, although rarely tested to its full complexity (Forrier et al., 2018), social exchange theory (Cropanzano and Mitchell, 2005) has been used in employability studies to explain how employers and employees may jointly contribute to employability development as a result of economic and socioemotional exchanges at work. For example, Fugate et al. (2021) proposed using a social exchange perspective with a strategic human resource management (HRM) framework to understand how employability may develop within employer–employee relationships. A second dominant theoretical paradigm has been human capital theory (Becker, 1964), often as a foundation for the value of various personal strengths. The basic idea is that investments in one’s employability will result in favorable work and career outcomes, such as career success (Forrier et al., 2018; Fugate et al., 2021). Third, studies have increasingly leveraged the conservation of resources (COR) theory (Hobfoll, 1989; Hobfoll et al., 2018) to study worker employability. COR theory aligns with the notion that employability is a personal resource that individuals can protect and enhance to deal with adversity and increase their well-being (De Cuyper et al., 2012; Forrier et al., 2018; Jabeen et al., 2022). These theoretical paradigms have been criticized for being overly agentic (Forrier et al., 2018). Recently, theories considering the role and interplay of agency (individual action and capability to enact change) and structure (external factors such as labor market conditions) are gaining ground in the worker employability literature, such as Bourdieu’s theory of practice (see, e.g. Delva et al., 2021). These theories highlight how worker employability is always contextually embedded. The graduate employability literature has also been criticized for being a-theoretical (Healy, 2023), perhaps reflective of the applied nature of many of the higher education journals. Yet, various theoretical approaches are deployed, and several are highly prominent. Employability research: a promising but divided scholarly area Although we acknowledge that we may be positively biased toward these initiatives as we have been involved in quite a few of them, we believe these recent attempts to connect the two disciplines are highly promising. As such, our main purpose in this article is to formulate opportunities for doing precisely that. To do so, we will first critically reflect on similarities and differences in (1) theorizing and (2) conceptualizing employability across the two literature streams. In each of those sections, we will briefly reflect on recent developments in each field, after which we will reconcile them and offer thoughts on progressing research in this specific area. Then, we will formulate a more extensive agenda for future research to connect them. In the final part of this article, we will also turn to our colleagues – as authors, reviewers and editors – to facilitate such connections in future research. 82 Theorizing employability h k l bili li First, human capital theory (Becker, 1964) is also a dominant theoretical paradigm in graduate employability studies. Recently, graduate employability researchers have extended and adapted human capital theory through the notion of employability capital (Clarke, 2018; Donald et al., 2019, 2023; Tomlinson, 2017). The employability capital approach retains the fundamental theoretical assumption of human capital theory, yet extends it by adopting a more holistic perspective of graduate employability to address concerns that human capital re-enforces pre-existing inequalities, cannot predict career success and fails to articulate how education augments productivity (Hooley and Sultana, 2019; Marginson, 2019). Connecting graduate and worker employability Second, COR theory (Hobfoll, 1989; Hobfoll et al., 2018) also features strongly and, similarly to worker employability research, there is an emphasis on developing personal resources to better position oneself to effectively navigate the transition from school to work (Jackson and Wilton, 2017; Vanhercke et al., 2014) and to increase well-being (Donald and Jackson, 2022; Nimmi et al., 2021). Third, perspectives that consider the role and interplay of agency and structure also feature prominently in graduate employability literature. These perspectives adopt a more relational focus on how students’ interpretation of and behaviors related to employability are inherently shaped by their personal background and dynamically negotiated with their macro context (Brown et al., 2004; Tholen, 2015). If a theory is leveraged in such articles, they usually mention Bourdieu’s theory of practice or Giddens’ theory of structuration (see Tholen, 2015). 83 Analyzing the two literature streams, we observe a remarkable overlap between the fields of graduate and worker employability research in how they theorize about employability. Indeed, both streams dominantly feature human capital theory and conservation of resources theory. Moreover, the theoretical arguments used are highly similar, as research in both streams supports the notion that employability is a form of capital or a resource that individuals can build to enhance their long-term work and career prospects. This significant overlap in theorizing about employability across the streams makes it all the more surprising – and problematic – that there is so little exchange of knowledge. Despite the similarities, we also see opportunities for mutual learning. First, although both areas are criticized for not being sufficiently theoretical, we would argue that worker employability research tends to focus on applying and advancing theory more prominently, perhaps as a consequence of career and psychology journals focusing more strongly on this issue as compared to educational journals. Theorizing employability h k l bili li As a result, the level of theoretical sophistication in the worker employability field could serve to stimulate more explicit theoretical advancement in graduate employability research. More importantly, though, both streams would benefit from more thorough theorizing, preferably using available knowledge from both of these streams. Second, although worker employability scholars have slowly started to incorporate contextual elements, we observe that the graduate employability field has made more significant developments in this area through its more prominent focus on labor market characteristics and the role of equity and inclusion. Hence, researchers in the worker employability domain could learn from these insights to more prominently feature such factors in their research ideas and designs. Conceptualizing employability A i d b f h k As mentioned before, the worker employability literature is fragmented, with employability being interpreted along three conceptual strands: (1) personal strengths, (2) perceived employability and (3) job transitions (Van Harten et al., 2022). This fragmentation hinders knowledge accumulation leading to calls for a more integrative employability approach (see, e.g. Guilbert et al., 2018). Interestingly and perhaps problematically, although Van Harten et al.’s (2022) review shows that empirical research has still not converged on an integrative approach yet, conceptual models have been developed to achieve more integration and bring clarity. Indeed, in 2009, Forrier et al. (2009) presented the employability process model that develops the relationship between personal strengths, perceived employability and job CDI 29,1 transitions and also adds contextual factors (such as structures of risk and opportunities, disruptive events, or opportunities to enhance one’s employability). In addition to this lack of integration between strands, the field of worker employability research is also characterized by fuzziness within strands (Van Harten et al., 2022). This is most particularly the case for research on personal strengths. Personal strengths come in many forms. They encompass dispositions (see, e.g. Fugate and Kinicki, 2008), attitudes (see, e.g. Van Dam, 2004) and competencies (see, e.g. Van der Heijde and Van der Heijden, 2006) and are classified in numerous ways. The movement capital classification into four dimensions, proposed by Forrier et al. (2009), encompasses the dimensions of human capital, social capital, adaptability and self-awareness. This classification has been used the most prominently among the personal strength models (Van Harten et al., 2022) and exhibits the most robust correlation with perceived employability (Harari et al., 2021). 84 Besides these conceptual issues, what is also problematic is the plethora of measurement instruments used. In their systematic review, Van Harten et al. (2022) identified 51 different instruments to measurepersonal strengths, 38 of whichwereused only once. These instruments often use different labels to measure the same or vice versa. Clearly, this is a significant problem for conceptualizing worker employability consistently and accurately. In line with Schreurs et al.’s (2022) recommendations for publishing quantitative research, we urge employability scholars to strive for more consistency in their use of measurement instruments and, at the very least, tobeexplicitaboutwhich instrumenttheyused and why(and,ifapplicable,which changes they made to established scales and why). After all, it will be difficult, if not impossible, to converge on a consolidated knowledge base (e.g. Conceptualizing employability A i d b f h k Although specific nuances vary across conceptual approaches, the basic underlying idea of movement or employability capital as a critical resource for graduates’ and workers’ employability is the same. Of course, there are differences too. For example, the conceptual models in the graduate employability literature tend to incorporate the role of stakeholders and the external environment more explicitly. Conversely, research on competence-based employability and movement capital integrates dimensions around movement and flexibility more deeply. Such differences are likely due to the specific field of research. For example, inequality and unequal opportunities are prominent topics in educational research, which has likely led to a stronger focus on structural factors impacting employability. Similarly, as worker employability research is often part of career research, this stream tends to focus on (career) transitions across the lifespan, hence focusing more strongly on adaptability and flexibility as key factors of employability. Still, despite those different nuances, the similarities are far greater than the differences. Hence, we argue that there are clear opportunities for the two fields to learn from each other in terms of conceptualizing employability. Connecting graduate and worker employability 85 p g p y y First, worker employability researchers could focus more strongly on the various stakeholders and contextual factors involved in employability development (see also Delva et al., 2021; Forrier et al., 2009, 2018). Second, graduate employability researchers could incorporate career-related factors related to movement and transitions (see also Donald et al., 2023; Healy, 2023; Healy et al., 2022). At the same time, we believe such research efforts should aim to consolidate and integrate the available knowledge instead of creating even more new conceptual frameworks that each include slightly different lists of elements or dimensions. Moreover, both streams – together – could start solving the conceptual “fuzziness” around concepts used in employability research. For example, it seems the term “capital” is not conceptualized consistently within and across streams. In particular, we observed that the worker employability research seems to have a narrower definition of (employability or movement) capital focusing on individual characteristics and personal strengths, whereas the graduate employability stream also considers contextual factors to be forms of capital. More consistency and clarity in defining and conceptualizing such critical terms would be an important step forward. Conceptualizing employability A i d b f h k through meta-analysis) of employability if we continue to use so many different scales for the same concept. The graduate employability literature also continued to face conceptual issues related to presenting a plethora of different capital and competency-based models. More specifically, Tomlinson (2017), Clarke (2018) and Donald et al. (2019) conceptualized three influential capital-based graduate employability models. First, Tomlinson’s model featured human, social, cultural, identity and psychosocial capital (or, as he called it, resources). Second, Clarke distinguished six forms of employability capital: human capital, social capital, individual attributes, individual behaviors, perceived employability and labor market factors. Third, Donald and colleagues proposed human capital (as a composite of six forms of capital, including social, cultural, psychological, scholastic, market-value and skills), career guidance and career ownership as determinants of self-perceived employability. Although the similarities are apparent, these models have tended to develop and be empirically validated in parallel with limited integration of ideas (R€omgens et al., 2020). One exception is the recent integrative employability capital growth model presented by Donald et al. (2023). Based on a review of the graduate employability literature between 2016 and 2022, their model offers nine forms of employability capital: social, cultural, psychological, personal identity, health, scholastic, market-value, career identity and economic capital. It also encompasses external factors (e.g. access to career counseling and recruitment process bias) that sit beyond an individual’s agency and various personal outcomes (e.g. career progression, career satisfaction and well-being). Although the model has yet to be empirically tested since it was only just published at the time of writing this article, it encompasses the core dimensions of many of the capital models that have been empirically validated in the graduate employability literature. Consequently, we believe that it could offer a solid way forward to research the different types of capital valuable to graduate employability. There is perhaps no more obvious evidence of the lack of interdisciplinary connectivity in our research domains than the existing work on employability-related capital. It is not difficult to see the similarities between concepts used in the worker and graduate employability literature streams. To illustrate, all of those models contain some form of social capital and human capital (although sometimes conceptualized in different ways and sometimes implicitly rather than explicitly using that exact terminology). Conceptualizing employability A i d b f h k p p Overall, the past few years have seen a steep increase in employability research, resulting in many different frameworks and models across the worker and graduate employability literature streams (e.g. dispositional employability, competence-based employability, movement capital and employability capital). Given the increasing maturity of both fields, we argue that now is the time to start testing and consolidating that knowledge to critique and refine our understanding of employability among graduates and workers. Future research agenda In the previous sections on theorizing and conceptualizing employability, we have provided several suggestions for connecting worker and graduate employability scholarship. Here, we formulate additional ideas for doing so. But before doing so, we want to take a moment and reflect on one major conclusion we have drawn. After writing this article, we are even more surprised that our fields are still considered separate research streams despite the fact that we have similar research interests, leverage similar theories and also use and develop similar conceptual frameworks. Perhaps the ultimate example of this is the significant degree of overlap in conceptual models focused on employability-related capital, such as Forrier et al.’s (2009) movement capital concept and Donald et al.’s (2023) employability capital concept. And yet, we still do not collaborate much! This lack of exchange is likely due to the academic communities we are part of and the journals we read and publish in. Still, we hope that this CDI 29,1 article will show others, as it has taught us, that connections between our research fields must be developed more strongly. article will show others, as it has taught us, that connections between our research fields must be developed more strongly. Connections through collaboration Our first suggestion for research bridging the two literature streams is as straightforward as it is crucial: let us start building connections by actively collaborating on research projects related to employability. It is becoming increasingly clear that we need an interdisciplinary approach to our research and research teams. Indeed, during the writing process of this article, we have all learned much about “the other side” of employability research through our conversations, our sharing of relevant literature and writing the paper. Similarly, each of the authors of this paper has experienced rich and highly meaningful learning opportunities through collaborations with scholars from other institutions, geographical locations and fields of study, helping to advance our understanding of employability around the initial transition into work. Such collaborations show that although our fields are, until now, mostly disconnected, we can start making those connections simply by seeking each other out. In other words, as employability scholars, we each need to play a part in breaking down these research silos based on disciplines (i.e. graduate versus worker employability) but also those related to, for example, geographic and demographic factors. Future research agenda 86 Seeking collaborations also means that we can learn from the unique strengths and insights of each literature stream. For example, scholars in the worker employability literature have regularly observed a lack of solid contextualization (e.g. supply and demand mechanisms) and practical implications of employability research (e.g. Forrier et al., 2018). Related to this, research on how HRM policies and practices can influence employability is still limited (for an exception, see the recent special issue edited by Van Harten et al., 2020, in the International Journal of Human Resource Management). We see a clear opportunity for worker employability researchers to benefit from the considerable expertise and experience that graduate employability scholars possess related to these matters related to, for example, policy and institutional practices (e.g. Bridgstock and Jackson, 2019; Minocha et al., 2017). Conversely, research in the worker employability domain generally seems to adopt more sophisticated research designs (e.g. multi-wave structural path models and person-centered approaches) and a stronger emphasis on theoretical implications. This trend may be due to the publication norms in the respective research areas and journals, as psychology and career journals tend to emphasize theory-building. In contrast, higher education journals often focus on the practical value of research insights due to the applied nature of many of the prominent journals. Still, the graduate employability field may benefit from the available knowledge on research design and methods, as well as the theory building in the worker employability field. g , y g p y y Content-wise, both streams could inspire each other and broaden the scope of research topics in joint projects. We suggest three potential tracks for future collaborative research. First, where graduate employability research has a particular focus on the preparation for the initial transition into work and worker employability on the career path after this first transition, an opportunity for joint research is collaboratively exploring the longer-term trajectories, experiences and outcomes of graduates at work, moving beyond higher education’s problematic focus on short-term job attainment metrics (Jackson and Bridgstock, 2018). Such studies would also enhance the field of school-to-work transition research, in which longitudinal studies spanning the entire transition into work are still rare (Blokker et al., 2023). A second potential track for joint research is employability development and, in particular, work-integrated learning. Development and learning are central to employability and are part of the research debate in both streams. Future research agenda In worker employability research, employability development and learning are seen as part of an evolving career over time. With a background in educational science, learning is central in graduate employability literature. Work-integrated learning of graduates in workplaces is where both fields’ interests in employability development and learning could meet (Jackson et al., 2023). Third, another potential avenue for future research is about changes in employability perceptions throughout the transition from school to work and over time. There are clear indications that such perceptions can change over time and across individuals. For example, Grosemans et al. (2023) identified three different patterns of employability perception changes among graduates. Similarly, Farrugia (2021) showed how the meaning of work and careers differs across graduates with or without work experience and unemployment. It is also likely that employability perceptions change as a result of disruptive events – or: career shocks – during the school-to-work transition (Akkermans et al., 2021b). Building on these studies, we believe it would be highly interesting to research how perceptions of employability may develop before, during and after the initial transition into work, and which structural factors and disruptive events may impact these processes. Connecting graduate and worker employability 87 Sustainable careers as an overarching perspective Sustainable careers as an overarching perspective Researchers in graduate and worker employability fields have leveraged the sustainable career perspective to study and understand employability development. This theoretical perspective argues that, in an increasingly flexible and dynamic career landscape, building a sustainable career is about creating a career path that allows resources to be protected and renewed instead of depleted (De Vos et al., 2020). According to De Vos et al., achieving such a person-career fit is characterized by happiness, health and productivity, and these three elements should be somewhat balanced across the lifespan. Moreover, career sustainability is not only about individual agency but also contextual (e.g. organizational, national and cultural context) and temporal (e.g. events and changes) factors. As the sustainable career perspective offers a systemic and dynamic take on employability development, it seems particularly suited for research bridging the graduate and worker employability fields. Indeed, we have done so in several of our recent articles. For example, two recent book chapters by Akkermans et al. (2021a) and De Vos et al. Future research agenda (2019) used the sustainable career perspective to explain how the school-to-work transition has changed from a one-off decision to an ongoing series of learning cycles. Employability is a core factor in this dynamic perspective on the initial transition to work. This was confirmed in a recent systematic literature review of the school-to-work transition literature by Blokker et al. (2023), which used sustainable careers as an organizing framework and included employability as one of the outcomes of a successful transition into work. From the graduate employability perspective, Donald et al. (2020) and Donald and Jackson (2023) also incorporated the sustainable career perspective as one of the theoretical foundations in their work on graduate employability and initial career entry. These works emphasize the development of personal capital resources, career ownership and a commitment to lifewide and lifelong learning during university years as critical antecedents to sustainable careers, coupled with the ability to signal their employability through a coherent and appealing employability narrative that connects them with prospective employers. Overall, though we certainly do not advocate using only one theoretical perspective in our research on employability, we believe the sustainable career perspective may be a helpful way of reconciling the graduate and worker employability research streams. Graduate and worker employability among underrepresented groups O i h h d h i hi i l il i A turn to our colleagues In this final section of our article, we turn to our fellow authors, reviewers and editors who are active in the area of employability research. After all, building stronger connections between graduate and worker employability research starts with the people conducting and assessing the research. Therefore, we end this article by highlighting several observations and suggestions that we hope will lead to more fruitful collaborations and cross-over of knowledge. g First, we encourage you all – including us! – to look beyond our usual and comfortable research silos when conducting employability research. We realize there is a lot of new research emerging even within each stream, let alone across the two streams, making it difficult to keep track of the latest insights. Still, our article shows there is considerable overlap in how we theorize and conceptualize employability, thereby offering many opportunities for mutual learning. Therefore, we believe it would be helpful and enriching for our research if we would actively try to search for (new) employability research outside of the “usual suspect” journals that we may follow. For example, educational researchers could consult the Journal of Vocational Behavior or Career Development International, whereas career researchers could examine Studies in Higher Education. More broadly speaking, using varied keywords and being open to research from a domain that might be less familiar would be an important first step toward finding each other’s work and being able to use it to advance our knowledge of employability. This argument also applies to reviewers and editors, who are likely used to a certain theoretical or conceptual perspective on employability. Yet, when you review or edit a manuscript leveraging insights from “the other” side of employability research, please be open to them and consider whether it might enrich the current understanding of employability in your specific area. Also, for editors specifically, it would be valuable to invite reviewers from both streams when manuscripts on employability are submitted, as it would enhance the odds of receiving balanced reviews that incorporate insights from both areas. On a related note, we want to call attention to another issue that prevents the effective sharing of knowledge between the graduate and worker employability streams. When preparing and writing this article, we became increasingly aware of some differences in research approaches between the two fields. Graduate and worker employability among underrepresented groups While employability-building activities such as work-integrated learning and peer mentoring can be transformational for everyone, systemic barriers may inhibit their engagement and there is a critical need for more inclusive practice and greater stakeholder support (Donald and Ford, 2023; Jackson et al., 2023). Therefore, it is critical to adopt a broader focus to understand employability development among underrepresented and potentially disadvantaged groups, as there may be an increasing “employability gap” between the “haves” and the “have nots” if left unstudied and unsupported (see, e.g. Forrier et al., 2018). 88 Graduate and worker employability among underrepresented groups One issue we have not touched upon much in this article until now is the dominant focus in both graduate and worker employability research on theoretically trained individuals. This trend is clearly visible in graduate employability research, which is often published in higher CDI 29,1 education journals, thereby almost by default excluding non-highly educated (or, as we prefer to say, theoretically schooled) individuals. Though not as explicitly visible in journal names, the worker employability literature also heavily leans on research among so-called white- collar workers. However, employability research among practically schooled or vocationally trained people is much less common. In addition to vocational and educational differences, there are many groups underrepresented so far in the employability research domain. For example, graduate and worker employability scholars could collaborate to better understand employability development among people from less privileged socioeconomic backgrounds, individuals with mental or physical disabilities, and those who are pursuing non-standard career paths characterized by, for example, entrepreneurship, freelancing and gig work. While employability-building activities such as work-integrated learning and peer mentoring can be transformational for everyone, systemic barriers may inhibit their engagement and there is a critical need for more inclusive practice and greater stakeholder support (Donald and Ford, 2023; Jackson et al., 2023). Therefore, it is critical to adopt a broader focus to understand employability development among underrepresented and potentially disadvantaged groups, as there may be an increasing “employability gap” between the “haves” and the “have nots” if left unstudied and unsupported (see, e.g. Forrier et al., 2018). education journals, thereby almost by default excluding non-highly educated (or, as we prefer to say, theoretically schooled) individuals. Though not as explicitly visible in journal names, the worker employability literature also heavily leans on research among so-called white- collar workers. However, employability research among practically schooled or vocationally trained people is much less common. In addition to vocational and educational differences, there are many groups underrepresented so far in the employability research domain. For example, graduate and worker employability scholars could collaborate to better understand employability development among people from less privileged socioeconomic backgrounds, individuals with mental or physical disabilities, and those who are pursuing non-standard career paths characterized by, for example, entrepreneurship, freelancing and gig work. Notes 1. Though some reviews of the graduate employability are available, they typically focus on specific themes within this literature, such as stakeholder perspectives (Cheng et al., 2022), transnational education (Schueller, 2023) or employability capital (Donald et al., 2023). 1. Though some reviews of the graduate employability are available, they typically focus on specific themes within this literature, such as stakeholder perspectives (Cheng et al., 2022), transnational education (Schueller, 2023) or employability capital (Donald et al., 2023). Conclusion B f i Before setting out to write this article, we felt that stronger connections were necessary between the graduate and worker employability streams. During the process, we became even more convinced of this. Indeed, through our conversations and writing process, we have learned much from each other. In that sense, this project embodies exactly what we hope will happen more broadly in research on employability: more conversations and collaborations between graduate and worker employability researchers. This could happen through joint research, special issues, conferences and many other ways. But they all start with the same thing: making connections. So let’s connect! A turn to our colleagues More specifically, it seems that there are different perceptions of “what good research is” and “how research should be done” between the educational (i.e. graduate employability) and career and psychology (i.e. worker employability) journals. These differences can be quite fundamental (e.g. the focus on theoretical versus practical implications) but also seemingly small (e.g. the choice of words and writing style). Hence, although we share an interest in a similar topic (with a somewhat different focus) and study it in similar ways (and experience the same issues – e.g. conceptual fuzziness), integration is and will remain difficult as long as the rules of the game are different. It is almost as if we play a similar game in a different league. Therefore, this is an open invitation to journal editors and reviewers to question their own rules of the game and be open to different “ways of doing”. Moreover, it is, once again, a call for more collaboration between researchers, as the best way of getting to know each other’s rules of the game is by actively discussing them and working together on projects. Connecting graduate and worker employability 89 References Akkermans, J. and Kubash, S. 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Van der Heijde, C.M. and Van der Heijden, B.I.J.M. (2006), “A competence-based and multidimensional operationalization and measurement of employability”, Human Resource Management, Vol. 45 No. 3, pp. 449-476, doi: 10.1002/hrm.20119. Van Harten, J., De Cuyper, N., Guest, D., Fugate, M., Knies, E. and Forrier, A. (2020), “Introduction to special issue on HRM and employability: mutual gains or conflicting outcomes?”, The International Journal of Human Resource Management, Vol. 31 No. 9, pp. 1095-1105, doi: 10. 1080/09585192.2020.1740457. Van Harten, J., De Cuyper, N., Knies, E. and Forrier, A. (2022), “Taking the temperature of employability research: a systematic review of interrelationships across and within conceptual strands”, European Journal of Work and Organizational Psychology, Vol. 31 No. 1, pp. 145-159, doi: 10.1080/1359432X.2021.1942847. Vanhercke, D., De Cuyper, N., Peeters, E. and De Witte, H. (2014), “Defining perceived employability: a psychological approach”, Personnel Review, Vol. 43 No. 4, pp. 592-605, doi: 10.1108/PR-07-2012-0110. Corresponding author Corresponding author Jos Akkermans can be contacted at: j.akkermans@vu.nl For instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com
https://openalex.org/W2920895711	https://zaguan.unizar.es/record/85406/files/texto_completo.pdf	Spanish; Castilian	null	Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto	Fotocinema	2,019	cc-by-sa	6,881	E-ISSN 2172-0150 Nº 18 (2019) REVISTA CIENTÍFICA DE CINE Y FOTOGRAFÍA Recibido 12-01-2018 / Aceptado 28-08-2018 Publicado 25/01/2019 E-ISSN 2172-0150 Nº 18 (2019) REVISTA CIENTÍFICA DE CINE Y FOTOGRAFÍA Recibido 12-01-2018 / Aceptado 28-08-2018 Publicado 25/01/2019 E-ISSN 2172-0150 Nº 18 (2019) REVISTA CIENTÍFICA DE CINE Y FOTOGRAFÍA Recibido 12-01-2018 / Aceptado 28-08-2018 Publicado 25/01/2019 Recibido 12-01-2018 / Aceptado 28-08-2018 Publicado 25/01/2019 Recibido 12-01-2018 / Aceptado 28-08-2018 Publicado 25/01/2019 Resumen: El presente artículo analiza la incorporación de la fotografía como miembro de pleno derecho en el arte contemporáneo, a partir de la perspectiva que la sitúa en la vertiente documental y extremadamente banalizada de los territorios del arte de concepto. Para ello, en primer lugar, se aborda la fotografía en su realineamiento con lo cotidiano y lo anodino de autores como Ed Ruscha que sumergen a la fotografía en una nueva dimensión discursiva nunca antes planteada. Después, se analiza el discurso artístico de L. Sultan y M. Mandel donde, a través de una estética de extrañamiento, consiguen situar a la fotografía en el foco de la vertiente documental del arte de concepto. Para llegar al punto de cómo, desde las estrategias del amateurismo, la foto consigue forzar la mirada desde sus propias formas de aparecer, en una nueva imagen alejada de lo documental y asentada definitivamente en los territorios de lo artístico. Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto About the discursive dimension of photography in the sixties: a genealogy of the documentary side of concept art Víctor Murillo Ligorred Universidad de Zaragoza, España vml@unizar.es Víctor Murillo Ligorred Universidad de Zaragoza, España vml@unizar.es Palabras clave: Palabras clave: Fotografía; indiferencia visual; arte de concepto; narrativas; arte contemporáne Keywords: Abstract: The present article analyzed the incorporation of photography as a full member in contemporary art, from the perspective that places it in the documentary aspect and extremely banalized of the concept art territories. To do this, photography is firstly addressed in its realignment with the everyday and the anodyne of authors like Ed Ruscha who immerse photography in a new discursive dimension never before raised. Then, the artistic discourse of L. Sultan and M. Mandel is analyzed, where, through an aesthetic of estrangement, they achieve to situate photography in the focus of the documentary aspect of concept art. To get to the point of how, from the strategies of amateurism, the photo reach to force the gaze from its own forms of appearance, in a new image far from the documentary and definitively settled in the artistics territories. Keywords: y Photography; visual indifference; conceptual art; narratives; contemporary art. 215 FOTOCINEMA, nº 18 (2019), E-ISSN: 2172-0150 215 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 1. El espacio de la fotografía desde su nueva dimensión discursiva E-ISSN: 2172-0150 216 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto por tanto, entendida de este modo, se muestra como un producto moderno sujeto al cambio. En la época que la tratamos a partir de estos años, es cuando comienza a reivindicarse como un medio específico. la foto intenta buscar su sitio dentro de los cauces del arte contemporáneo y, es entonces, cuando emerge en los avatares de la liquidación de la historia que, al igual que el arte, son entendidos en un proceso de finitud. Danto aclara que no se refiere a la muerte del arte, sino más bien, a su fin dentro de un paradigma histórico (Danto, 2002, 31). Es el tiempo que sucede a los grandes relatos de la modernidad como explica Lyotard (2006). La posmodernidad se sitúa en el colapso de las estructuras basadas en las creencias de los paradigmas históricos (Padilla Córdova, 2014). En esta disolución de las categorías y los paradigmas lo interesante es que la fotografía, equiparada al mismo nivel que el resto de praxis, es elevada al mismo estadio de crítica que el resto de disciplinas, entrando así en los cauces artísticos y entendida la imagen foto como un producto artístico, más allá de lo técnico. Para entender este proceso, debemos comprender la evolución de lo cotidiano en el arte hasta constituir un tema autónomo y vinculado a lo fotográfico. Así, la mirada de los artistas hacia temas cotidianos comienza a finales del s. XIX, dentro de la pintura primero, y hacia el interés en lo fotográfico después. Unas relaciones que se han sucedido hasta el momento tratado, con una práctica sobre la búsqueda y refuerzo de lo anodino, muy en auge en los años sesenta (Crow, 2002, p. 131). Así, la fotografía comienza a mirar hacia el mundo de lo cotidiano y lo banal, en una continuada y reiterada búsqueda de lo anodino como también advierte Buchloh (Buchloh, 2004, p. 1. El espacio de la fotografía desde su nueva dimensión discursiva La fotografía en los años sesenta promueve una serie de intentos por legitimarse como práctica artística de pleno derecho. Esta situación emerge sin un claro mito originario o fundacional. Este hecho, sumado a una serie de políticas culturales proclives a que así sucediese, se presenta en una suerte de soldadura imposible en numerosos casos (Del Río García, 2008, p. 83). En tal situación, la fotografía es la que intenta esa escalada hasta la cumbre de las instituciones como miembro legitimador de discursos, al igual que lo eran el resto de prácticas artísticas. En este contexto, instituciones como el Moma de Nueva York iniciaron una andadura de grandes exposiciones fotográficas que atrajeron gran número de público a las salas de los museos, equiparando las cifras de visitantes a las grandes citas de pintura. Curadores como Edward Steichen, quien creó la exposición The Family of Man en 1953, o más tarde, el propio John Szarkowski con su célebre New documents de 1967, iniciaron una tradición expositiva en torno a lo fotográfico, anterior a la del resto de galerías de arte que, en aquella época, todavía no mostraban este tipo de obra en sus paredes (Szarkowski, 2010). El arte conceptual en sus inicios, en la denominada vertiente fotográfica, plantea problemáticas desde las estrategias de camuflaje mediante las que inscribe en los cauces de lo artístico. Trabajos artísticos a medio camino del pop y a medio camino de la negación de lo artístico forman parte de las vías mediante las cuales la fotografía consigue pertenecer a la institución artística. Los primeros proyectos fotográficos de los sesenta se constituyen con el paso del tiempo como obras fundacionales de un género poco articulado por la crítica del momento. Estas obras, inscritas en el pop, con un marcado carácter conceptual, transgreden los límites de lo establecido para la propia fotografía centrando el discurso en el análisis de la cultura de su tiempo, desde la fragmentación, el documentalismo, el amateurismo expresado en ellas mismas. Estas obras, lejos de ser reconocidas como obras de arte autónomas, pertenecientes a una nueva corriente artística, los álbumes de fotografías y las series de los fotógrafos como Ruscha o Graham eran catalogadas como obras descriptivas del paisaje americano, o como libros de viaje. La fotografía, hasta los años sesenta, era considerada como un producto consecuencia directa del desarrollo de la técnica, FOTOCINEMA, nº 18 (2019). FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 1. El espacio de la fotografía desde su nueva dimensión discursiva 180) y, como escribe Crow, es en el denominado fotorrealismo de los sesenta cuando, los paisajes suburbanos gozan de gran predilección no sólo por su aspecto insulso característico, sino también, por el enfoque vernáculo de la práctica pictórica, preferido por gran número de sus habitantes, práctica que los artistas profesionales de este género han llevado a un alto nivel de competencia técnica (Crow, 2002, p. 108). Algunos artistas americanos como es el caso de Edward Ruscha, pintor del denominado pop americano y gran aficionado de la fotografía, comienzan a explorar los nuevos caminos de la imagen fotográfica. A partir de esta afición, en el año 1962 decidió embarcarse en un proyecto en el que se fotografiaban veintiséis gasolineras. Las imágenes resultantes de aquella selección se recogían FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 217 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto en un pequeño libro de tirada corta (Jean, 2008). Las imágenes no destacaban por nada particularmente; más bien, la cotidianeidad de las escenas, la banalidad de lo representado y lo anodino de lo paisajístico era el denominador común de aquellas instantáneas. Este trabajo se tituló Twenty Six Gasoline Stations. Esta obra supone una suerte de corte y sutura al combinar una serie de paisajes triviales y anodinos como son las gasolineras, con una fuerte connotación documental. El resultado de esta obra supone la aproximación desde una concepción pop, a una obra de marcado carácter conceptual. En la temática de las gasolineras o en las series de las piscinas, Ruscha crea un ambiente con una omisión total de sujeto (Marchan Fiz, 2002, p. 41). Como señala Crow, el arte culto de los sesenta explora y persigue una condición de producción casi anónima (Crow, 2002, p. 107). Solamente importan en estas representaciones las construcciones sin estilo, los paisajes anodinos y las escenas cotidianas que no tienen nada de características. Parece que estas obras respondían a “la personalidad hippie” de Ruscha según Díez Cabrera, puesto que combinaba su amateurismo artístico con las inquietudes intelectuales, donde se sitúa en los símbolos de vida urbana con precisión perfecta y los reemplaza en un contexto predominentemente rural. 1. El espacio de la fotografía desde su nueva dimensión discursiva Su obra tuvo gran repercusión en Europa. Su imaginario abre una ventana al mundo, donde se transporta el espectador en un bombardeo constante de imágenes e información fragmentada. El lenguaje de Ruscha tiene numerosas capas de significado y apela a los sentidos y poderes de asociación del observador (Díaz Cabrera, 2005, p. 523). El propio Jill Lloyd comenta, parafraseando al crítico Peter Schjeldahl lo siguiente: Así como Manet era el artista por excelencia de París, Ruscha lo era de la ciudad de Los Ángeles (como nos lo demuestra en sus libros de fotografías a través del fotomontaje durante la década de los sesenta y setenta, que toman mayoritariamente temas e imágenes de la ciudad (Lloyd, 1998, p. 3). En el mismo sentido, Hulten compara a Ruscha con De Chirico o Marinetti, en tanto que éste se sitúa como el gran artista californiano, a pesar de que su infancia y juventud transcuriese en Oklahoma, al igual que sucedió con los dos grandes artistas italianos mencionados nacidos fuera de Italia (Hulten, 1990, p. 19). FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 218 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Aunque exageradas estas comparaciones, cierto es que Ruscha puede ser considerado como un gran artista. Su ímpetu, en la reiterada búsqueda de lo anodino, tambaleó los cimientos de la fotografía alzándola como práctica cotidiana del arte contemporáneo dentro de los territorios del arte de concepto. F1. Edward Ruscha. Twenty six gasoline stations, 1962. Disponible en: https://artmattermagazine.com/ed-ruscha-books-co-gagosian-gallery/ F1. Edward Ruscha. Twenty six gasoline stations, 1962. Disponible en: https://artmattermagazine.com/ed-ruscha-books-co-gagosian-gallery/ F1. Edward Ruscha. Twenty six gasoline stations, 1962. Disponible en: https://artmattermagazine.com/ed-ruscha-books-co-gagosian-gallery/ De ahí que la obra de Ruscha se encuadre en la ruptura de unos moldes preestablecidos y redescubra un espíritu dadaísta, al huir de la etiqueta de pop como hiciese en su momento Duchamp, no dejándose clasificar y mostrando su obra en diversos soportes como cuadros, fotografías o libros que cogieron por sorpresa a la crítica del momento (Díez Cabrera, 2005, p. 517). Concretamente, la obra presentada en formato de libros de este autor nos sorprende al comprobar que se trata de un documento de reducidas dimensiones. 1 Ruscha, Edward. Twenty six gasolina stations. Obra artística pero también Catálogo. Mucho tiempo se clasificó como catálogo de gasolineras en diversos depósitos y bibliotecas estando desubicada y erróneamente catalogada. FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 1. El espacio de la fotografía desde su nueva dimensión discursiva Este pequeño libro fue editado con una corta tirada de ejemplares cuya portada titulaba: Twenty Six Gasoline Stations1. Al abrir las páginas, el libro nos muestra lo que el título de la obra reza con total literalidad. Se trata de las fotografías de veintiséis gasolineras del condado de Los Ángeles, en la costa oeste de Estados Unidos. Como señala Jeff Wall: “los libros de Ruscha destruyen el género de libro de fotografías, ese formato clásico mediante el cual la fotografía artística manifiesta su clara independencia” (Wall, 2003, p. 246). Lo que aparece en el libro de fotografías de Ruscha son unos paisajes contemporáneos con elementos iconográficos de algunas edificaciones de Los FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 219 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Ángeles, así como escenas triviales y cotidianas que inciden en esa reiterada búsqueda de lo anodino. Lucy R. Lippard escribe: “Los libros de ‘antifotografía’ de Ruscha tuvieron una gran influencia desde su aparición en 1962” (Lippard, 2004, p. 42). Esta definición de “antifotografía” hecha por Lippard es corroborada por el propio Ruscha en una entrevista realizada por Coplans en la que señala: No me interesan realmente los libros como tales, pero sí las publicaciones inusuales (…). En primer lugar, las fotografías que utilizo no son artísticas bajo ningún concepto. Pienso que la fotografía está muerta en tanto que arte; su único lugar está en el mundo comercial, al servicio de la técnica o la información. Por tanto, (Small Fires) no es un libro que albergue una colección de fotografías de arte; son datos técnicos, como la fotografía industrial (…). Uno de los propósitos de mi libro es realizar un objeto fabricado de serie. El producto final tiene un sentido muy comercial, muy profesional (…). He eliminado completamente el texto de mis libros. Quiero únicamente un material neutro. Mis fotografías no son particularmente interesantes, ni tampoco lo es el tema. Son simplemente una colección de “hechos”; mi libro se parece más a una colección de “ready-mades” (Coplans, 1965). FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 1. El espacio de la fotografía desde su nueva dimensión discursiva Además de las citadas gasolineras, Ruscha también realizó otro tipo de investigaciones en forma de obras fotográficas que muestran imágenes de piscinas (serie Nine Swiming pool, 1968). La capa de agua azulada, así como sus alrededores en entornos cotidianos muestran siempre ese pesado carácter anodino. Los apartamentos como espacios privados también son representados en pro de ese anonimato de la escena cotidiana. El carácter trivial de este tipo de fotografías, prolongado hasta la época actual, parece comenzar con este tipo de obras en los años sesenta, sumergidas dentro de la corriente del pop-art, con la peculiaridad de deslizarse y emerger como obras fundacionales del arte conceptual. La característica de Ruscha de excluir al sujeto de las escenas lo distingue de otro tipo de artistas como puede ser el caso de Lee Friedlander, quien expresa todo lo contrario. Así, Friedlander se incluye siempre en la imagen, bien de forma implícita, con una sombra, o una indicación que le sitúa en el momento y hace referencia a su persona, o bien de forma explícita apareciendo él en las fotografías. En Ruscha, lo singular es que siempre mantiene el anonimato de las FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 220 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto escenas no dando mayores pistas de los lugares, sino todo lo contrario, representando los no lugares (Augé, 2000, p. 83). Obras como las casas seriadas de Dan Graham, serie titulada Homes for America, señalan un tipo de fotografía que suscita el debate abierto por Ruscha de una asociación entre la fotografía y el arte conceptual (Graham, 1967). La especulación con este tipo de lugares anónimos y comunes a todo el mundo, como las gasolineras, los apartamentos o las piscinas entran a formar parte de los lugares “que no pueden definirse ni como espacios de identidad, ni como relacionales, ni como históricos” (Augé, 2000, p. 83). 1. El espacio de la fotografía desde su nueva dimensión discursiva Este tipo de espacios son capaces de presentarse idénticos a sí mismos en múltiples localizaciones, un concepto en el análisis contemporáneo, el cual, evidencia que “la sobremodernidad es productora de no lugares” (Augé, 2000, p. 83). La existencia de espacios vacíos, en los que la presencia de las figuras está ausente, pero al mismo tiempo, representada en el paisaje de la cotidianeidad de forma fugaz (Martín Hernández, 2014, p. 5), supone que, esos mismos lugares representados son los que han de ocupar las figuras ausentes. Este tipo de imágenes inspirarían a un joven artista, Jeff Wall, quien en 1973 realizaría su serie titulada Landscape Manual, obra gestada con anterioridad a las fotografías de gran formato, por las cuales verdaderamente sería conocido tiempo después. La obra de Dan Graham en la representación y la seriación de los módulos de algunos edificios de la periferia de algunas ciudades recuerdan a las estructuras minimalistas. La reiteración de lo cotidiano se muestra en el despojo de toda estética y toda aura poética. Este marcado carácter, cercano a lo documental, se encamina en una suerte de disfraz camuflado del arte, en relación con una auto-negación propia como señalamos, más cercana al arte conceptual que al pop-art propiamente establecido. Además del carácter documental expresado anteriormente, el carácter serial es otra de las condiciones que se dan en este tipo de obras. Los libros catálogos de Ruscha, así como lo son los de Bruce Nauman, Barry Flanagan o Richard Long se tornan como obras conceptuales, en tanto que la idea, el refuerzo del anonimato, lo anodino de las representaciones o la banalidad de las temáticas se sitúan como la parte más destacable de las mismas (Marchán Fiz, 2002, p. 384). La insistencia de lo trivial, lo cotidiano y lo vacío resultan FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 221 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto inquietantes. El señalamiento reiterativo del refuerzo del anonimato se comprueba con la desaparición del sujeto de la escena. Espacios plenamente reconocibles, deshabitados. 1. El espacio de la fotografía desde su nueva dimensión discursiva E-ISSN: 2172-0150 222 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Estas afirmaciones de Graham, así como de los denominados artistas conceptuales de los años sesenta como J. Kosuth, entran a formar parte de esa interesada y reiterada auto negación artística en su vertiente fotográfica y documental. Más allá de esta idea de auto negación, la obra de Graham adquiere un firme compromiso con las tradiciones clásicas del reportaje y el foto- periodismo, cualidad que se identifica habitualmente con el arte de concepto. Sus fotografías de arquitecturas, proporcionan un fundamento social para los modelos estructurales de la experiencia intersubjetiva que ha desarrollado a través de textos, vídeos, performances y piezas escultóricas ambientales (Wall, 2003, p. 230). En definitiva, también la obra de Ed Ruscha se muestra en una suerte de camuflaje que pasa desapercibida ante los cauces corrientes de lo artístico como explica Dan Cameron (Cameron, 1990, p. 10). Durante mucho tiempo, estas imágenes estuvieron confundidas con los libros de viajes y no identificadas con los libros de arte; o peor, ordenadas en otras categorías menos afortunadas. Se trata de una suerte de negación artística de lo estético, en la que se mezclan temas ajenos al propio arte como son lo real y lo cotidiano, camuflados en este tipo de obras enciclopédicas (Cameron, 1990, p. 10). En este mismo contexto, a partir del discurso elaborado por Ruscha aparecen otro tipo de obras como las presentadas por Dan Graham y, posteriormente, las obras realizadas por dos autores que interesan en esta investigación, como son Larry Sultan y Mike Mandel. 1. El espacio de la fotografía desde su nueva dimensión discursiva Piscinas en absoluta calma, carreteras por donde no circula ningún vehículo, edificios y casas sin personas. Las fotografías a las que nos referimos exceden su condición de imagen documental centrando el debate artístico, en un punto, en el que nunca antes había estado. La imagen fotográfica se refuerza en muchos casos con el texto, que sitúa a la fotografía en una lectura de lo cotidiano despojada de cualquier aura poética, o idealización estética, asentada en la indiferencia y el anonimato de los objetos o la condición de escenas ya vistas y reconocibles cuyos reencuadres artísticos resultan casi irónicos como señala Del Río (2008). El principio descontextualizador y de búsqueda del anonimato permanece en los proyectos fotográficos en torno a lo cotidiano, pero aquí las fotografías de archivo, separadas de su contexto original, pierden la capacidad de ser entendidas de forma instrumental; dejan, en definitiva, de ser documentos, para relacionarse con el imaginario del arte (Del Río, 2008, p. 23). La estética uniforme en la presentación de cosas comunes tiene una reescritura en clave romántica en el caso de Robert Smithson en sus Passaic de 1967, donde, en un recorrido por la periferia, destaca numerosas construcciones como grandes ruinas monumentales. Sin embargo, esas edificaciones son vulgares paisajes del extrarradio en plena construcción. La ironía, el camuflaje, la descontextualización o el anonimato son parte de las estrategias a través de las cuales la fotografía se integra en los discursos de lo artístico, alejada de su condición documental primitiva. En este sentido, el propio Dan Graham insiste en alimentar el mito de la pretendida auto-exclusión artística, en el momento que describe su inserción en los cauces artísticos como casual, de la siguiente forma: Me vi envuelto en el sistema del arte de manera accidental, cuando unos amigos míos sugirieron abrir una galería. (…) El otoño siguiente al cierre de la galería, yo mismo empecé a experimentar con obras de arte que podían ser leídas como una reacción contra la experiencia de la galería, pero también como una respuesta a contradicciones que yo entreví en los artistas de galería (Graham, 1997, p. 61). FOTOCINEMA, nº 18 (2019). FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 y Mandel Lo interesante de la nueva dimensión discursiva de la fotografía se centra en la superación de una serie de determinaciones formales de géneros y tradiciones que la definían hasta ese momento. Esta situación traerá al frente un realineamiento del discurso artístico, en su vertiente teórica, concretamente en la crítica cultural (Lippard, 2004, p. 171). A raíz de los planteamientos abiertos por Ruscha o Graham, la obra de Sultan y Mandel integra imágenes personales y narrativas que explican aspectos usuales de la vida americana contemporánea (Silberstein, 2000, p. 20). Asimismo, las imágenes que aglutinan Larry Sultan y Mike Mandel principalmente FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 223 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto corresponden a extractos de archivos de grandes empresas americanas, archivos policiales, institutos de investigación o agencias gubernamentales. Al igual que con el ejemplo de Ruscha, Sultan y Mandel desarrollan un tipo de fotografía destinada a aparecer impresa en libros. La publicación de libros de fotografías iniciada en la década de los años sesenta, según advierte Horacio Fernández, está inserta en la publicación en libros entendidos como catálogos de imágenes fotográficas (Fernández, 1999, p. 87). A pesar de pertenecer a las poéticas de los archivos detectivescos, o a la indiferencia mostrada en ejemplos de autores anteriores, las fotografías de Larry Sultan y Mike Mandel escapan a la tónica del resto de investigaciones fotográficas de este periodo. El concepto de fotoperiodismo utilizado por Jeff Wall es válido para las mencionadas investigaciones, puesto que, su fragmentación, cripticismo y la idea basada en el extrañamiento son comunes en la obra señalada. Así, una característica compartida por este tipo de proyectos fotográficos se sitúa en la autocrítica del arte asociada a la tradición vanguardista, un enfoque de la fotografía rupturista con su tradición inmediata. Esta publicación de imágenes en libros impresos tiene su origen más cercano en la obra de Edward Ruscha. Lo anónimo, lo trivial y lo cotidiano tienen cabida en este tipo de búsqueda reiterativa de lo anodino y lo indiferente. FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 y Mandel La obra de Sultan y Mandel huye de la modulación de las estructuras y arquitecturas urbanas de Dan Graham mostradas en sus Homes For America. Desde estas premisas, las obras de Sultan y Mandel se presentan como una suerte de documentos encontrados de indescifrable discernimiento, como una excepción, en relación a todos los casos anteriormente descritos (Monteiro Schenkel, 2013, p. 335). Se fundamentan en el extrañamiento de las formas que representan, tanto las acciones de las personas que las ejecutan en el momento mismo de su desarrollo, como en los objetos de laboratorio que aparecen. El concepto de extrañamiento llevado al límite se sitúa en esta dialéctica del panorama detectivesco, tan de moda en las prácticas artísticas de la segunda mitad del siglo XX. Las escenas de crímenes, archivos policiales, asesinatos, víctimas, sangre, datos encriptados o causas desconocidas, son las motivaciones de este tipo de imaginario (Biber, 2011, p. 577). La estética de vestigios encontrados puede rastrearse en la obra de Benjamin, “al establecer una asociación entre el paradigma detectivesco y el del nuevo espectador de la FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 224 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto realidad, que reconstruye alegóricamente el relato de los hechos” (Benjamin, 2001, p. 57). Un ejemplo concreto de este tipo de representaciones sucede con dos de las escenas de la serie Evidence de Sultan y Mandel. A la izquierda, se observa un mecanismo de arrastre o cabestrantes, similar a los utilizados por los cuerpos de bomberos. Su parcialidad y descontextualización nos impide ver qué acción se realiza con él. F2. Larry Sultan y Mike Mandel. Evidence, 1977. Disponible en: http://larrysultan.com/gallery/evidence/ F2. Larry Sultan y Mike Mandel. Evidence, 1977. Disponible en: http://larrysultan.com/gallery/evidence/ En la imagen derecha, nos encontramos con una escena que representa la realización de una proyección radiológica del casco de un caballo. Concretamente es “una proyección palmaroproximal-palmarodistal a 45º del hueso sesamoideo distal impar.”2 A pesar de la descripción científica de la imagen, la fragmentación de la misma impide averiguar el resto de información que debería ofrecer la escena, como es la situación, el ejemplar, el entorno o la acción de una forma más documental. 2 Fuente: Facultad de Veterinaria. Departamento de patologia animal. Universidad de Zaragoza. FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 225 traído por el sesgo informacional y descontextualizador que presentan (Monteiro Schenkel, 2013, p. 335). traído por el sesgo informacional y descontextualizador que presentan (Monteiro Schenkel, 2013, p. 335). Las fotografías seleccionadas presentan vestigios de presencia humana en los escenarios fragmentados en los que sólo vemos un lapso de una escena clave para algunos tipos de informes o procesos al que acceder sin ninguna referencia orientativa a nuestra lectura (2013, p. 336) La fragmentación, sumada a la descontextualización, hacen que estas imágenes pierdan su función instrumental, es decir, abandonan su condición de documento, para pasar a relacionarse directamente con lo artístico. Por su parte, Wollen sugiere que este tipo de prácticas, en esta estética, se sitúa en el epicentro del arte conceptual: Esta estetización del documento neutro tiene sus raíces en el arte conceptual desarrollado a su vez a partir del minimalismo. El conceptualismo pone el documento en el centro de la práctica artística contemporánea, desplazando al dibujo y a la pintura de su pedestal (Wollen, 2000, p. 22). Esta estetización del documento neutro tiene sus raíces en el arte conceptual desarrollado a su vez a partir del minimalismo. El conceptualismo pone el documento en el centro de la práctica artística contemporánea, desplazando al dibujo y a la pintura de su pedestal (Wollen, 2000, p. 22). y Mandel A partir del señalamiento anterior en las dos imágenes, la característica común se sitúa en la falta de discernimiento y en la descontextualización de los elementos formales que aparecen en ella, así como en la parcialidad manifiesta de las instantáneas. Las imágenes de Sultan y Mandel responden a ese pesado carácter anónimo y atemorizante, en muchos casos, FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 225 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 3. Conclusiones Los distintos tratamientos del documento por el arte conceptual sitúan, de este modo, en primer plano a la fotografía como miembro de pleno derecho en las prácticas artísticas de la segunda mitad del siglo XX. El arte conceptual desempeñó un papel importante en la evolución de los términos y las condiciones mediante los que la fotografía artística se definía a sí misma y en su relación con las otras artes, como señala Wall (2003) que, en su transformación, se convirtió en una forma moderna institucionalizada que evoluciona explícitamente en una dinámica de autocrítica. En este proceso, y a través de su integración en discursos artísticos, como en el caso de la apropiación y estetización que se hace de ella, la fotografía comienza a desarrollarse más allá de su función referencial-real, hacia una salida en la ficción como la presentada por Sultan y Mandel. Los nuevos significados adoptados por el medio se adaptan en forma de extrañamiento como en el caso descrito, de Imágenes encontradas. Esta colección de fotografías presentadas por Sultan y Mandel se muestran como una excepción y una extraña variedad de visiones cuyo origen, como señala Del Río (2008), resultan tan indescifrable como inquietante. Las imágenes se tornan ajenas a lo cotidiano y FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 226 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto fundamentadas en este caso en el extrañamiento provocado por los objetos científicos o los lugares investigados, así como las acciones de las personas que las fotos registran en el momento en que éstas se producen. fundamentadas en este caso en el extrañamiento provocado por los objetos científicos o los lugares investigados, así como las acciones de las personas que las fotos registran en el momento en que éstas se producen. El planteamiento del proyecto de Sultan y Mandel enlaza con algunos de los caminos explorados por los territorios del arte en la segunda mitad del s. FOTOCINEMA, nº 18 (2019). E-ISSN: 2172-0150 3. Conclusiones E-ISSN: 2172-0150 227 Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto Víctor Murillo Ligorred, Sobre la dimensión discursiva de la fotografía en los sesenta: una genealogía de la vertiente documental del arte de concepto lo mostrado, consiguen reactivar la imagen a partir de una revolución en el visor –o la visión-, esta vez, centrada en la estética de lo cotidiano. Desde esta nueva formulación, la fotografía fuerza la mirada del espectador en la convencionalidad de lo representado, y surgen en ello como emblemas fundacionales de una nueva forma de hacer alejada de la actitud conservadora asumida por la fotografía desde sus inicios. Es a partir de este entendimiento de lo fotográfico, en su realineamiento semántico de las imágenes hacia lo social (Del Río García, 2008: p. 26), donde la fotografía se incorpora a los territorios de lo artístico en los cauces del arte contemporáneo. Dicho imaginario de la década de los sesenta y setenta trasciende de su estética de documento, a través de las estrategias de camuflaje acogidas en el arte de concepto, un tipo de imagen artística tangencial a la cultura de masas, reintegrando la propia representación, el tema y la narración. Una nueva dialéctica que trata de sostenerse simultáneamente en el carácter documental de la imagen foto, su vínculo mítico con una realidad acontecida, así como también la fuga de las imágenes hacia una ficción. En el arte de los años sesenta y setenta se constatan una reintegración de los discursos artísticos de fotografías científicas o descriptivas que liberan nuevos significados y se adaptan a una forma de extrañamiento, como sucede con el caso de las imágenes encontradas de Larry Sultan y Mike Mandel arrojando nuevas lecturas sobre lo icónico en los albores de la postmodernidad. 3. Conclusiones XX, como el paradigma detectivesco o las escenas de los crímenes, señaladas por Jeff Wall en su apreciación del fotoperiodismo como antesala de lo artístico en los sesenta y que traen al frente unas narrativas basadas en una estética emanada del encriptamiento de datos. La fragmentación de las escenas con un sesgo en el material presentado que, a su vez, ahondan en el principio inquietante de las imágenes, precisamente, por el desconocimiento de las causas que las motivaron. Las imágenes archivo, desligadas de su contexto original, pierden esa capacidad de ser comprendidas de forma instrumental, abandonan su función de documento, para pasar a relacionarse con el imaginario del arte. Por tanto, a través de los ejemplos señalados, los proyectos artísticos en los años sesenta y setenta en forma de fotografía se presentan como la posibilidad de ser incorporados a lo artístico desde su condición de nueva imagen con el característico sesgo banal. De hecho, los discursos traspasan la problemática de la cognición, para postular derivas representacionales principalmente en dos campos; en primer lugar, en el de la política y, en segundo lugar, en el de la estetización del documento. La obra de Ruscha, de Graham o Frank exploran esos otros caminos de lo fotográfico inaugurando así un nuevo territorio de la dimensión discursiva de la fotografía desde el amateurismo que los caracteriza. La sombra del reduccionismo, en forma de amateurismo estaba presente de un modo u otro en la experimentación con lo “anestético”, con la apariencia de “no-arte” o la pérdida de lo visual como explica Wall (2003: p. 238). Esta nueva forma de mirar, asentada no en lo insólito, sino en la banalidad y lo anodino, reflejan la preocupación por romper los moldes de un tipo de representación atrapada en la documentalidad de su propio género. A través de estas fotografías donde las imágenes no persiguen una simple documentación sino, más bien, pretenden fraccionar, inquietar, preguntar y cuestionar, así como relacionarse con aspectos concretos de la cultura, a través de la problemática de FOTOCINEMA, nº 18 (2019). Referencias bibliográficas Arnheim, R. (1980). Arte y entropía. Madrid. Ed. Alianza Forma. Augé, M. (2000). Los “No lugares”, espacios del anonimato: una antropología de la modernidad. Barcelona. Ed. Gedisa. Benjamin, W. (2001). Poesía y capitalismo. Iluminaciones II. Madrid. Ed. 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https://openalex.org/W2752373348	https://europepmc.org/articles/pmc5619849?pdf=render	English	null	Start-to-end simulation of single-particle imaging using ultra-short pulses at the European X-ray Free-Electron Laser	IUCrJ	2,017	cc-by	7,721	IUCrJ Carsten Fortmann-Grote,a* Alexey Buzmakov,b Zoltan Jurek,c,d Ne-Te Duane Loh,e,f,g Liubov Samoylova,a Robin Santra,c,d,h Evgeny A. Schneidmiller,i Thomas Tschentscher,a Sergey Yakubov,i Chun Hong Yoon,j Michael V. Yurkov,i Beata Ziaja-Motykac,d,k and Adrian P. Mancusoa Carsten Fortmann-Grote,a* Alexey Buzmakov,b Zoltan Jurek,c,d Ne-Te Duane Loh,e,f,g Liubov Samoylova,a Robin Santra,c,d,h Evgeny A. Schneidmiller,i Thomas Tschentscher,a Sergey Yakubov,i Chun Hong Yoon,j Michael V. Yurkov,i Beata Ziaja-Motykac,d,k and Adrian P. Mancusoa Received 21 March 2017 Accepted 26 June 2017 Received 21 March 2017 Accepted 26 June 2017 aEuropean XFEL GmbH, Holzkoppel 4, 22869 Schenefeld, Germany, bFSRC ‘Crystallography and Photonics’, Russian Academy of Sciences, Moscow, Russian Federation, cCenter for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany, dThe Hamburg Center for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany, eCentre for Bio-Imaging Sciences, National University of Singapore, Singapore, fDepartment of Biological Sciences, National University of Singapore, Singapore, gDepartment of Physics, National University of Singapore, Singapore, hDepartment of Physics, University of Hamburg, Jungiusstrasse 9, 20355 Hamburg, Germany, iDESY, Notkestrasse 85, 22607 Hamburg, Germany, jLinac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park CA 94025, USA, and kInstitute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Krakow, Poland. *Correspondence e-mail: carsten.grote@xfel.eu Keywords: single-particle imaging; X-ray free- electron lasers; simulations; diffraction; scattering. Single-particle imaging with X-ray free-electron lasers (XFELs) has the potential to provide structural information at atomic resolution for non- crystalline biomolecules. This potential exists because ultra-short intense pulses can produce interpretable diffraction data notwithstanding radiation damage. This paper explores the impact of pulse duration on the interpretability of diffraction data using comprehensive and realistic simulations of an imaging experiment at the European X-ray Free-Electron Laser. It is found that the optimal pulse duration for molecules with a few thousand atoms at 5 keV lies between 3 and 9 fs. research letters Start-to-end simulation of single-particle imaging using ultra-short pulses at the European X-ray Free-Electron Laser Keywords: single-particle imaging; X-ray free- electron lasers; simulations; diffraction; scattering. 560 https://doi.org/10.1107/S2052252517009496 1.1. Radiation damage Electronic radiation damage or ionization begins with the very ﬁrst photons hitting the sample, producing K-shell photoelectrons with a kinetic energy of a few hundred to a few thousand electronvolts (Cryan et al., 2010; see also Berrah, 2016). This process is followed by Auger decay (Hau-Riege et al., 2004; Moribayashi & Kai, 2009; Lorenz et al., 2012); Auger lifetimes of the most abundant atoms in biomolecules lie between 4.9 fs (oxygen) and 10.7 fs (carbon) (Ziaja et al., 2015; Hubbell et al., 1994). The immediate effect of ionization is a decrease in the number of coherently scattered photons as the elastic scattering cross section scales with the square of the number of bound electrons, whereas ionized electrons predominantly scatter incoherently, thus contributing to the background signal (Slowik et al., 2014; Gorobtsov et al., 2015). Auger electrons from L or M shells leave the atom with an energy of a few hundred electronvolts, triggering an avalanche of secondary impact ionization (Kai & Moribayashi, 2009a,b) on time scales of roughly 10–100 fs, creating the strong repulsive forces between ions responsible for Coulomb expansion (Hau-Riege et al., 2004; Ziaja et al., 2006). Typical ion velocities in the sample reach of the order 0.1 A˚ fs1, hence already limiting the resolution to ’10 A˚ levels after tens of femtoseconds. The onset of plasma expansion is related to the effect of electrostatic trapping (Hau-Riege et al., 2004), when the positive charge of the ionized molecule is so high that further ionized electrons can no longer escape from the system, and leads to a drastic increase in the impact ionization rate. Electronic radiation damage or ionization begins with the very ﬁrst photons hitting the sample, producing K-shell photoelectrons with a kinetic energy of a few hundred to a few thousand electronvolts (Cryan et al., 2010; see also Berrah, 2016). This process is followed by Auger decay (Hau-Riege et al., 2004; Moribayashi & Kai, 2009; Lorenz et al., 2012); Auger lifetimes of the most abundant atoms in biomolecules lie between 4.9 fs (oxygen) and 10.7 fs (carbon) (Ziaja et al., 2015; Hubbell et al., 1994). The immediate effect of ionization is a decrease in the number of coherently scattered photons as the elastic scattering cross section scales with the square of the number of bound electrons, whereas ionized electrons predominantly scatter incoherently, thus contributing to the background signal (Slowik et al., 2014; Gorobtsov et al., 2015). research letters Electron-density reconstruction of the three-dimensional electron density from experimental SPI data in the soft X-ray regime (Ekeberg et al., 2015; Seibert et al., 2011) has so far achieved resolutions in the regime of a few tens of nano- metres. Diffraction data at a theoretical resolution of 5.6 A˚ were measured (Munke et al., 2016) but not enough patterns were recorded for reconstruction. A comprehensive summary of SPI results from the Linac Coherent Light Source (LCLS) is given by Barty (2016), along with several references to work on imaging of larger particles (e.g. cells) using synchrotrons and two-dimensional imaging. Despite these encouraging results, SPI at a resolution of a few a˚ngstro¨ms is still regarded as a severe challenge (Aquila et al., 2015), in particular with respect to delivering the sample molecules at a high repetition rate (Daurer et al., 2017) and with a narrow size distribution. A discussion of these and other challenges can be found in the literature [e.g. Aquila et al. (2015), Ziaja et al. (2015) and Barty (2016)]. Theoretical treatments of radiation damage fall into two categories: atomistic ﬁrst-principles simulations (Moribayashi, 2010; Son et al., 2011; Lorenz et al., 2012; Gorobtsov et al., 2015; Jurek et al., 2016; Ho & Knight, 2017) describing the sample dynamics on the level of individual particles including the quantum electrodynamics of electrons in intense X-ray ﬁelds, and continuum models operating on distribution func- tions and densities rather than particles. Continuum models (Hau-Riege et al., 2004; Ziaja et al., 2006; Moribayashi, 2008; Quiney & Nugent, 2010; Kai et al., 2013) are numerically less expensive, allowing simulations of large systems on modest computer hardware. However, if kept on the level of single- particle densities, they neglect the correlations between particles. For a treatment of two-particle correlations, see Jurek et al. (2012). Among these challenges, a detailed understanding of the radiation damage incurred by the sample, and of the perfor- mance of the reconstruction algorithms applied to low signal- to-noise diffraction patterns, has recently received increased attention and is also the focus of the present paper. 1.2. Orientation recovery The expand–maximize–compress (EMC) algorithm (Loh & Elser, 2009) is often used in the SPI community, not least thanks to its user-friendly and open-source implementation (Ayyer et al., 2016). EMC is an extension of the expectation– maximization technique described by Dempster et al. (1977). Similar reconstruction algorithms that apply Bayesian infer- ence are used in three-dimensional cryoelectron microscopy as well (Scheres et al., 2007). Generative topographical mapping (GTM; Svensen, 1998) has also been applied to a partial subspace of a full three-dimensional rotation group (Fung et al., 2009). A formal comparison of EMC and GTM can be found in the work by Moths & Ourmazd (2011). 1.1. Radiation damage 1. Introduction Resolving the atomic structure of biologically relevant macromolecules on length scales of a few a˚ngstro¨ms (1010 m) is a key challenge in structural biology. X-ray free-electron lasers (XFELs) are expected to advance this ﬁeld due to their unprecedented levels of X-ray ﬂuence and peak brightness and, simultaneously, their ultra-short pulse duration from a few up to a few tens of femtoseconds. These intense pulses are capable of probing the sample before radiation damage processes signiﬁcantly alter and ultimately destroy it (Neutze et al., 2000) and, due to their extreme intensity, they can compensate for the inherently weak scattering efﬁciency of a single molecule, such that diffraction patterns with sufﬁcient signal and signal-to-noise levels for the reconstruction of three-dimensional structures can be observed. During a single- particle imaging (SPI) experiment, a large number of two- dimensional diffraction patterns from individual particles (e.g. molecules, clusters, or biological specimens like cells or viruses) are recorded. Since the orientation of the sample with respect to the beam and the detector is unknown, the indivi- dual patterns must be oriented and merged into a three- dimensional diffraction volume before the three-dimensional electron-density map is reconstructed via phase retrieval (Fienup, 1982). IUCrJ (2017). 4, 560–568 0 https://doi.org/10.1107/S2052252517009496 1.3. Scope of this paper 1.3. Scope of this paper 2.1. XFEL source and wave propagation to the sample The robustness and ﬁdelity of orientation and phasing algorithms depends on the signal-to-noise level of the measured diffraction patterns. Previous theoretical predic- tions (Son et al., 2011; Gorobtsov et al., 2015) indicate that the resolution of SPI should increase with decreasing FEL pulse duration at a ﬁxed ﬂuence (number of photons per surface area) of the incoming X-ray pulse; see also the discussions in the articles by Aquila et al. (2015) and Ziaja et al. (2015). In this work, we take a closer look at the question of preferential experimental parameters for SPI, taking into account available machine parameters. In particular, the maximum available X-ray pulse energy in an FEL based on self-ampliﬁcation of spontaneous emission (SASE) decreases at shorter pulse durations (Schmu¨ser et al., 2009; Pellegrini et al., 2016), so these properties cannot be varied independently of each other. The XFEL Photon Pulses Database (XPD, https://in.xfel.eu/ xpd/), operated by European XFEL GmbH, provides precomputed pulses at the undulator exit for a large range of accelerator energies, bunch charges, undulator lengths and photon energies at the European XFEL. The database is populated with results from the FAST code (Saldin et al., 1999). Here, we pick 4.96 keV X-ray photons emitted from 12 GeV electrons, the same parameters as used by Yoon et al. (2016). The X-ray pulse durations are 3, 9 and 30 fs, containing approximately 1 1011, 5 1011 and 1 1012 photons per pulse, respectively. We query 40 pulses from the database to sample the shot- to-shot ﬂuctuations of the temporal structure of SASE pulses. We propagate the X-ray laser pulses through the SASE1 beamline and the focusing optics at the SPB/SFX instrument (Mancuso et al., 2013; Bean et al., 2016) using the Fourier optical wave propagation code WPG (Chubar et al., 2002; Samoylova et al., 2016), which yields the intensity and phase distribution as a function of time at the sample position. We investigate the impact of pulse duration on simulated diffraction patterns exploiting comprehensive simulations (Yoon et al., 2016) of an imaging experiment at the European XFEL (Altarelli, 2015) under realistic conditions. Our simu- lations track the X-ray ﬁelds from their generation in the FEL’s undulator structure through the X-ray optical beamline to the sample interaction point. 1.3. Scope of this paper Subsequently, we model the X-ray interaction with the sample and scattering from it, including time-dependent effects and their eventual registra- tion in the detector. Orientation and phasing (Loh & Elser, 2009) of the simulated diffraction patterns are also part of the simulation workﬂow. From the propagated pulse data, we convert the time- dependent X-ray intensity into a photon number, which is then used in the subsequent simulation steps. Other pulse properties, such as the curvature of the wavefront, the pointing stability and the related hit statistics, are neglected, i.e. we assume that each sample molecule is fully exposed to the brightest part of the X-ray pulse. The ﬁne structure of the source spectrum is also neglected as it only becomes important in the vicinity of an absorption edge or a resonance line of one of the sample elements, which is not the case at our photon energy of 5 keV. Yoon et al. (2016) showed that reducing the pulse duration from 30 to 9 fs markedly improves the speckle contrast in diffraction patterns and the consistency of oriented diffraction volumes, and ultimately the agreement of reconstructed electron densities with crystallographic data. A rather small sample molecule (PDB entry 2nip) was used in that study. These results support the theoretical argument in favour of ultra-short pulses of only a few femtoseconds duration being capable of probing the sample before atomic displacement reaches a level that becomes prohibitive for a˚ngstro¨m-level resolution. Here, we study whether we can further improve the signal level and signal-to-noise ratio, and thereby in turn the consistency of oriented diffraction volumes, by reducing the pulse duration to 3 fs, i.e. shorter than the Auger lifetime of typical biomolecule constituents. We use the same study molecule and compare our predictions with the earlier results of Yoon et al. (2016). 1.1. Radiation damage EMC starts from a random initialization of a three-dimen- sional diffraction volume, the reference model, which is then iteratively updated to maximize overlap with the measured (simulated) two-dimensional patterns until the relative change in voxel intensities stays below a given threshold in two subsequent iterations. The use of a reference model has the advantage that EMC’s complexity is OðMÞ, where M is the number of recorded diffraction patterns. Alternative methods classify patterns based on their mutual cross correlation (Huldt et al., 2003; Bortel & Faigel, 2007), giving OðM2Þ complexity. Patterns of the same class are then averaged to amplify the signal-to-noise ratio and a three-dimensional diffraction pattern is assembled using the ‘common-arc’ method, as described by Huldt et al. (2003) and demonstrated by Bortel & Tegze (2011). The latter authors have also developed an orientation scheme suitable for large molecules and noisy patterns (Tegze & Bortel, 2012). A graph-theore- tical manifold-embedding technique is described by Giannakis et al. (2012) and applied by Schwander et al. (2012). Quiney & Nugent (2010) show a way of orienting the measured patterns and directly reconstructing the atomic positions without the need for determining a three-dimensional electron distribu- tion ﬁrst. This method makes use of the fact that the disturbed electron distribution imprints features of a partially coherent waveﬁeld on the scattered radiation, allowing the treatment of Auger electrons from L or M shells leave the atom with an energy of a few hundred electronvolts, triggering an avalanche of secondary impact ionization (Kai & Moribayashi, 2009a,b) on time scales of roughly 10–100 fs, creating the strong repulsive forces between ions responsible for Coulomb expansion (Hau-Riege et al., 2004; Ziaja et al., 2006). Typical ion velocities in the sample reach of the order 0.1 A˚ fs1, hence already limiting the resolution to ’10 A˚ levels after tens of femtoseconds. The onset of plasma expansion is related to the effect of electrostatic trapping (Hau-Riege et al., 2004), when the positive charge of the ionized molecule is so high that further ionized electrons can no longer escape from the system, and leads to a drastic increase in the impact ionization rate. Carsten Fortmann-Grote et al. Simulation of single-particle imaging 561 IUCrJ (2017). 4, 560–568 562 Carsten Fortmann-Grote et al. Simulation of single-particle imaging research letters duration) within this ﬁxed framework. Whether and to what extent our results change if different algorithms are employed is an important open question that will be addressed else- where. electronic radiation damage and orientation recovery within a uniﬁed framework. Multi-tiered iterative phasing (Donatelli et al., 2015) is a rather novel method that combines the orien- tation and phasing steps of coherent diffraction imaging into one algorithm. 2.3. Radiation damage and diffraction 2.3. Radiation damage and diffraction Our study aims to assess the interpretability of diffraction patterns and the potential for reconstruction of the molecular structure at atomic resolution, hence we employ a molecular dynamics (MD) scheme to describe the sample and its inter- action with the X-ray pulse. This provides the required atomistic spatial accuracy. We use the code package XRAYPAC (Centre for Free Electron Laser Science Theory Division, 2016), which combines the MD code XMDYN (Murphy et al., 2014; Jurek et al., 2016) for electron and ion real-space dynamics with a Monte Carlo code modelling inner- shell electronic transitions and subsequent inelastic electron scattering and recombination events. Rates and cross sections are read from the tabulated output of the ab initio electronic structure code XATOM (Son et al., 2011), which is also part of XRAYPAC. Other implementations of this atomistic approach to radiation damage are presented by Moribayashi (2010) and, more recently, by Ho & Knight (2017). XMDYN and XATOM have been successfully used to interpret spec- troscopy experiments (Rudek et al., 2012; Fukuzawa et al., 2013; Murphy et al., 2014; Tachibana et al., 2015). The rate equation approach underlying XATOM has also been applied in investigations of radiation damage in biological samples (Lorenz et al., 2012; Gorobtsov et al., 2015) that completely neglected atomic displacement for pulse durations shorter than 40 fs, citing self-termination of diffraction on these time scales observed in nanocrystallographic diffraction measure- ments by Barty et al. (2011). IðqÞ ¼ dThðÞ d X i I0ðtiÞ Fðq; tiÞ 2þSðq; tiÞ þ NðtiÞ h i t: ð1Þ IðqÞ ¼ dThðÞ d X i I0ðtiÞ Fðq; tiÞ 2þSðq; tiÞ þ NðtiÞ h i t: ð1Þ The wavevector q is determined by the distance of the assumed pixel area detector from the sample and the pixel coordinates in the detector plane, is the solid angle spanned by the respective detector pixel, dTh/d is the differential Thomson cross section, I0(t) is the FEL intensity as a function of time, which we take from the X-ray propagation results, F(q, t) is the bound-electron form factor for coherent scat- tering, S(q, t) and N(q, t) denote the incoherent contributions from bound and free electrons, respectively, ti is the time stamp of the ith snapshot, and t is the time step of the simulation. From each trajectory we calculate 200 diffraction patterns. 2.3. Radiation damage and diffraction Every pattern calculation starts from a random rigid rotation of the sample’s atom coordinates to simulate the erratic a priori unknown and uncontrolled orientation of sample molecules in the X-ray beam. In our simulations, the detector is represented by a square pixel array (80 80 pixels) in a plane perpendicular to the beam axis located at a distance of 13 cm downstream from the sample. The pixel size is 1200 mm. Hence, one pixel of our simulated detector corresponds to a 6 6 pixel array in the AGIP detector (AGIPD) (Allahgholi et al., 2015) planned for the SPB/SFX instrument (Mancuso et al., 2013). These ﬁgures result in a half-period resolution of 3.6 A˚ at the detector edge. The total scattered intensity at each pixel is divided by the central photon energy to yield a photon count n0,j, where j indexes the detector pixel. Poisson noise is added by drawing the detected photon count nj from a Poisson distribution Pn0;jðnjÞ with n0,j P nj njPn0;jðnjÞ. In our simulations, the detector is represented by a square pixel array (80 80 pixels) in a plane perpendicular to the beam axis located at a distance of 13 cm downstream from the sample. The pixel size is 1200 mm. Hence, one pixel of our simulated detector corresponds to a 6 6 pixel array in the AGIP detector (AGIPD) (Allahgholi et al., 2015) planned for the SPB/SFX instrument (Mancuso et al., 2013). These ﬁgures result in a half-period resolution of 3.6 A˚ at the detector edge. It should be noted that there are important differences between serial femtosecond crystallography (SFX) and SPI, which make this assumption questionable. In SFX (Chapman, 2015; Schlichting, 2015) the gating effect applies, i.e. as soon as the crystalline lattice is destroyed, Bragg diffraction, the dominant contribution to the overall signal, is ‘switched off’. Only incoherent scattering remains, enhancing the back- ground, but always at levels which are small compared with the already accumulated Bragg signal. In SPI, there is no lattice to start with and such self-gating does not apply. Furthermore, while electronic damage to crystalline samples occurs on similar time scales as in isolated molecules, atomic displacement is signiﬁcantly reduced due to the conﬁning crystal potential. In short, our results for radiation damage must not be transferred literally to the SFX case. 2.3. Radiation damage and diffraction The total scattered intensity at each pixel is divided by the central photon energy to yield a photon count n0,j, where j indexes the detector pixel. Poisson noise is added by drawing the detected photon count nj from a Poisson distribution Pn0;jðnjÞ with n0,j P nj njPn0;jðnjÞ. research letters counteracting effects. On the one hand, theory predicted (Hau-Riege et al., 2004, 2007; Jurek & Faigel, 2008) and experiments conﬁrmed (Hau-Riege et al., 2010) a tampering effect of the solvent layer, mitigating the effect of radiation damage. On the other hand, the solvent layer increases the background scattering, thus reducing the signal-to-noise ratio. Both effects are size dependent and it can be expected that an optimal solvent layer thickness exists, which mitigates radia- tion damage as much as possible while keeping the diffraction background tolerable. Simulations that investigate this aspect are in progress. For each pulse, we carry out 25 MD simulations, giving a total of 1000 MD runs. The simulation time is ﬁxed to three times the FWHM pulse duration. 100 snapshots of each trajectory, i.e. atom positions and electron-density distribu- tions, are saved during each run. At each time step during the simulation, we calculate the X-ray intensity scattered by the sample. The instantaneous scattering is given by the momentary distributions of electrons and X-ray pulse intensity. We then calculate a diffraction pattern by integrating the instantaneous scattering over the pulse duration and over the solid angle covered by each pixel. For a given electronic conﬁguration, the time-integrated scattered intensity, including coherent (elastic) scattering from bound electrons and incoherent Compton scattering from bound and free electrons, reads 2.3. Radiation damage and diffraction 2.2. The sample Our simulated sample is the two-nitrogenase iron protein (2nip). Diffraction from 2nip for 9 and 30 fs pulse durations at 4.96 keV was simulated by Yoon et al. (2016). We compare these reference data to our new results for diffraction of 3 fs X-ray pulses. All other X-ray pulse parameters (photon energy, active undulator length of 35 m, focusing optics and detector geometry) are the same as used by Yoon et al. (2016). Note that the rather small 2nip (7 nm in diameter) is not a typical candidate for SPI experiments at the European XFEL. It is studied here mainly for the pragmatic reason that simu- lations of much larger particles with our techniques become numerically expensive and a pulse duration scan as presented in this work would not be possible within a reasonable time on our current computing infrastructure. We employ only one method or algorithm for each simulation step and explore the variation in experimental observables (diffraction patterns and their orientation) as a function of experimental parameters (in particular pulse We ignore the fact that the sample is typically embedded in a solvent (see e.g. Wang et al., 2011). The solvent has two 562 Carsten Fortmann-Grote et al. Simulation of single-particle imaging IUCrJ (2017). 4, 560–568 research letters resolution shell q:jqj ¼ q and divided by the number of voxels in the resolution shell Mq. This metric uses the simulated data alone and no a priori information such as the original sample position. Hence it may be applied to experimental data, where the original structure and sample orientation are truly unknown, as opposed to e.g. the misorientation angle (Tegze & Bortel, 2012; Morawiec, 2004), which calculates the angular distance between the recovered orientation and the original sample orientation. An alternative metric not requiring the true orientation is the so-called correlation C factor (Tegze & Bortel, 2016). originated only from different sample orientations, not from noise, each EMC run would yield the same three-dimensional output. Hence, a normalized root-mean-square (r.m.s.) varia- tion of all EMC runs is a suitable ﬁgure of merit to measure the consistency of the oriented three-dimensional data and the likelihood that EMC ﬁnds the true orientation of noisy and shot-to-shot ﬂuctuating individual two-dimensional patterns. The coefﬁcient of variation v as a function of resolution q = \|q\| was deﬁned by Yoon et al. (2016) as originated only from different sample orientations, not from noise, each EMC run would yield the same three-dimensional output. Hence, a normalized root-mean-square (r.m.s.) varia- tion of all EMC runs is a suitable ﬁgure of merit to measure the consistency of the oriented three-dimensional data and the likelihood that EMC ﬁnds the true orientation of noisy and shot-to-shot ﬂuctuating individual two-dimensional patterns. The coefﬁcient of variation v as a function of resolution q = \|q\| was deﬁned by Yoon et al. (2016) as vðqÞ ¼ 1 Mq X q:jqj¼q 1 N P N i¼1 IiðqÞ hIðqÞiN 2 1=2 hIðqÞiN : ð2Þ ð2Þ 3. Results and discussion 2.4. Orientation Lastly, the simulated diffraction patterns are fed into the EMC algorithm to generate a three-dimensional diffraction volume. We calculate multiple such three-dimensional diffraction volumes (typically ﬁve), starting each EMC run from a different random initialization. If the input data had zero noise, i.e. if the differences between diffraction patterns Carsten Fortmann-Grote et al. Simulation of single-particle imaging 563 IUCrJ (2017). 4, 560–568 research letters 3. Results and discussion The inner sum is the mean-square deviation from their average over N orientation runs. The r.m.s., normalized to the average intensity, is then summed over all voxels within a Fig. 1 shows the temporal intensity variation of one repre- sentative X-ray pulse simulation from the output of the X-ray source simulation. Underneath, we show the evolution of the Carsten Fortmann-Grote et al. Simulation of single-particle imaging IUCrJ (2017). 4, 560–568 oral structure of the simulated X-ray pulse, the average number of bound electrons (Zbound, dashed curves) and the average atomic ents (solid curves) in the 2nip sample as a function of time for pulse durations of (a) 3 fs, (b) 9 fs and (c) 30 fs. Figure 1 The temporal structure of the simulated X-ray pulse, the average number of bound electrons (Zbound, dashed curves) and the average atomic displacements (solid curves) in the 2nip sample as a function of time for pulse durations of (a) 3 fs, (b) 9 fs and (c) 30 fs. 564 Carsten Fortmann-Grote et al. Simulation of single-particle imaging 564 Carsten Fortmann-Grote et al. Simulation of single-particle imaging IUCrJ (2017). 4, 560–568 research letters balanced by an increase in scattering efﬁciency due to the lower degree of ionization. balanced by an increase in scattering efﬁciency due to the lower degree of ionization. number of bound electrons (dashed curves) and atomic displacement from initial positions (solid curves) as a function of time. Both quantities are averaged over all atoms of a given species and over all sample trajectories. As expected, the electronic and ionic radiation damage becomes more severe if the pulse duration is increased. At the shortest pulse duration, the ionization stays below a level of 30%, even for the heaviest species S and Fe. The average displacement is below 0.1 A˚ over the entire duration of the pulse, which is negligible compared with the displacement of a few a˚ngstro¨ms in the 9 fs pulse and that of >10 A˚ in the case of the 30 fs pulse. We now turn to the question of how the simulation results inﬂuence the consistency of oriented diffraction data measured by the coefﬁcient of variation [equation (2)]. Fig. 3(a) shows v as a function of the radial detector pixel coordinate (lower x axis) and as a function of the half-period resolution (upper x axis). The error bars represent the r.m.s. deviation over the resolution shell. 3. Results and discussion Red and blue circles represent diffraction data from simulations using 30 and 9 fs pulses, respectively, taken from Yoon et al. (2016), and green circles correspond to the 3 fs simulations. The 30 and 9 fs diffraction data yield nearly identical variations down to resolutions of >10 A˚ (14 pixels). For lower resolutions (larger pixel numbers), the 30 fs curve increases more quickly than the 9 fs curve. This 10 A˚ length scale agrees with the order of average atomic displacement found in the MD simulation of the sample towards the end of the 30 fs pulse (Fig. 1). The shorter 9 fs pulse clearly mitigates ionic damage since the coefﬁcient of variation rises much more slowly as a function of radial pixel coordinate beyond 10 A˚ resolution. These ﬁndings also correspond with the improved electron-density recon- struction from the 9 fs data compared with the 30 fs data, as demonstrated by Yoon et al. (2016). While low-resolution From the propagated pulse data, the radiation damage results and the diffraction patterns, we extract the average number of photons per pulse Nph,pulse (top left in Fig. 2), the number of detected photons per simulated diffraction pattern Nph,det = P6400 j¼1 nj averaged over all simulated patterns (bottom left), the number of bound electrons in the sample Ne,bound at the middle of the pulse averaged over all simulated sample trajectories (top right), and the observed scattering efﬁciency, taken here as the ratio Nph,det/Nph,pulse (bottom right). p p p For a pulse duration of 3 fs, Nph,det is reduced by more than a factor of three compared with both the 9 fs and 30 fs cases. At 9 fs, Nph,det is approximately equal to the 30 fs case because, coincidentally, the lower ﬂuence in the 9 fs pulses is counter- Figure 2 (Top left) The number of photons per pulse incident on the sample (Nph,pulse) as a function of pulse duration. (Bottom left) The number of detected photons per diffraction pattern (Nph,det). (Top right) The square of the average number of bound electrons in the sample molecule (N2 e;bound) in the middle of the pulse. (Bottom right) The scattering efﬁciency Nph,det/Nph,pulse. The decrease in Ne,bound as a consequence of ionization processes results in a reduced scattering efﬁciency with increasing pulse duration. Nevertheless, the total number of detected photons increases, since the longer pulses contain more photons. research letters Figure 3 (a) The coefﬁcient of variation of oriented three-dimensional diffraction volumes for pulse durations of 3, 9 and 30 fs. (b) The coefﬁcient of variation for a pulse duration of 9 fs and re-scaled coefﬁcients for 3 fs. Triangles: every pattern has been multiplied by a constant factor of 3.3 to match the average photon count in the 9 fs patterns. Squares: every 3 fs pattern has been multiplied by an individual factor such that the average and r.m.s. photon counts match the 9 fs data. features (e.g. the size and shape of the molecule) are recov- ered equally well in both cases, ﬁner structures and the sample surface are resolved more markedly in the 9 fs reconstructions. features (e.g. the size and shape of the molecule) are recov- ered equally well in both cases, ﬁner structures and the sample surface are resolved more markedly in the 9 fs reconstructions. This underlines the usefulness of the coefﬁcient of variation as a measure of data quality as it indicates down to which length scales electron-density reconstructions are trustworthy. In our example, v > 0.2 indicates a loss of accuracy at the 10 A˚ length scale. At this point we would like to remind the reader that our analysis applies to the case of SPI, whereas SFX is much less affected by radiation damage due to the self-gating effect (Barty et al., 2011), as discussed above. Fi 3 Analyzing the 3 fs curves in Fig. 3(a), we ﬁnd that v is more than a factor of two larger than in the 9 fs case over the entire range of q values and quickly approaches v = 1, indicating that variations between individual orientation runs are of the same magnitude as the average. Also, the variation in a given resolution shell (vertical error bars) is nearly twice as large as in the 9 fs case. Despite the low ionization and negligible atomic displacement, the signal-to-noise level for the 3 fs diffraction data is insufﬁcient for the orientation algorithm to work consistently and robustly. i y y Fig. 3(b) represents additional analysis. The green triangles mark the variation coefﬁcient of ﬁve EMC runs, starting from the simulated 3 fs diffraction data after multiplying each pixel value by a factor 3.3, such that the resulting average total photon count equals the average for the 9 fs case. 3. Results and discussion Figure 2 (Top left) The number of photons per pulse incident on the sample (Nph,pulse) as a function of pulse duration. (Bottom left) The number of detected photons per diffraction pattern (Nph,det). (Top right) The square of the average number of bound electrons in the sample molecule (N2 e;bound) in the middle of the pulse. (Bottom right) The scattering efﬁciency Nph,det/Nph,pulse. The decrease in Ne,bound as a consequence of ionization processes results in a reduced scattering efﬁciency with increasing pulse duration. Nevertheless, the total number of detected photons increases, since the longer pulses contain more photons. Carsten Fortmann-Grote et al. Simulation of single-particle imaging 565 IUCrJ (2017). 4, 560–568 research letters The green squares represent the orientations obtained after scaling the 3 fs data such that we obtain both the average photon count and the r.m.s. photon count of the 9 fs diffraction data. The resulting coefﬁcient of variation agrees well with the 9 fs data (blue circles) within the error bars, while the green triangles lie systematically above the 9 fs data. This analysis shows that the quality of the 3 fs data cannot be improved by simply increasing the number of simulated diffraction patterns, since this would not reduce the signal-to-noise ratio. These results make it clear that any electron-density reconstructions from our 3 fs data would be meaningless and we did not carry out this last step of the simulation pipeline. Figure 3 (a) The coefﬁcient of variation of oriented three-dimensional diffraction volumes for pulse durations of 3, 9 and 30 fs. (b) The coefﬁcient of variation for a pulse duration of 9 fs and re-scaled coefﬁcients for 3 fs. Triangles: every pattern has been multiplied by a constant factor of 3.3 to match the average photon count in the 9 fs patterns. Squares: every 3 fs pattern has been multiplied by an individual factor such that the average and r.m.s. photon counts match the 9 fs data. 566 Carsten Fortmann-Grote et al. Simulation of single-particle imaging References Allahgholi, A. et al. (2015). J. Instrum. 10, C01023. Altarelli, M. (2015). High Power Laser Sci. Eng. 3, e18. Aquila, A. et al. (2015). Struct. Dyn. 2, 041701. Aquila, A. et al. (2015). Struct. Dyn. 2, 041701. Ayyer, K., Lan, T.-Y., Elser, V. & Loh, N. D. (2016). J. Appl. Cryst. 49, 1320–1335. Barty, A. (2016). Curr. Opin. Struct. Biol. 40, 186–194. 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Another algorithm presented by Tegze & Bortel (2012) overcomes this limitation and also shows better scaling compared with EMC in the dimensionless parameter R = D/d, where D is the diameter of the sample and d is the desired spatial resolution [R5logR versus R6 R8 (Moths & Ourmazd, 2011)]. A comparison of this algorithm with EMC, using noisy diffraction data, is, to the best of our knowledge, outstanding and would make an important contribution to the ﬁeld. Hau-Riege, S. P., Boutet, S., Barty, A., Bajt, S., Bogan, M. J., Frank, M., Andreasson, J., Iwan, B., Seibert, M. 4. Conclusions and outlook 4. Conclusions and outlook In conclusion, our realistic start-to-end simulations indicate that 3 fs pulses contain too few photons to allow consistent orientation of diffraction patterns from our sample protein 2nip. Over the range of resolutions studied here (3–15 A˚ ), the optimal pulse duration for molecules of comparable size is closer to 9 fs than to 3 fs. Although shorter pulses mitigate electronic radiation damage (ionization), longer pulses are preferable as they contain ﬁve times more photons, which leads to much better photon statistics in the detected diffraction patterns and hence allows a more accurate recon- struction of the three-dimensional diffraction data. point. In particular, radiation damage time scales, such as the onset of electrostatic trapping and nano-plasma expansion, differ signiﬁcantly for small and large samples (Hau-Riege et al., 2004). The availability of simulation data for only one small molecular sample does not justify their extrapolation to larger particles, as strong nonlinearities can be expected at such scaling. Simulations for larger particles and various photon energies are planned as the next step towards a simulation database which will also allow the development and tuning of analytical models, e.g. for radiation damage time scales (Hau-Riege et al., 2004), and the inference of the minimum number of detected Our results for the optimal pulse duration for single-particle imaging of a given sample and a given experimental conﬁg- uration are, of course, strictly valid only for molecules of a size comparable with 2nip (< 10 nm). Any conclusion regarding larger, more realistic, samples could only be speculative at this 566 Carsten Fortmann-Grote et al. Simulation of single-particle imaging IUCrJ (2017). 4, 560–568 research letters photons needed for the reconstruction of a molecule of a certain size and at a targeted resolution. Such an expression will help to deﬁne the requirements for experimental para- meters (pulse duration and ﬂuence) for the measurement of unknown structures. 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https://openalex.org/W2950909637	https://europepmc.org/articles/pmc6667404?pdf=render	English	null	Using Plantago major and Plantago lanceolata in environmental pollution research in an urban area of Southern Poland	Environmental science and pollution research international	2,019	cc-by	10,790	Iryna Skrynetska1 & Jagna Karcz2 & Gabriela Barczyk1 & Marta Kandziora-Ciupa1 & Ryszard Ciepał1 & Aleksandra Nadgórska-Socha1 Received: 17 December 2018 /Accepted: 21 May 2019 /Published online: 14 June 2019 # The Author(s) 2019 Abstract The aim of this study was to perform a complex assessment of changes in the elements of an ecosystem that are caused by environmental pollution in industrial and urban biotopes. The study focused on three sites: a park, a road and the site of the metallurgical plant “Pokój” in the city of Ruda Śląska (Southern Poland), which are each under a different level of anthropogenic load. Soil and plant material samples (Plantago major and Plantago lanceolata leaves) were investigated by performing biochemical, ecophysiological and scanning electron microscopy (SEM) analyses. A significant difference was observed in all of the study samples. The content of Pb, Zn and Cd in the soil samples that had been collected at the site of the metallurgical plant exceeded the permitted limits (Cd > 4 mg kg−1, Pb > 100 mg kg−1, Zn > 300 mg kg−1). The content of Fe, Mn, Pb, Cd and Zn in the plant material was much higher in unwashed samples than in washed samples. The concentrations of potentially toxic elements (PTEs) were below the permitted level in the leaves of Plantago lanceolata for Cd (> 5 mg kg−1) and in the leaves of Plantago major for Zn (> 100 mg kg−1). The SEM observations revealed a significant decrease in the stomata pore length (SPL) in the Plantago lanceolata leaves that had been collected at the road site compared with the plants from the park site. The elemental content on the leaf surface was also determined using X-ray microanalysis. The total chlorophyll (Chl) content, ascorbic acid (AA), proline, guaiacol peroxidase (GPX) activity, pH, relative water content (RWC) and air pollution tolerance index (APTI) were evaluated. The APTI for the investigated species ranged from 5.6 to 7.4, which demonstrated that the studied plant species are sensitive to air pollutants. Keywords SEM-EDX . APTI . Potentially toxic metals (PTMs) . Plantago . * Iryna Skrynetska i.skrynetska@gmail.com Environmental Science and Pollution Research (2019) 26:23359–23371 https://doi.org/10.1007/s11356-019-05535-x Environmental Science and Pollution Research (2019) 26:23359–23371 https://doi.org/10.1007/s11356-019-05535-x RESEARCH ARTICLE Abstract Soil pollution Abbreviations AA Ascorbic acid APTI Air pollution tolerance index Chl Total chlorophyll GPX Guaiacol peroxidase RWC Relative water content SEM Scanning electron microscopy SEM-EDX Scanning electron microscopy with energy-dispersive X-ray spectroscopy SPL Stomata pore length Introduction Over the last several decades, the quality of the environ has undergone a significant deterioration, which was prim due to rapid developments in industry as well as urbanis Environmental pollution has become a factor that is resp ble for many negative effects on the health of fauna and as well as on the ecosystem as a whole because of poten toxic metals that do not degrade and accumulate in the ronment, most of which have long-term toxic effects on Responsible editor: Roberto Terzano Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-019-05535-x) contains supplementary material, which is available to authorized users. * Iryna Skrynetska i.skrynetska@gmail.com 1 Department of Ecology, Faculty of Biology and Environmental Protection, University of Silesia, Bankowa 9, 40-007 Katowice, Poland 2 Scanning Electron Microscopy Laboratory, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland Abbreviations AA Ascorbic acid APTI Air pollution tolerance index Chl Total chlorophyll GPX Guaiacol peroxidase RWC Relative water content SEM Scanning electron microscopy SEM-EDX Scanning electron microscopy with energy-dispersive X-ray spectroscopy SPL Stomata pore length Introduction Responsible editor: Roberto Terzano Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-019-05535-x) contains supplementary material, which is available to authorized users. Responsible editor: Roberto Terzano Introduction * Iryna Skrynetska i.skrynetska@gmail.com Over the last several decades, the quality of the environment has undergone a significant deterioration, which was primarily due to rapid developments in industry as well as urbanisation. Environmental pollution has become a factor that is responsi- ble for many negative effects on the health of fauna and flora as well as on the ecosystem as a whole because of potentially toxic metals that do not degrade and accumulate in the envi- ronment, most of which have long-term toxic effects on living 1 Department of Ecology, Faculty of Biology and Environmental Protection, University of Silesia, Bankowa 9, 40-007 Katowice, Poland 23360 Environ Sci Pollut Res (2019) 26:23359–23371 organisms (Kardel et al. 2010; Remon et al. 2013; Muszyńska et al. 2018). However, the effects of this interaction on the function and structure of the elements of an urban ecosystem have not yet been adequately quantified and are poorly understood. possible application in soil phytostabilisation and revegetation in industrial areas that have been contaminated with potential- ly toxic metals (Serbula et al. 2012; Nadgórska-Socha et al. 2013; Romeh et al. 2016). These results may be useful in evaluating the adaptive properties of these plants to harsh environmental conditions as well as their use in ecological risk assessment (Djingova et al. 2004; Przedpełska and Wierzbicka 2007; Słomka et al. 2008). In flora, the epidermis is the first site of interaction with atmospheric pollution because pollutants first pass through the stomata of the epidermal tissues. The stomata, which regulate the flow of gases entering into or escaping out of leaves, are an excellent site to study the interaction between plants and their environment because they are the first to be affected by air pollution, which may cause changes in their morphology (Robinson et al. 1998; Kardel et al. 2010; Uka et al. 2017). There are many different biochemical and physiological mechanisms that help plants adapt to pollutants, and their efficiency can be assessed by a number of parameters such as the total chlorophyll (Chl) content, ascorbic acid (AA) con- tent, pH and relative water content (RWC). All of these index- es make up the so-called air pollution tolerance index (APTI). The value of the APTI defines a plant’s tolerance to pollution because these parameters determine a plant’s adaptation to the environment and thus predetermine the sensitivity or resis- tance of a species to pollution (Lakshmi et al. Study area The investigated areas represented a variety of habitats (green belts, squares, lawns and park) with ruderal and invasive spe- cies such as Robinia pseudoacacia, Solidago canadensis and Reynoutria japonica. Ruderal species were represented by Taraxacum officinale, Achillea millefolium, Bellis perennis, Trifolium repens, Poa annua, Medicago lupulina and others. The study sites were located in the city of Ruda Śląska (Upper Silesian Industrial District, Southern Poland). For the study, three locations were selected: a road (50°15′17.9″ N, 18°51′17.1″ E), a metallurgical plant (50°17′30.5″ N, 18°52′ 25.7″ E) and a park (50°16′28.4″ N, 18°50′12.8″ E) (Fig. 1). The aim of this study was to perform a complex assessment of changes in the elements of an ecosystem that are caused by environmental pollution in industrial and urban biotopes. Two ruderal species, Plantago major and Plantago lanceolata, were selected for this study. The Plantago species has been used as a traditional medicinal plant in many parts of the world for centuries (Abd El-Gawad et al. 2015; Gomes de Andrade et al. 2018). Plantago lanceolata and Plantago major are easy to recognize and are very common in urban environments and in the countryside. Previous studies have indicated that the Plantago major and Plantago lanceolata species contain sig- nificant levels of trace elements (Tinkov et al. 2016; Nadgórska-Socha et al. 2017; Skrynetska et al. 2018). The “Strzelnica” Park is a recreational and leisure area and is considered to be a potentially “clean” area. The road is the intersection of the A4 expressway and the provincial road 925, which has intensive road traffic. The metallurgical plant “Pokój,” where steel products are produced and distributed, is a site with a high level of environmental pollution. Introduction 2008; Prajapati and Tripathi 2008). Additionally, a biochemical as- sessment of variations in metabolites could be helpful in de- fining the tolerance of a species. Proline accumulation is regarded as an indicator of heavy metal stress and enzymatic antioxidant components such as GPX may be used as an in- dicator of environmental stress for an ecosystem (Kandziora- Ciupa et al. 2017; Nadgórska-Socha et al. 2017). The following hypotheses were evaluated: – Metal pollution contributes to changes in the ecophysio- logical and morphological properties of selected species within polluted sites compared with plants from a non- contaminated area. – Plantago species may be useful biological indicators for industrialised urban areas. – Plantago species may be useful biological indicators for industrialised urban areas. Soil and plant material collection Soil samples were taken from the top layer at 0–10 cm depth from five locations at each site. The samples were collected during the vegetation season in late June and early July 2016. The soil and plant material samples were collected in five replicates at each site (i.e. a total of 15 soil samples and 15 plant material samples). The objective of this study was to perform a comparative analysis of selected ecophysiological and biochemical param- eters and to determine the metal concentrations in soils and plants in samples that had been collected from three areas with different levels of the anthropogenic load. The data obtained enabled us to observe any differences in the morphology and physiological parameters, to analyse the air pollution tolerance indexes and to assess the potential use of the tested species as a bioindicator in an urban biotope. The tolerance of these plants to metal toxicity was established in order to determine their Plant materials from herbaceous lawns were selected: greater plantain (Plantago major) and narrow leaf plantain (Plantago lanceolata), which are species of Plantago, family Plantaginaceae. These two ruderal species are common and Environ Sci Pollut Res (2019) 26:23359–23371 23361 Fig. 1 Location of the study sites in Ruda Śląska: the road (expressway A4); the site of the metallurgical plant “Pokój” and “Strzelnica” Park Fig. 1 Location of the study sites in Ruda Śląska: the road (expressway A4); the site of the metallurgical plant “Pokój” and “Strzelnica” Park e site of the metallurgical plant “Pokój” and “Strzelnica” Par working conditions were 5 kVor 15 kVaccelerating voltages, a working distance (WD) ranging from 8 to 25 mm. widespread and are also well known as good biological indi- cators (Kurteva 2009, Nadgórska-Socha et al. 2013, Romeh et al. 2016, Giacomino et al. 2016). The plant material for the biochemical analysis was frozen immediately after collection and kept frozen until the analysis. Energy-dispersive X-ray microanalysis (EDX) with a de- tection limit of 0.1% of weight and beam penetration of 2– 5 μm was used to identify the elemental content on the leaf surface using dry plant material that had not been fixed in GA. The parts of the leaves were mounted on aluminium stubs with double-sided adhesive carbon tape and sputter coated with gold. Soil and plant material collection The specimens were examined using a field emission scanning electron microscope (FESEM) and a Thermo Scientific NORAN System 7 energy-dispersive spectrometer (Thermo Fisher Scientific, Madison, WI, USA). Background and element specific peak spectra were analysed with NSS 3 X-ray Microanalysis software (Thermo Fisher Scientific). SEM mode microanalysis was carried out at a 15-kV acceler- ation and the acquisition time was set to 60 s. Analyses were performed at × 500–× 1100 magnifications on 1–5 points of ten randomly selected pieces of the leaves from all of the investigated sites. Scanning electron microscopy with energy-dispersive X-ray spectroscopy analysis SEM was used to investigate the micromorphology of the leaf surfaces and stomata size. Leaves from plants of about the same age were taken randomly. Small pieces of fresh leaves near the central nerve (0.5 × 1 cm2) were cut from the same area of the leaf lamina, fixed in 3% glutaraldehyde in a 0.1 M sodium phosphate buffer, washed three times with the same buffer and then dehydrated with ethanol. In the next step, the samples were critical-point dried in a Pelco CPD2 apparatus (Ted Pella Inc., Redding, CA, USA) and then mounted on aluminium stubs with double-sided adhesive carbon tape and at lastly sputter coated in a Pelco SC-6 sputter coater (Ted Pella Inc.) with a 20 nm layer of gold in order to improve the electrical conductivity properties of the samples. All spec- imens were imaged using a field emission scanning electron microscope (Hitachi SU8010 FESEM; Hitachi High- Technologies Corporation, Tokyo, Japan), which was equipped with a secondary electron detector (ESD). The Soil analysis Soil pollution, particularly due to potentially toxic metal con- tamination, has been widely investigated by researchers around the world as one of the major environmental problems that can affect plant productivity, the environment and human health (Ross 1994; Alloway 1997; Kabata-Pendias and Pendias 2001; Kandziora-Ciupa et al. 2016). Previous soil metal accumulation researches that have been conducted in the urban areas of Upper Silesia (Miasteczko Śląskie, Chorzów, Piekary Śląskie, Sosnowiec, Dąbrowa Górnicza) have also reported excessive concentrations of Pb, Cd and Zn especially in soil samples that had been collected from areas near metallurgical plants (Nadgórska-Socha et al. 2013, 2016; Kandziora-Ciupa et al. 2013; Dziubanek et al. 2015; Skrynetska et al. 2018). Most of these studies were based on the fractions of the extracted elements. According to Zheljazkov et al. (2008), while the pseudo-total or HNO3 extractable soil metal concentrations are important, the phyto- available forms of specific metals in the soil are the ones to which plant roots are actually exposed. Amoakwah et al. (2013) noted that CaCl2 mobilises both Cd and Zn because of the combined effect of complexation by the chloride anion and cation exchange. Metal content analysis The metal content of the soil was determined as pseudo-total HNO3 extractable fraction as was described in detail by Zheljazkov and Nielsen (1996). Additionally, metals were al- so extracted from the soil samples with 0.01 M CaCl2 (poten- tially available elements) according to Wójcik et al. (2014). 23362 Environ Sci Pollut Res (2019) 26:23359–23371 Statistical analyses The metal content was measured in the filtered extracts using atomic absorption spectroscopy (Thermo Fisher Scientific iCE 3500). All of the statistical calculations were performed using Statistica version 13 (StatSoft Inc., Tulsa, OK, USA). The observations were replicated five times for each parameter. The mean standard error was also calculated. Significant sta- tistical differences were estimated using Tukey’s test. The Pearson coefficient of correlation for assessing estimated pa- rameters was also calculated. Analysis of variance (ANOVA) helped to determine the variables that were significantly dif- ferent among the soil and plant materials. Soil pH was determined using a standard method (Ostrowska et al. 1991) using a 1:2.5 soil to water ratio. Organic matter content (expressed in %) was estimated fol- lowing the method of Ostrowska et al. (1991). The content of trace elements in the plants was measured using atomic absorption spectrometry (Thermo Fisher Scientific iCE 3500). The plant samples were divided into two groups and analysed as “washed” and “unwashed” sam- ples. The “washed” plants were thoroughly washed with dis- tilled water in an ultrasonic bath (ULTRON, Olsztyn, Poland) for 10 min at 20 °C to remove any dust deposits and then rinsed twice with distilled water. The plant samples were dried at 105 °C and then ground in a stainless steel mill; then, 0.25 g of the samples was wet digested in concentrated HNO3 at a maximum of 120 °C and finally diluted to 25 ml with deionised water (Lin et al. 2008). Biochemical analyses Root viability was determined by measuring the GPX activity according to Fang and Kao (2000). Proline accumulation in the leaves was determined using the acid ninhydrin method (Bates et al. 1973). The RWC for the plant samples was de- termined according to Pathak et al. (2011). The pH value of the leaves was determined using a pH meter after homogenising 5 g f.w. of the leaves in 10 ml deionised water (Nadgórska-Socha et al. 2017). The content of total chloro- phyll in the samples was quantitatively determined (Prajapati and Tripathi 2008) in accordance with Arnon (1949). The quantitative determination of ascorbic acid was performed ac- cording to Keller and Schwanger (1977) and as described in detail in Nadgórska-Socha et al. (2016). The calculation of the air pollution tolerance index enables the degree of a plant’s tolerance to environmental pollution to be defined. The APTI was calculated according to Prajapati and Tripathi’s (2008) formula: Taking into consideration both points, in our study, we elected to use both methods of metal extraction. According to the Regulation by the Minister of Environment (2002), the metal concentrations in the soil pseudo-total fraction, particu- larly cadmium, lead and zinc, exceeded the permissible con- centrations at the site of the metallurgical plant site (4 mg kg−1, 100 mg kg−1 and 300 mg kg−1, respectively). The potentially toxic elements are usually extracted to a great- er extent using HNO3 extraction rather than CaCl2 extraction, which was confirmed by our study. In most cases, the poten- tially bioavailable toxic metal content was below 1% of the estimated content of the elements in the soil fraction that had been extracted using HNO3. In the CaCl2 extracted concentra- tions, the highest content of Mn, Zn and Cd was recorded at APTI ¼ A T þ P ð Þ þ R 10 10 where A is the ascorbic acid content (mg g−1 fresh weight); T is the total leaf chlorophyll content (mg g−1 fresh weight); P is the pH of leaf extract; R is the relative water content (%). According to Singh and Rao (1983), plants with APTI < 10 are sensitive; 10 < APTI < 16 are medium sensitive and APTI > 17 are resistant to air pollution. Biochemical analyses where A is the ascorbic acid content (mg g−1 fresh weight); T is the total leaf chlorophyll content (mg g−1 fresh weight); P is the pH of leaf extract; R is the relative water content (%). According to Singh and Rao (1983), plants with APTI < 10 are sensitive; 10 < APTI < 16 are medium sensitive and APTI > 17 are resistant to air pollution. Extra material about the methodology that was used is in- cluded in the supplementary material (Online Resource 1). Environ Sci Pollut Res (2019) 26:23359–23371 23363 Table 1 Analysis of the soil samples. Stand Extraction Mn (mg kg−1) Fe (mg kg−1) Pb (mg kg−1) Cd (mg kg−1) Zn (mg kg−1) Organic matter (%) pH Road HNO3 236 ± 68a 6198 ± 1124a 34.7 ± 2.4a 0.7 ± 0.1a 133 ± 5a 6.0 ± 0.1a 7.0 ± 0.3b CaCl2 0.8 ± 0.2a 0.3 ± 0.1a 0.5 ± 0.1a 0.1 ± 0.0a 0.1 ± 0.0a Park HNO3 820 ± 41b 8765 ± 342bc 51.2 ± 6.5a 2.1 ± 0.1b 237 ± 17a 8.1 ± 0.1b 5.5 ± 0.1a CaCl2 27 ± 4ab 0.4 ± 0.1a 0.5 ± 0.1a 0.5 ± 0.0b 15 ± 1b Metallurgical plant HNO3 596 ± 40a 42,320 ± 1678c 693 ± 63b 7.2 ± 0.6c 2222 ± 228b 13.0 ± 0.1bc 7.2 ± 0.6c CaCl2 0.9 ± 0.1a 0.4 ± 0.1a 0.6 ± 0.1a 0.1 ± 0.0a 0.5 ± 0.1a Data is expressed as the mean ± SD. The different letters denote significant differences between specific metal concentrations in the fraction that had been extracted with HNO3 and CaCl2, organic matter content, and pH (p < 0.05) Table 1 Analysis of the soil samples. Data is expressed as the mean ± SD. The different letters denote significant differences between specific metal concentrations in the fraction that had been extracted with HNO3 and CaCl2, organic matter content, and pH (p < 0.05) serrations. In both of the investigated species, the abaxial surface of the leaves is lighter than the adaxial surface. No epicuticular waxes were present on the surfaces of the leaves. Fine deposits with irregular shapes and of different sizes were seen on the surfaces of the leaves in a polluted environment (the area near the road and the site of the metallurgical plant) (Fig. 2b, c, e, and f). At the road, the stomata were mostly closed and blocked by dust (Fig. Biochemical analyses 2b, e). Single trichomes were rarely observed on surfaces of the leaves from all of the investigated sites (Fig. 2b, c, and f). the park, which may be connected with low pH. By contrast, the CaCl2 extracted fraction had a comparable level of lead with an average of 0.5 mg kg−1 and iron content with an average 0.4 mg kg−1 for all of the investigated sites (Table 1). Our results are similar to a study in the nearby Miasteczko Śląskie, Poland (Nadgórska-Socha et al. 2016). According to Meers et al. (2007), the 0.01 M CaCl2 extraction procedure proved to be the most versatile because it provided a good indication of phytoavailability. The results that were obtained from the investigated loca- tions provide clear information about the impact of pollution on a natural environment that is under pressure from industrialisation and urbanisation. Soils in a city are characterised by a high level of acidity and show a high level of mechanical damage as a result of human activity. Despite this, in our study, the pH of the surface soils at the road and the site of the metallurgical plant were nearly neutral, thus confirming the efficiency of the revitalisation programmes that began in 2015 (The Local Revitalisation Programme of the City of Ruda Śląska until 2030 (2015)). The study of the selected sites showed that the average level of organic matter was 9%. The lowest content was found at the road (Table 1). Amphistomatous leaves and stomata occurred on both sides of the leaves in all of the Plantago lanceolata and Plantago major plants that were observed. The study of the micromorphology and anatomy of the Plantago lanceolata leaves using SEM revealed differences in the SPL in the area that has heavy traffic and near the site of the metal- lurgical plant compared with the park. The highest SPL values were found in the Plantago lanceolata (24.04 ± 1.26 μm) leaves at the park. Despite the fact that the SPL values in the leaves of Plantago major were much lower at the park (16.5 ± 0.75 μm), the lowest values for Plantago lanceolata (13.58 ± 0.95 μm) and for Plantago major (14.53 ± 0.65 μm) were recorded at the road. At the site of the metallurgical plant, the average SPL was 16.4 ± 0.91 μm and 18.23 ± 0.6 μm for Plantago lanceolata and Plantago major, respectively. Biochemical analyses The leaves of Plantago major had a comparable SPL at all of the investigated sites. A strong positive correlation was observed between the SPL and RWC, total chlorophyll content and APTI (r2 = 0.7, r2 = 0.55 and r2 = 0.7, respectively) and a negative correlation was observed between the SPL and ascorbic acid and proline content (r2 = −0.58 and r2 = −0.81, re- spectively). No correlation was observed between the SPL and metal content in either the washed or unwashed plant samples. SEM observation Accumulation of particles on surface of leaves depends on physico-chemical nature of the particulates and the charac- teristics of the contact surface (Bussotti et al. 1995; Liang et al. 2017). The interaction between plants and the atmo- sphere occurs mainly via the stomata and therefore can be considered to be an air quality indicator. A study of the stomatal characteristics is an inexpensive and easy way to obtain relevant results (Kardel et al. 2010). A preliminary examination of the leaves was performed using light microscopy. Plantago major leaves have a blunt apex, 3–9 nerves, are sometimes slightly serrated, a naked or slightly hairy surface and a round shape. The leaves of Plantago lanceolata have a lanceolata or elliptic shape. Its leaf blade is usually full and rarely has a few Wagoner (1975) reported no differences in the size of the stomata between polluted and unpolluted sites. Alves et al. (2008) described that an increase in stomatal density together with a decrease in stomatal size leads to an opti- mal adjustment for the control of gas exchange and the Environ Sci Pollut Res (2019) 26:23359–23371 23364 Fig. 2 Representative SEM images of the adaxial surfaces of the leaves. a Plantago lanceolata at the park. b Plantago lanceolata at the road. c Plantago lanceolata at the site of the metallurgical plant. d Plantago major at the park. e Plantago major at the road. f Plantago major at the site of the metallurgical plant major at the park. e Plantago major at the road. f Plantago major at the site of the metallurgical plant Fig. 2 Representative SEM images of the adaxial surfaces of the leaves. a Plantago lanceolata at the park. b Plantago lanceolata at the road. c Plantago lanceolata at the site of the metallurgical plant. d Plantago Almost the same elemental content was recorded on the surfaces in Bignoniaceae family leaves that had been collected from different areas of the Pune District, India (Kedar et al. 2018). In research conducted by Weerakkody et al. (2018) near a busy road, the amounts of C and O, in addition to Fe and Cl, were considerably larger compared with the other elements in PM10, and Ca, K, Si, Mg and S were present in particles of various sizes distributed on the leaves of all inves- tigated species. According to Weerakkody et al. SEM observation (2018), a high content of C and O can also indicate the presence of carcino- genic polycyclic aromatic hydrocarbons, primarily from fuel exhausts and tyre wear. Trace amounts of Ca, Ba, Mn, K, Mg and Zn can also be present in vehicle exhausts bound to or- ganic components (Lin et al. 2005; Sharma et al. 2013). In addition to the dust that originates from road traffic, PM10 containing Al, Ca, Na, Si, Cl, F and N can originate from soil dust (Maher et al. 2013; Weerakkody et al. 2018). entrance of pollutants through the stomata. Moreover, Kardel et al. (2010) noticed a decrease in both the adaxial and abaxial stomata sizes in the leaves of Plantago lanceolata that acts as a mechanism for adapting to pollu- tion stress in unsuitable habitats. The formation of smaller stomata in the leaf epidermis of trees was also recorded in Lublin, Poland (Chwil et al. 2015). X-ray microanalysis Quantitative EDX analysis only provided information on the distribution of the elements and was not sensitive in depicting low concentrations of the elements (below the detection limit (> 0.1% weight)). We also analysed the elemental composi- tion of the particles on the adaxial leaf surfaces. X-ray micro- analysis revealed the presence of Si, Fe, S, Na, Ca, Mg, Cl, O, K and Al over the entire leaf surface sections that were exam- ined. The results are presented as the averages of the spectra that were obtained at the study sites (Fig. 3). The gold (Au) signals can be considered to have originated from the sputter coating. Analysis of the metal content in the plant material In the study area, the highest concentrations of Zn were found in the unwashed samples at the site of the metallurgical plant site for both of the species that were studied. The average was 61 mg kg−1 for Plantago major and 43 mg kg−1 for Plantago lanceolata in the washed samples, and the average was 106 mg kg−1 for Plantago major and 88 mg kg−1 for Plantago lanceolata in the unwashed samples (Table 2). The iron content in the study area ranged from 51 to 391 mg kg−1 for the washed samples and from 308 to 2830 mg kg−1 for the unwashed samples. The highest concen- trations were observed in both study species at the site of the metallurgical plant. The Pb concentration was a few times greater in the unwashed than in the washed plants. The highest manganese concentration was recorded in the leaves of Plantago major in the unwashed samples (792 mg kg−1) at the site of the metallurgical plant, which is eight times higher than the concentration in the washed samples (Table 2). Potentially toxic concentrations of Cd (> 5 mg kg−1) were found in all of the samples of Plantago lanceolata leaves from Weerakkody et al. 2018). In the washed plant material, the highest accumulation of Mn, Fe and Pb was recorded in the leaves of Plantago lanceolata at the site of the metallurgical plant (Table 2). Weerakkody et al. 2018). In the washed plant material, the highest accumulation of Mn, Fe and Pb was recorded in the leaves of Plantago lanceolata at the site of the metallurgical plant (Table 2). The iron content in the study area ranged from 51 to 391 mg kg−1 for the washed samples and from 308 to 2830 mg kg−1 for the unwashed samples. The highest concen- trations were observed in both study species at the site of the metallurgical plant. The Pb concentration was a few times greater in the unwashed than in the washed plants. The highest manganese concentration was recorded in the leaves of Plantago major in the unwashed samples (792 mg kg−1) at the site of the metallurgical plant, which is eight times higher than the concentration in the washed samples (Table 2). In the study area, the highest concentrations of Zn were found in the unwashed samples at the site of the metallurgical plant site for both of the species that were studied. Analysis of the metal content in the plant material According to other researchers, the foliar metal uptake is mainly due to the soil–root pathway in urban and industrial environments (Schreck et al. 2012; Dao et al. 2014; Kandziora-Ciupa et al. 2017). The concentrations of the ele- ments (Fe, Mn, Pb, Cd, Zn) were investigated in both washed and unwashed leaves of Plantago major and Plantago lanceolata. Higher metal concentrations were found in the unwashed samples, which was predictable as the dust on the surfaces of leaves can also contain metals (Maher et al. 2013; The element content on the adaxial leaf surfaces from the park was mainly represented by O, C, K, Cl, Ca and Mg (Fig. 3a, b), while at the road and the site of the metallurgical plant, additional elements were present (Na, Al, Si and Fe) (Fig. 3c– f). The highest peaks of Si and Al were recorded at the road, and a higher Fe content was recorded at the site of the metal- lurgical plant, along with the presence of Mn and fungal spores (not shown). Environ Sci Pollut Res (2019) 26:23359–23371 23365 23365 hed plant material, the Pb was recorded in the site of the metallurgical centrations of Zn were site of the metallurgical The iron content in the study area ranged from 51 to 391 mg kg−1 for the washed samples and from 308 to 2830 mg kg−1 for the unwashed samples. The highest concen- trations were observed in both study species at the site of the metallurgical plant. The Pb concentration was a few times greater in the unwashed than in the washed plants. The highest Fig. 3 Selected SEM-EDX images of Plantago leaf samples with the chemical compositions of the most frequently identified elements on the adaxial surfaces. a, b The park. c, d The road. e, f The site of the metallurgical plant Fig. 3 Selected SEM-EDX images of Plantago leaf samples with the chemical compositions of the most frequently identified elements on the adaxial surfaces. a, b The park. c, d The road. e, f The site of the metallurgical plant Weerakkody et al. 2018). In the washed plant material, the highest accumulation of Mn, Fe and Pb was recorded in the leaves of Plantago lanceolata at the site of the metallurgical plant (Table 2). Analysis of the metal content in the plant material The different letters denote significant differences between the metal concentra washed and unwashed samples (p < 0.05). ascorbic acid, relative water content and leaf pH were deter- mined. The GPX activity was also analysed (Table 3). the entire study area, according to the limits reported by Kabata-Pendias and Pendias (2001). Moreover, the permissi- ble Cd content was exceeded in almost all of the unwashed samples, except for the leaves of Plantago major at the road (Table 2). Cd accumulation in edible plants has been found at significantly lower concentrations, i.e. 0.8 to 0.1 mg kg−1 (Dziubanek et al. 2015) compared with our results. A field study conducted by Nadgórska-Socha et al. (2017) also re- ported a lower metal content in Taraxacum officinale, Plantago lanceolata, Betula pendula and Robinia pseudoacacia leaves. Stafford et al. (2016) noted that the Cd accumulation in Plantago lanceolata ranged from 0.44 to 0.89 mg kg−1. In our study, the highest concentration of Zn in the washed samples, which exceeded the permissible con- centration 100 mg kg−1 (Kabata-Pendias and Pendias 2001), was found in the leaves of Plantago major at the park site. By contrast, the study conducted by Kurteva (2009) recorded a higher Zn accumulation in the leaves of Plantago lanceolata. Our investigation of the accumulation of potentially toxic metals found much higher metal concentrations in the soil and leaves of Plantago major compared with the study in Cluj-Napoca, Romania, of Levei et al. (2018). Proline accumulation is considered to be a common phys- iological response of many plants to environmental stress fac- tors (Verbruggen and Hermans 2008; Tantrey and Agnihotri 2010). Moreover, researchers have found a significant amount of proline in the reproductive parts of different plant species, which raises the possibility that the accumulation of this ami- no acid may also occur in non-stressed physiological condi- tions (Mattioli et al. 2009). Numerous studies have also noted a higher content of proline in samples from contaminated areas compared with potentially clean sites (Tantrey and Agnihotri 2010; Kumar et al. 2010; Kandziora-Ciupa et al. 2016; Kandziora-Ciupa et al. 2017). In our investigation, the highest proline content was recorded at the road site for both of the study species (Table 3). The average proline contents for the Plantago major and Plantago lanceolata leaves were 7.8 μmol g−1 and 8.5 μmol g−1 f.w., respectively (Table 3). Analysis of the metal content in the plant material An increase in the proline level during environmental contamina- tion was also found in Philadelphus coronarius leaves by Kafel et al. (2010) and confirmed in Taraxacum officinale, Plantago lanceolata, Betula pendula and Robinia pseudoacacia leaves because of urban environmental traffic contamination by Nadgórska-Socha et al. (2017). In a field study near the site of a smelter, a higher proline content was also recorded in the leaves of Vaccinium murtillus (Kandziora- Ciupa et al. 2017). After averaging the data that was obtained in the measure- ments, the concentrations of the elements in the washed sam- ples can be ranked in the following descending order for Plantago lanceolata: Fe > Mn > Zn > Cd > Pb and for Plantago major: Fe > Zn > Mn > Pb > Cd as was also record- ed for Plantago major in Sosnowiec (Skrynetska et al. 2018). The order of the concentrations in the unwashed samples for both Plantago species was the same: Fe > Mn > Zn > Pb > Cd. This fact supports the crucial point that the plant samples that are used in biomonitoring studies must be washed. GPX activity is significant for plant growth and develop- ment. The activity of antioxidant enzymes changes under bi- otic and abiotic stress conditions and can be used as a potential indicator of metal toxicity and other stress factors (Verma and Dubey 2003; Doğanlar and Atmaca 2011; Kandziora-Ciupa et al. 2017). According to the obtained results, a higher level of GPX activity was recorded in the leaves of Plantago major at the site of the metallurgical plant (1254 tetra-guaiacol g−1 f.w.), while the lowest was recorded in the Plantago lanceolata leaves (348 tetra-guaiacol g−1 f.w.) at the road (Table 3). In our study, a lower GPX activity was recorded Analysis of the metal content in the plant material The average was 61 mg kg−1 for Plantago major and 43 mg kg−1 for Plantago lanceolata in the washed samples, and the average was 106 mg kg−1 for Plantago major and 88 mg kg−1 for Plantago lanceolata in the unwashed samples (Table 2). Potentially toxic concentrations of Cd (> 5 mg kg−1) were found in all of the samples of Plantago lanceolata leaves from Environ Sci Pollut Res (2019) 26:23359–23371 23366 Table 2 Analysis of the metal content in Plantago lanceolata (Pl) and Plantago major (Pm) Mn (mg kg−1) Fe (mg kg−1) Pb (mg kg−1) Cd (mg kg−1) Zn (mg kg−1) Washed Unwashed Washed Unwashed Washed Unwashed Washed Unwashed Washed Unwashed Road Pl 14 ± 1a 41 ± 4a 76 ± 3a 408 ± 24a 0.4 ± 0.1a 4.0 ± 0.2a 7.9 ± 0.2de 10.0 ± 0.3bc 16.1 ± 3.2a 27.6 ± 2.9a Pm 20 ± 1a 25 ± 3a 90 ± 3a 308 ± 34a 0.9 ± 0.1d 4.7 ± 0.2a 0.1 ± 0.0a 0.5 ± 0.0a 25.3 ± 2.1ab 33 ± 3a Park Pl 18 ± 2a 46 ± 2a 51 ± 4a 443 ± 9a 0.4 ± 0.0abc 5.1 ± 0.1a 8.4 ± 0.1f 10.4 ± 0.3bc 38.6 ± 3.8b 70.8 ± 8.9a Pm 61 ± 3b 84 ± 4a 92 ± 8a 410 ± 35a 1.1 ± 0.0c 3.3 ± 0.1a 1.6 ± 0.1c 12.2 ± 0.5c 130 ± 6d 138 ± 9a Metallurgical plant Pl 133 ± 6d 307 ± 31b 391 ± 46b 1667 ± 110b 9.0 ± 0.3e 26 ± 3b 8.0 ± 0.1e 9.9 ± 0.4bc 73.8 ± 5.2c 165 ± 2b Pm 93 ± 8c 792 ± 4c 314 ± 17b 2830 ± 139c 0.6 ± 0.0b 27 ± 1b 0.8 ± 0.1b 10.2 ± 0.6c 27.8 ± 1.3ab 148 ± 7a Data are expressed as the mean ± SD. The different letters denote significant differences between the metal concentrations in the various plants in the washed and unwashed samples (p < 0.05). Table 2 Analysis of the metal content in Plantago lanceolata (Pl) and Plantago major (Pm) Data are expressed as the mean ± SD. The different letters denote significant differences between the metal concentrations in the various plants in the washed and unwashed samples (p < 0.05). Data are expressed as the mean ± SD. Analysis of the biochemical parameters Previous field studies have recorded a higher total chlorophyll content in the leaves of Plantago major and Plantago lanceolata in Sosnowiec, Poland (Skrynetska et al. 2018) and in the leaves of Plantago lanceolata in Dąbrowa Górnicza, Poland (Nadgórska-Socha et al. 2017). The content of Chl can be affected by high temperature, drought, salt stress, light inten- sity, gaseous pollutants and potentially toxic metal contami- nation (Pandey et al. 2015; Zhang et al. 2016). Relative water content (RWC) is the level of water that is required in plants to maintain a physiological balance (Rai and Panda 2014). According to Krishnaveni (2013), RWC is one of ecophysiological indicators of environmental stress in plants. In our study, the average relative water content in the leaves of Plantago major was nearly 64% and was nearly 67% for Plantago lanceolata, which confirms that the selected plants are resistant to water stress. The lowest values for both species were observed at the road (Table 3). Another important indicator of physiological condition of a plant is the content of ascorbic acid (AA), which is a strong reducing agent that activates many defence mechanisms in plants, whereby increased ascorbic acid content enhances pol- lution tolerance (Pandey et al. 2015; Zhang et al. 2016; Nadgórska-Socha et al. 2017). Ascorbic acid is located mainly in the chloroplast and plays an important role in the synthesis of the cell walls, cell division and the processes that are asso- ciated with detoxification (Ogunkunle et al. 2015). In our study, the average AA content in the leaves of Plantago major was 0.46 mg g−1 f.w., while for Plantago lanceolata, it was 0.37 mg g−1 f.w. The lowest AA content was observed in the leaves of Plantago lanceolate at the park (Table 3). A much lower AA content was found in the leaves of Plantago major in a field study in Sosnowiec, Poland (Skrynetska et al. 2018). As was reported by Tripathi and Gautam (2007), an increase in the AA content in all plant species may be due to the in- creased rate of the production of reactive oxygen species. In a field study conducted by Nadgórska-Socha et al. (2016), a decreasing tendency was found in the leaves of R. pseudoacacia, and an increase in the AA content was found in the leaves of M. album at contaminated sites. Analysis of the biochemical parameters To estimate the state of the environment, in addition to the above-mentioned results, the total chlorophyll, proline, Environ Sci Pollut Res (2019) 26:23359–23371 23367 Table 3 Analysis of the biochemical parameters of Plantago lanceolata (Pl) and Plantago major (Pm) GPX (μg g−1 f.w.) Proline (μmol g−1 f.w.) RWC (%) Chl (mg g−1 f.w.) pH AA (mg g−1 f.w.) APTI Road Pl 348 ± 4a 9.8 ± 0.5b 60 ± 3a 0.13 ± 0.01ab 4.5 ± 0.1a 0.48 ± 0.02a 6.2 ± 0.1b Pm 357 ± 4a 11.2 ± 2.2bc 54 ± 4a 0.09 ± 0.01a 5.0 ± 0.2 b 0.40 ± 0.08a 5.6 ± 0.6a Park Pl 572 ± 6a 6.9 ± 0.4ab 72 ± 4a 0.17 ± 0.02b 5.2 ± 0.1 bc 0.22 ± 0.04a 7.4 ± 0.5b Pm 434 ± 5a 8.3 ± 1.4b 69 ± 3a 0.16 ± 0.01b 5.6 ± 0.1c 0.51 ± 0.07a 7.2 ± 0.4b Metallurgical plant Pl 581 ± 6a 8.8 ± 0.2b 68 ± 3a 0.09 ± 0.01a 5.3 ± 0.1c 0.42 ± 0.04a 7.0 ± 0.3b Pm 1254 ± 63b 3.8 ± 0.6a 70 ± 2a 0.18 ± 0.01bc 5.0 ± 0.1bc 0.46 ± 0.08a 7.2 ± 0.2b Data are expressed as the mean ± SD. The different letters denote significant differences between the contents of AA, Chl, RWC, pH, GPX, and proline (p < 0.05) Table 3 Analysis of the biochemical parameters of Plantago lanceolata (Pl) and Plantago major (Pm) Data are expressed as the mean ± SD. The different letters denote significant differences between the contents of AA, Chl, RWC, pH, GPX, and proline (p < 0.05) at the road and was positively correlated with the Fe and Mn content in the washed samples and with Pb, Zn, Fe and Mg in the unwashed samples. A similar dependence was also found in studies that were conducted by Kandziora-Ciupa et al. (2013, 2017). Many authors have reported increased GPX activity in response to elevated potentially toxic metal concen- trations (Verma and Dubey 2003; Kafel et al. 2010; Doğanlar and Atmaca 2011; Nadgórska-Socha et al. 2013; Marchand et al. 2016). Ogunkunle et al. 2015; Nadgórska-Socha et al. 2017). We observed comparable results for Plantago major and Plantago lanceolata. The average contents in the leaves of Plantago major and Plantago lanceolata were 0.14 mg g−1 f.w. and 0.13 mg g−1 f.w., respectively (Table 3). Conclusions The examinations of the leaves of Plantago major and Plantago lanceolata showed anatomical, biochemical and ecophysiological changes in the plant samples that had been collected from an industrialized urban area. Strong correla- tions were found between the SPL and the ecophysiological parameters (RWC, APTI, Chl, AA, proline content). The met- al content also correlated with the biochemical and ecophys- iological indexes to different degrees depending on the specif- ic element. The difference in metal concentrations between the washed and unwashed plant material is an essential distinction. The statistical analysis demonstrated the necessity of washing the plant material that is used in metal bioaccumulation studies because this factor affects the experimental accuracy. A plant species with a higher APTI can be used in green belts and should be given priority for replantation in urban and industrial areas in order to reduce the effects of air pollution (Sisodia and Dutta 2016; Achakzai et al. 2017; Bharti et al. 2018). A plant species with a lower APTI can be recommend- ed as a bioindicator and for environmental monitoring (Nadgórska-Socha et al. 2016, 2017; Bharti et al. 2018). The results that were obtained indicate that Plantago major and Plantago lanceolata species can be classified as being sensi- tive to air pollution and can be recommended for bioindicative research in urban and industrial areas. According to the SEM-EDX results, a higher content of Al, Fe, Si and Mn was observed on the adaxial leaf surfaces at the road and metallurgical plant sites. During such an analysis, researchers should take into account the detection limit and depth of the beam penetration because tracking the trace ele- ment content in particles >2 μm is quite difficult. Therefore, for bioaccumulation studies, SEM-EDX analysis with an ad- ditional analysis of the metal concentration (AAS, ICP etc.) is recommended. A clear correlation was found between the pH value and the content of Mn and Zn (r2 = 0.5 and r2 = 0.87, respectively) and between the Chl and Pb content (r2 = −0.85) in the washed plant material. Much stronger correlations were ob- served in the unwashed material. A correlation was found between the dehydrogenase activity and the content of Pb, Fe, Mn and Zn (r2 = 0.76; r2 = 0.92; r2 = 0.96 and r2 = 0.57, respectively). Analysis of the biochemical parameters Some studies have also reported a high concentration of AA at industrial sites (Agbaire and Esiefarienrhe 2009; Meerabai et al. 2012; Ogunkunle et al. 2015). The leaf pH, which is a common physiological parameter, is also suggested to be an indicator of plant stress (Krishnaveni et al. 2013; Husson et al. 2018). The pH of the extracts from the leaves in the study area ranged from 4.5 to 5.6. The lowest pH was recorded in both study species at the road. The aver- age pH for the Plantago major leaves was 5.21, and it was 4.98 for Plantago lanceolata (Table 3). The values of the leaf pH that were obtained were lower for both species than the results of a field study in Sosnowiec (Poland) (Skrynetska et al. 2018). Krishnaveni et al. (2013) also recorded a decrease in the leaf pH values at polluted sites. By contrast, a laboratory study conducted by Cornelissen et al. (2011) reported that leaf pH is largely a species-specific trait, and therefore, the inves- tigated species could maintain a leaf pH independently from the soil environment. Studies conducted by Sharma et al. (2013), Zhang et al. (2016) and Bharti et al. (2018) emphasised that a lower leaf pH is connected with the pres- ence of SOx and NOx in the air. This fact suggested us to conclude that the leaf pH depends directly on air quality. Studies on chlorophyll content are considered to be rele- vant as its level is connected with tolerance in contaminated environments (Pathak et al. 2011; Rai and Panda 2014; Calculating the air pollution tolerance index (APTI) en- ables the tolerance of plants to air pollution to be determined 23368 Environ Sci Pollut Res (2019) 26:23359–23371 and the biochemical parameters that are responsible for resis- tance to environmental stress factors to be found. In our study, Plantago major and Plantago lanceolata had a narrow range of tolerance in the APTI index (5.6 to 7.4). It was found that the relative APTI average of Plantago major was 6.7 while it was 6.8 for Plantago lanceolata, thus indicating that both are sensitive to air pollution. The road site had the lowest APTI values for both of the study species (Table 3). According to the classification of Singh and Rao (1983), the investigated plants are species that are sensitive to air pollution. Analysis of the biochemical parameters Low values of APTI were also noted in both contaminated and convention- ally clean sites in Sosnowiec, Poland (Skrynetska et al. 2018). In Dąbrowa Górnicza, Poland, the APTI of Plantago lanceolata was higher (8.43–14.57), especially at non- contaminated sites compared with contaminated sites (Nadgórska-Socha et al. 2017). Another study in Southern Poland (Miasteczko Śląskie, Katowice, Jaworzno) using Robinia pseudoacacia and Melandrium album at potentially toxic metal-contaminated sites recorded a mean APTI value for all of the investigated sites at 9.4 for R. pseudoacacia and 8.7 for M. album (Nadgórska-Socha et al. 2016). Zhang et al. (2016) identified species that are tolerant to air pollution (Magnolia denudata, Diospyros kaki, Ailanthus altissima, Fraxinus chinensis and Rosa chinensis), which had been col- lected from two heavy traffic roadside sites and one suburban site in Beijing and recommended it to be planted at locations where there is heavy traffic. Bharti et al. 2018 estimated the APTI of 25 plant species that were growing at the Talkatora Industrial Area, India, and determined that Polythalia longifolia was the species that was most sensitive to air pollution. Zn (r2 = 0.72). Negative correlations were observed between proline content and the content of Pb, Fe, Zn and Cd in the unwashed samples (r2 = −0.61; r2 = −0.76; r2 = −0.58 and r2 = −0.56, respectively). 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https://openalex.org/W3128655047	http://scielo.pt/pdf/rpe/v33n1/v33n1a07.pdf	Portuguese	null	PISA, TIMSS e PIRLS em Portugal: Uma análise comparativa	Revista portuguesa de educação/Revista portuguesa de educação	2,020	cc-by-sa	12,848	RESUMO Neste artigo apresentamos uma análise comparativa entre três questio nários internacionais: o PISA, conduzido pela OCDE, e o TIMSS e o PIRLS, realizados pela IEA, tendo em conta os seus objetivos gerais e correlações entre os respetivos dados referentes a Portugal (por regiões da NUTS III). A revisão bibliográfica e documental mostrou como os resultados podem ser reformulados ou reinterpretados pelos diferentes atores sociais e que são vistos pelos decisores políticos e pelas organizações como uma ferramenta de comparação dos sistemas escolares, reveladora de pontos fortes e pon tos fracos. A análise dos resultados gerais evidencia que Portugal tem vindo a melhorar os seus resultados no PISA, nos três domínios avaliados (leitura, matemática e ciências), que melhorou no TIMSS em matemática e piorou em leitura no PIRLS. A análise comparativa por regiões revelou que existe uma diferenciação dos resultados por regiões e que, enquanto o fator domí nio não se apresenta como relevante, o fator objeto de avaliação em cada estudo parece determinante para os resultados dos questionários. Revelou, ainda, que há uma relação direta entre o PIB/habitante e os resultados dos questionários nas regiões com valores extremos de PIB/habitante, mas que essa relação não existe para a generalidade das regiões. Vítor Rosa i Universidade Lusófona de Humanidades e Tecnologias, Portugal João Sampaio Maia ii Universidade Lusófona do Porto, Portugal Daniela Mascarenhas iii Instituto Politécnico do Porto, Portugal António Teodoro iv Universidade Lusófona de Humanidades e Tecnologias, Portugal Palavras-chave: Regiões NUTS III; PISA; TIMSS; PIRLS Palavras-chave: Regiões NUTS III; PISA; TIMSS; PIRLS Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 1. INTRODUÇÃO Os estudos internacionais desenvolvidos a partir de questionários são cada vez mais numerosos e abrangem diferentes domínios. Os da educação dizem respeito a diversos níveis de ensino (da educação básica à universidade) e também aos adultos não escolarizados. Várias revistas e obras especializadas fazem eco destes estudos, insistindo sobre os seus objetivos, os métodos utilizados e os seus resultados. O Programme for International Student Assessment (PISA), conduzido pela Organização para a Cooperação e Desenvolvimento Económico (OCDE) e que incide tradicionalmente sobre a leitura, as ciências e a matemática, e mais recentemente inclui a resolução colaborativa de problemas e a literacia financeira, o Trends in International Mathematics and Science Study (TIMSS), que abrange a matemática e a física, Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 95 PISA, TIMSS e PIRLS em Portugal 95 e o Progress in International Reading Literacy Study (PIRLS), que incide sobre a leitura, ambos realizados pela International Association for the Evaluation of Educational Achievement (IEA), são, certamente, os mais conhecidos e tornaram-se uma referência para a governação nos campos da educação e da formação. Dado que todos estes estudos se centram essencialmente na avaliação dos alunos, foram designados pelo acrónimo ILSAs, que significa International Large-Scale Assessments. Vários documentos e bases de dados, de acesso livre, podem ser encontrados(as) na internet e, ao longo dos anos, todos eles se dotaram de recursos humanos, metodológicos e tecnológicos importantes. Será que estes questionários são um instrumento útil para um diag nóstico dos sistemas educativos? Eles pretendem funcionar como um instru mento de controle dos níveis das competências adquiridas pelos estudantes. As referências utilizadas para definir a escala em que são medidos os níveis de competência são precisamente aquelas que fornecem a distribuição dos resultados do conjunto de alunos que participaram nos testes (a média é fixa da em 500 e o desvio-padrão em 100). A comparação entre esses resultados permite dizer se Portugal tem o mesmo nível de outros países 1. Ao lermos a imprensa sobre estes questionários, verifica-se, muitas vezes, que incidem sobre as comparações internacionais, mas a apresentação dos níveis de lite racia (de leitura, matemática, ciências, ou as mais recentes, financeira e de resolução de problemas em contexto colaborativo) é sumária, limitando-se a breves descrições. 1. De notar que a IEA não tem a OCDE como referência nos seus estudos e que o PISA inclui vários países que não são membros da OCDE. 2. As NUTS estão organizadas em três níveis. O NUTS III é constituído pelas 25 sub-regiões (23 no continente e 2 nas Regiões Autónomas dos Açores e da Madeira), que correspondem a entidades intermunicipais e em que se dividem as sete regiões do NUTS II (5 no Continente e as 2 Regiões Autónomas dos Açores e Madeira). O nível NUTS I é constituído por três unidades: Continente, Região Autónoma da Madeira e Região Autónoma dos Açores. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 2. ASPETOS GERAIS DO PISA, DO TIMSS E DO PIRLS 2.1. PISA 2.1. PISA A missão da OCDE é a de promover políticas que melhorem o bem-estar económico e social em todo o mundo. Convencida da importância da educação no desenvolvimento económico dos países e no bem-estar dos indivíduos, não poderia deixar de se interessar por este domínio, tendo-se tornado uma das principais organizações internacionais de análise da educação em todo o mundo (Rizvi & Lingard, 2006; Teodoro, 2010, 2019, 2020). A adesão de países a esta organização tem vindo a crescer ao longo dos vários ciclos do estudo. No quadro da sua reflexão, “e motivados pela vontade de aplicar o rigor da pesquisa às políticas de educação” (Schleicher, 2018, p. 17), a OCDE lançou o programa PISA. Com ele, e avaliando os alunos de 15 anos de idade 3 e que frequentam, pelo menos, o 7º ano de escolaridade, pretende verificar se as escolas de cada país participante preparam os seus jovens para exercerem o papel de cidadãos na sociedade contemporânea. Não pretendem avaliar os currículos diretamente, mas as competências que os alunos adquiriram para a vida ativa. Este estudo internacional pretende avaliar como os alunos conseguem mobilizar as suas competências em três domínios de literacia: leitura, matemática e ciências. Procura estudar também a resolução colaborativa de problemas e a literacia financeira. Três dimensões foram tomadas em conta para elaborar o material de avaliação do PISA: os tipos de tarefas de leitura, os tipos de texto e o contexto. As tarefas de leitura (questões) definem a natureza das capacidades a avaliar e situam-se a um “nível macro”. Podem ser agrupadas em três categorias: 1) encontrar informações em materiais variados (informar-se); 2) interpretar o que se lê (interpretar); 3) refletir sobre o que se leu e reagir (refletir). Os con teúdos são organizados em categorias. Por exemplo, as categorias referentes à matemática são as seguintes: transformações e relações; espaço e forma; quantidade; incerteza e dados. As categorias não têm sido alteradas desde 2000. No entanto, e sem modificar o espírito, a sua apresentação evoluiu para melhor colocar em evidência a importância dos conteúdos ensinados e a ade quação existente entre as categorias e os objetivos definidos pelos programas curriculares dos países participantes. Querendo inscrever as questões de avaliação da literacia no mundo real e na vida dos alunos, o PISA distingue e hierarquiza os tipos de situações nas quais os problemas são encontrados. 3. Os alunos desta idade encontram-se no final da escolaridade obrigatória na maioria dos países da OCDE. 1. INTRODUÇÃO Os trabalhos sobre a participação de Portugal no PISA, no TIMSS e no PIRLS são poucos e muito restritos nos seus campos de abordagem (Carvalho & Costa, 2009; Conselho Nacional de Educação [CNE], 2013; Marôco, 2020). Com este artigo, pretendemos alargar o espaço de informação sobre esses questionários e saber se é possível compará-los, tendo em conta os seus ob jetivos. Fazemo-lo a partir dos dados globais conhecidos dos países partici pantes, com um olhar particular para os dados de Portugal, mas numa pers petiva diferente dos autores atrás referidos. Enquanto, por exemplo, Marôco (2020) compara os resultados globais de Portugal nos estudos do PISA, do TIMSS e do PIRLS com os de outros países na sua evolução ao longo do tem po, neste artigo, além de fazermos referência à evolução dos resultados glo bais de Portugal nesses estudos, olhamos para os resultados discriminados por regiões da Nomenclatura das Unidades Territoriais para Fins Estatísticos (NUTS) III 2 em cada um dos estudos referidos, num período restrito de tem po, e comparamo-los entre si e com o Produto Interno Bruto (PIB) por habi tante. Isto é, fazemos uma análise comparativa correlacional dos resultados regionais obtidos pelos estudantes portugueses naqueles estudos e que não encontrámos em nenhum outro artigo. 2. As NUTS estão organizadas em três níveis. O NUTS III é constituído pelas 25 sub-regiões (23 no continente e 2 nas Regiões Autónomas dos Açores e da Madeira), que correspondem a entidades intermunicipais e em que se dividem as sete regiões do NUTS II (5 no Continente e as 2 Regiões Autónomas dos Açores e Madeira). O nível NUTS I é constituído por três unidades: Continente, Região Autónoma da Madeira e Região Autónoma dos Açores. Assim, resumindo, temos como objetivos comparar os resultados de Portugal nos estudos do PISA, do TIMSS e do PIRLS no que respeita às respeti vas evoluções e a algumas das suas caraterísticas e fazer uma análise correla cional entre esses resultados, tendo como base as regiões da NUTS III. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 96 96 PISA, TIMSS e PIRLS em Portugal Tabela 1 as categorias P Síntese das categorias PISA-2015 em literacia matemática g Fonte: Adaptado de OCDE (2013). Fonte: Adaptado da OC Conteúdos ç Espaço e forma Q tid d Em cada ano do questionário são avaliados os três domínios (leitura, matemática e ciências), mas um deles é considerado como “maior” (M), ao qual são consagrados dois terços da duração dos testes; os dois outros domí nios são considerados “menores” (m). Na Tabela 2, e a sombreado, salienta-se o enfoque principal colocado nos diferentes tipos de literacia avaliados nas edições do PISA (2000 a 2018). Tabela 2 Enfoque nos tipos de literacia avaliados nas edições do PISA Anos Literacia de Leitura Literacia Matemática Literacia Científica 2000 M m m 2003 m M m 2006 m m M Incerteza e dados Contextos Pessoais Profissionais Societais Científicos Fonte: Adaptado da OCDE (2013) Tabela 2 Enfoque nos tipos de literacia avaliados nas edições do PISA 2012 m M m 2015 m m M 2018 M m m Tabela 4 TIMSS, médias para a matemática e para a ciência, 4.º ano de escolaridade, Portugal, 1995 e 2015 Média para a Matemática Média para a Ciência Tabela 2 Enfoque nos tipos de literacia avaliados nas edições do PISA Anos Literacia de Leitura Literacia Matemática Literacia Científica 2000 M m m 2003 m M m 2006 m m M 2009 M m m 2012 m M m 2015 m m M 2018 M m m Enfoque nos tipos de literacia avaliados nas edições do PISA 15 m m 18 M tipos de literacia avaliados nas edições do PISA Portugal participou em todas as edições (2000, 2003, 2009, 2012, 2015 e 2018). Na edição do PISA-2018, numa lista ordenada de 79 partici pantes, Portugal ficou em 24º lugar na literacia científica, 24º na literacia de leitura e 22º na literacia matemática, com 492 pontos em cada domínio, fican do acima da média da OCDE em todos os domínios 4. Desde a edição do ano 2000, Portugal tem registado uma tendência de melhoria dos resultados nos três domínios analisados, conforme se mostra na Tabela 3 e nos pequenos gráficos (sparkline). No entanto, de 2015 para 2018 houve uma pequena des cida, não estatisticamente significativa, a leitura e a ciências. (4. ano de Escolaridade) (4. 4. Os resultados obtidos são expressos numa escala de 0 a 1000, sendo a média de 500. 2. ASPETOS GERAIS DO PISA, DO TIMSS E DO PIRLS Até 2012, o quadro de referência apontava para que as situações se podiam enquadrar numa grande variedade de contextos: • Situação próxima do aluno: a sua vida pessoal e familiar; • Situação afastada do aluno: a sua vida escolar, o mundo do trabalho e os lazeres; • Situação ainda mais afastada do aluno: a comunidade local e a socie dade nacional ou mundial; • Situação extremamente afastada do aluno: as situações de natureza científica. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal PISA, TIMSS e PIRLS em Portugal 97 97 No quadro de referência de 2012, os tipos de situações tornam-se em categorias de contexto: pessoais, profissionais, societais e científicos. A Tabela 1 apresenta, como exemplo, as categorias do PISA-2015 para a literacia matemática. Tabela 1 Síntese das categorias PISA-2015 em literacia matemática Fonte: Adaptado de OCDE (2013). Tabela1 Síntese das categorias PISA-2015 em literacia matemática Domínios de Avaliação Categorias de cada um dos domínios avaliados Processos Formular Empregar Interpretar Conteúdos Transformações e relações Espaço e forma Quantidade Incerteza e dados Contextos Pessoais Profissionais Societais Científicos Fonte: Adaptado da OCDE (2013) abela1 íntese das categorias PISA-2015 em literacia matemática Domínios de Avaliação Categorias de cada um dos domínios avaliados Processos Formular Empregar Interpretar Conteúdos Transformações e relações Espaço e forma Quantidade Tabela 1 as categorias P Tabela 1 Tabela1 Síntese das categorias P Tabela 1 as categorias P ano de escolaridade) 1995 475 3.5 480 4.0 2011 532 3.4 522 3.9 2015 541 2.2 508 2.2 Fonte: Marôco, Gonçalves, Lourenço e Mendes (2016a); Martin e Kelly (1997) abela 4 IMSS, médias para a matemática e para a ciência, 4.º ano de escolaridade, Portugal, 1995 e 015 Anos Média para a Matemática (4.º ano de Escolaridade) Erro-Padrão Média para a Ciência (4.º ano de escolaridade) Erro-Padrão 1995 475 3.5 480 4.0 2011 532 3.4 522 3.9 2015 541 2 2 508 2 2 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 98 PISA, TIMSS e PIRLS em Portugal 98 Tabela 3 PISA, literacias de leitura, matemática e ciências (média e erro-padrão da média), 2000-2018 Fonte: OCDE (1999, 2003, 2013, 2019a, 2019b); Lemos (2014). Tabela 3 PISA, literacias de leitura, matemática e ciências (média e erro-padrão da média), 2000-2018 Fonte: OCDE (1999, 2003, 2013, 2019a, 2019b); Lemos (2014). Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 2.2. TIMSS O TIMSS, promovido sob a égide da IEA, realiza-se de quatro em quatro anos e é uma avaliação internacional da literacia em matemática e em ciências, dos 4º, 8º anos e, recentemente, do 12º ano de escolaridade. Procura avaliar o desempenho dos alunos em diversos contextos de aprendizagem, tentando medir o que designam por eficácia escolar ou competências académicas. Distingue vários níveis de currículos, interessa-se pelos conteúdos de ensino e a sua presença nos programas escolares, pelos contextos familiares e escolares, e analisa a aprendizagem dos alunos dentro de cada contexto. Isto é, o TIMSS diferencia-se do PISA porque se centra nos currículos, recursos e ambientes familiares e escolares e no sucesso dos alunos na escola (Martin et al., 2013), enquanto este pretende avaliar essencialmente competências para a vida. Este questionário internacional inscreve-se na história da IEA, que se inicia em 1964 com o First International Mathematics Study (FIMS) e depois com o Second International Mathematics Study (SIMS), em 1982. O TIMSS, de 1995 a 1999, designou-se Third International Mathematics and Science Study, assumindo o acrónimo por que é conhecido, para se tornar, em 2003, o Trends in International Mathematics and Science Study. O número de países que participam no TIMSS é irregular e muitos só participam em um ou dois níveis de escolaridade. Em 2015, 57 países partici param no estudo: 48 para o 4º ano de escolaridade, 40 para o 8º ano de esco laridade e 9 (EUA, França, Itália, Líbano, Noruega, Portugal, Rússia, Eslovénia e Suécia) para o nível terminal científico (12º ano) (Conseil National d’Évalua tion du Système Scolaire [CNESCO], 2016). Para o nível terminal, o estudo de signa-se TIMSS Advanced. Ele avalia a matemática e a física. Integrando o conjunto dos 46 países aderentes, Portugal participou na edição de 1995, a primeira, avaliando os alunos dos 3º e 4º anos de escola ridade (1º ciclo do ensino básico) e dos 7º e 8º anos (3º ciclo do ensino bási co). Depois de um hiato de 16 anos, correspondendo a três edições do TIMSS, voltou a participar em 2011 e em 2015 (Marôco, 2016b, 2016c), mas somen te com alunos do 4º ano de escolaridade. Em 2015, envolveu cerca de 5000 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 99 PISA, TIMSS e PIRLS em Portugal Anos Literacia de Leitura 99 alunos provenientes de 158 escolas. 2.2. TIMSS Portugal ficou em 13º lugar a matemática e em 32º lugar a ciências. Face aos resultados de 2011, subiu dois lugares no ranking (em matemática), mas desceu 14 pontos a ciências (em 2011 ocupa va o 19º lugar) (cf. Tabela 4). No TIMSS Advanced, e no reduzido universo de países participantes, Portugal ficou classificado em 5º lugar a matemática e em 4º lugar a física. 2003 m M m 2006 m m M 2009 M m m 2012 m M m 2015 m m M 2018 M m m Tabela 4 TIMSS, médias para a matemática e para a ciência, 4.º ano de escolaridade, Portugal, 1995 e 2015 édias para a matemática e para a ciência, 4.º ano de escolaridade, Portugal, 1995 e TIMSS, médias para a matemática e para a ciência, 4.º ano de escolaridade, Portugal, 1995 e 2015 édias para a matemática e para a ciência, 4.º ano de escolaridade, Portugal, 1995 e Portugal, 1995 e 2015 Fontes: Marôco (2016b); Martin e Kelly (1997). Anos Média para a Matemática (4.º ano de Escolaridade) Erro-Padrão Média para a Ciência (4.º ano de escolaridade) Erro-Padrão 1995 475 3.5 480 4.0 2011 532 3.4 522 3.9 2015 541 2.2 508 2.2 Fonte: Marôco, Gonçalves, Lourenço e Mendes (2016a); Martin e Kelly (1997) Fontes: Marôco (2016b); Martin e Kelly (1997). Fonte: Marôco, Gonçalves, Lourenço e Mendes (2016a); Martin e K Em 2019, decorreu o eTIMSS8, versão digital do estudo, que incluiu, também, os alunos do 8º ano de escolaridade. O Instituto de Avaliação Educativa (IAVE), organismo público de re gime especial sob a tutela do Ministério da Educação que assegura a coor denação e a aplicação dos estudos internacionais em Portugal, compilou, re centemente, um conjunto de itens utilizados em edições anteriores do estudo (IAVE, 2019). Devido ao processo de renovação cíclica, esses itens deixaram de integrar o teste, pelo que foram libertados pela IEA com o propósito de mostrar ao público a natureza das questões colocadas aos alunos. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 2.3. PIRLS Com início em 1995, e com uma periodicidade quinquenal, o PIRLS é outro estudo internacional promovido pela IEA, que avalia o desempenho em literacia de leitura dos alunos a frequentar o 4º ano de escolaridade e, como o TIMSS, centra-se nos currículos e outros fatores familiares e académicos e no sucesso escolar dos alunos. Como este, procura apreender também os elementos de contexto cultural, socioeconómico, pedagógico, familiar e individual, que podem estar associados às performances observadas, fazendo análises intra e interpaíses. Para o efeito, recolhe informações sobre as escolas, as classes e os contextos educativos através de questionários dirigidos aos diferentes interlocutores da escola: alunos, encarregados de educação, professores e diretores. O protocolo de avaliação (textos e itens) é baseado no cruzamento de duas dimensões: os processos de compreensão e os objetivos de leitura. Os questionários sobre os hábitos de leitura e atitudes sobre a escrita são submetidos aos alunos e aos encarregados de educação. Outros questionários procuram avaliar as práticas pedagógicas e a formação dos professores e dos diretores de escola. A amostragem, tal como para o TIMSS e o PISA, segue um proto colo rigoroso para este tipo de questionários internacionais, garantindo a Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 100 sua representatividade e que assegura a comparabilidade dos dados com outros países. Portugal participou na primeira edição, em 2011. No PIRLS-2016, introduziu-se uma novidade, procurando-se avaliar a competência de leitura online dos alunos (ePIRLS). Se olharmos para os dados médios dos países da OCDE e da União Europeia (UE), verifica-se que Portugal ficou abaixo 13 e 12 pontos, respetivamente (cf. Figura 1). Figura 1 Média global e erro-padrão da média, no PIRLS, segundo o agrupamento dos países, 2016 Fonte: IEA. Fonte: IEA. O grupo de especialistas do PIRLS definiu uma escala de compe tências em quatro níveis. O primeiro (baixo) corresponde a uma pontuação global entre 400 e 475. O segundo nível (intermediário) corresponde a uma pontuação entre 476 e 550. Os limites do terceiro nível (elevado) são 551 e 624, e uma pontuação global igual ou superior a 625 corresponde ao nível 4 (avançado). Relativamente ao nível de desempenho dos alunos portugueses e da UE, nos dois ciclos de avaliação PIRLS (2011 e 2016), a Tabela 5 mostra as per centagens acumuladas referentes a cada nível em Portugal e na UE. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 2.3. PIRLS Verifica-se que, em Portugal, apenas 9% e 7% conseguiram um nível avançado em 2011 e 2016, respetivamente, ou seja, que compreenderam os textos propostos, im plicando inferências para explicar a ligação entre as intenções, as ações, os acontecimentos, exprimir uma preferência e justificá-la. Constata-se também que a esmagadora maioria dos alunos em Portugal (98% em 2011; 97% em 2016) foi capaz de localizar e restituir um detalhe ou uma informação expli citamente mencionada e diretamente acessível no texto; isto é, somente 2% e 3%, respetivamente, não o conseguiram. De notar que as percentagens não estão muito afastadas das verificadas para a UE. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 101 Tabela 5 Nível de desempenho dos alunos no PIRLS, em %, 2011 e 2016 Fonte: IEA. Tabela 5 Nível de desempenho dos alunos no PIRLS, em %, 2011 e 2016 PIRLS 2016 Avançado (625) Elevado (550) Intermediário (475) Baixo (400) Portugal 2016 7% 38% 79% 97% 2011 9% 47% 84% 98% União Europeia 2016 12% 48% 82% 96% Fonte: IEA Tabela 5 Nível de desempenho dos alunos no PIRLS, em %, 2011 e 2016 sempenho dos alunos no PIRLS, em %, 2011 e 2016 De 2011 para 2016, os alunos (rapazes e raparigas) do 4º ano pio raram o seu desempenho. Dos 4558 alunos e 218 escolas, Portugal desceu 13 pontos (de 541 para 528 pontos), em relação às provas realizadas em 2011 (cf. Tabela 6), não se tendo verificado diferenças significativas entre os par ticipantes de cada sexo. Relativamente à literacia online (ePIRLS), Portugal ficou seis pontos abaixo dos resultados obtidos no PIRLS: os alunos portugue ses obtiveram 522 pontos, ocupando a 12ª posição. Tabela 6 Resultados do PIRLS em Portugal, 2011 e 2016 PIRLS – Resultados 2011 2016 Portugal 541 528 Raparigas - Pontuação Média 548 529 Rapazes - Pontuação Média 534 527 Raparigas (% de Alunos) 49 49 Rapazes (% de Alunos) 51 51 Nível de desempenho dos alunos no PIRLS, em %, 2011 e 2016 PIRLS 2016 Avançado (625) Elevado (550) Intermediário (475) Baixo (400) Portugal 2016 7% 38% 79% 97% 2011 9% 47% 84% 98% União Europeia 2016 12% 48% 82% 96% Fonte: IEA Tabela 6 Resultados do PIRLS em Portugal, 2011 e 2016 Fontes: IAVE (2017); Mullis et al. (2012). 2.3. PIRLS Fonte: Mullis, Martin, Foy e Drucker (2012); IAVE (2017) Tabela 7 Inquéritos internacionais e a participação de Portugal PISA PIRLS TIMSS TIMSS Advanced Pilotagem do Inquérito OCDE Organismo Intergovernamental IEA Cooperativa Internacional de Instituições de Investigação Data de Criação 2000 2001 1995 1995 T d 4 Tabela 6 Resultados do PIRLS em Portugal, 2011 e 2016 PIRLS – Resultados 2011 2016 Portugal 541 528 Raparigas - Pontuação Média 548 529 Rapazes - Pontuação Média 534 527 Raparigas (% de Alunos) 49 49 Rapazes (% de Alunos) 51 51 Fonte: Mullis, Martin, Foy e Drucker (2012); IAVE (2017) Tabela 6Fon d • Um estudo efetuado por Lafontaine (2008) permitiu apontar algu mas caraterísticas pedagógicas que distinguem os países com pon tuações mais baixas: riodicidade Todos os 3 anos Todos os 5 anos anos Irregular úmero de es/Regiões* 72 (34 da OCDE) 50 (26 da OCDE) 48 (26 da OCDE) 9 (7 da OCDE) Alunos de 15 anos ela 7 uéritos internacionais e a participação de Portugal PISA PIRLS TIMSS TIMSS Ad d • A tendência para se ensinar em vez de se promover a capacidade de compreensão; blico-Alvo Alunos de 15 anos de idade que frequentam, pelo 7 º d 4.º ano de escolaridade 4.º, 8.º anos de escolaridade 12.º ano de escolaridade PISA PIRLS TIMSS TIMSS Advanced lotagem do I é i OCDE Organismo IEA C i I i l d I i i õ d I i ã • O ensino das estratégias de compreensão está longe da prática do quotidiano das aulas; menos, o 7.º ano de escolaridade Literacia de Leitura nquérito Organismo Intergovernamental Cooperativa Internacional de Instituições de Investigação a de Criação 2000 2001 1995 1995 T d 4 • O pouco tempo consagrado ao ensino formal da leitura no 4º ano de escolaridade; omínios valiados Literacia Matemática Lit i Ci tífi Literacia de Leitura Matemática e Ciências Matemática e Física riodicidade Todos os 3 anos Todos os 5 anos Todos os 4 anos Irregular úmero de 48 (26 da • Os livros para crianças não são considerados como um material di dático essencial; Literacia Científica texto dos Itens Competências úteis ao futuro cidadão Competências de leitura Saberes disciplinares úmero de es/Regiões* 72 (34 da OCDE) 50 (26 da OCDE) 48 (26 da OCDE) 9 (7 da OCDE) Alunos de 15 anos • A irregularidade da leitura de livros mais extensos; xercícios Poucos Poucos Muitos blico Alvo de idade que frequentam pelo 4.º ano de 4.º, 8.º anos de 12.º ano de • A predominância das avaliações tradicionais do tipo questionário de escolha múltipla e questões abertas que exigem respostas escritas curtas. 3. METODOLOGIA Neste artigo, usamos dois tipos de análise metodológica. Nos três primeiros pontos da secção 3. Resultados, analisamos e comparamos a conceção de literacia em cada um dos estudos envolvidos, os respetivos conteúdos dos protocolos e tipo de tratamento de dados e os resultados globais obtidos pelos alunos portugueses nos três estudos e a sua evolução. Usamos uma abordagem qualitativa de caráter interpretativo, seguindo as perspetivas de IAVE (2019), Mullis et al. (2009) e Rémond (2006), entre outros. Nos dois pontos finais da secção 3. Resultados, fazemos uma aná lise quantitativa dos dados numéricos dos estudos em causa, como fizeram vários autores, como, por exemplo, Marôco (2018) e Mullis et al. (2012) e os autores da maioria dos capítulos de Monitoring Student Achievement in the 21st Century. European Policy Perspectives and Assessment Strategies, de Harju-Luukkainen et al. (2020). Apresentamos uma análise comparativa cor relacional entre os resultados médios (RM) de cada um dos três domínios do PISA-2015, dos dois domínios do TIMSS-2015, dos dois domínios do TIMSS Advanced-2015, do único domínio do PIRLS-2016 e do ePIRLS-2016, por re giões da NUTS III, e, ainda, uma análise comparativa correlacional entre estes RM e os valores médios do PIB/habitante dessas regiões em 2015. São, pois, estas as 10 variáveis que comparámos correlacionalmente. Dentro dos estu dos, dado que o PISA abrange três domínios, o TIMSS e o TIMSS Advanced dois cada um, e o PIRLS e o ePIRLS um cada, e que determinámos a correlação entre estas nove variáveis, duas a duas, calculámos trinta e seis correlações. Entre os RM de cada domínio de cada estudo (nove) e os valores médios da variável correspondente ao PIB/habitante, determinámos as nove (9×1) cor relações possíveis. O número de valores possíveis de cada das 10 variáveis são sempre 25, correspondentes às 25 regiões da NUTS III. No tratamento estatís tico destas variáveis e respetivas comparações para determinação das corre lações e do valor de foi usado como suporte o software IBM SPSS® Statistics. Embora já se tenha acesso aos RM do PISA de 2018 por NUTS III (Lourenço et al., 2019), para reduzir os efeitos de fatores externos aos resulta dos, reduzimos essa análise a um período temporal curto, o mais recente pos sível, mas que abrangesse todos os estudos. Esta opção levou-nos a escolher os anos de 2015, abrangendo o PISA e o TIMSS, e de 2016, que inclui o PIRLS e o ePIRLS. 2.3. PIRLS bertados Poucos Poucos Muitos cipação de ortugal 2000, 2003, 2006, 2009, 2012, 2015, 2018 2011, 2016 1995, 2011, 2015, 2019 2015 blico-Alvo frequentam, pelo menos, o 7.º ano de escolaridade escolaridade escolaridade escolaridade Literacia de Leitura Sobre a realidade portuguesa, e de uma forma mais abrangente, Benavente (2016) destaca vários constrangimentos a nível escolar: aulas expositivas, número elevado de alunos por classe, necessidades dissociadas da realidade das crianças e dos jovens, concorrência económica entre es colas, dispensa de milhares de professores e de funcionários, reformulação Avaliados Matemática Literacia Científica Leitura Ciências Física Contexto dos Itens Competências úteis ao futuro cidadão Competências de leitura Saberes disciplinares Exercícios Libertados Poucos Poucos Muitos Participação de 2000, 2003, 2006, 1995 2011* Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 102 curricular e programática, programas extensos e desadequados, obstáculos à integração de crianças e jovens com necessidades educativas específicas, menorização de outras disciplinas (desporto, educação artística, educação cí vica, educação ambiental). As políticas e as práticas são irregulares e mudam consoante os governos, os atores educativos e as escolas. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 3. METODOLOGIA Assim, a análise comparativa que apresentamos compreende os últimos resultados conhecidos do TIMSS, do PIRLS e do ePIRLS e o penúlti mo do PISA; no entanto, quando se justifica, os resultados do PISA-2018 são também referidos nessa análise. Em relação ao PIB/habitante, para que haja coerência nas comparações com os RM é o ano de 2015 que é considerado, pois é o que corresponde ao maior número de RM comparados. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 103 Nessa análise correlacional, assumimos que os valores apresentados nos relatórios nacionais sobre o PISA de 2015, de Marôco (2016a), sobre o TIMSS e o TIMSS Advanced de 2015, também de Marôco (2016b, 2016c), e sobre o PIRLS e o ePIRLS de 2016, do IAVE (2017), são fidedignos e que as amostras referidas e usadas nos estudos citados são representativas de cada região da NUTS III em cada um deles. Dado que os RM, por regiões da NUTS III, de quaisquer dois domínios de qualquer dos estudos, e o PIB/habitante são métricos e referentes às mes mas regiões, esses resultados são emparelhados, tendo sentido calcular se há correlação linear entre eles, usando um teste paramétrico. Para tal, usou-se o coeficiente de correlação linear de Pearson, r, que é o usado pelo SPSS em todas as situações com condições semelhantes às nossas. Para avaliarmos se as correlações lineares calculadas tinham ou não significado estatístico relativamente às respetivas populações, usamos o teste de hipóteses, H0: ρ = 0; H1: ρ ≠ 0, sobre a variável habitualmente usada em correlações lineares, tc= r n −2 1−r 2 , que segue a distribuição -Student com n-2 graus de liberdade, sendo n= número de regiões da NUTS III (25) e r = coeficiente de correlação de Pearson entre as amostras em causa. Note-se que é a expressão que o SPSS usa com as variáveis com caraterísticas semelhantes às nossas, e que está associada ao valor de prova, p. Para distinguirmos se a correlação calculada tem ou não significado estatístico para as respetivas populações, definimos o habitual nível de significância em estudos educativos: α = 5%. 3. METODOLOGIA Assim, se o valor de prova, p, for inferior a 0,05 há correlação estatisticamente significativa, positiva ou negativa, entre as populações correspondentes aos dados emparelhados dos resultados dos dois domínios dos estudos em causa, e se p for superior ou igual a 0,05 não há correlação estatisticamente signifi cativa, positiva ou negativa, entre as populações correspondentes aos dados emparelhados dos resultados comparados. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 4.1. AS DEFINIÇÕES DE LITERACIA SEGUNDO O PISA, O TIMSS E O PIRLS Dado que é a literacia, nas três áreas referidas, que está em foco no PISA, no TIMSS e no PIRLS, importa esclarecer o que se entende por este conceito nestes três questionários e quais as suas diferenças e semelhanças. Em relação ao PISA, no relatório sobre Portugal no PISA-2018, Lourenço et al. (2019) fazem um resumo da respetiva posição. Referem que a literacia científica é encarada como “a capacidade de um indivíduo se en volver em questões relacionadas com as ciências e de compreender as ideias científicas como um cidadão reflexivo sendo capaz de explicar fenómenos cientificamente, avaliar e conceber investigações científicas, interpretar da dos e evidências cientificamente” (Lourenço et al., 2019, p. 32). Em relação à literacia de leitura, dizem que é a “capacidade de um indivíduo compreender, utilizar, avaliar, refletir e de se envolver na leitura de textos escritos, com a finalidade de atingir os seus objetivos, de desenvolver os seus conhecimentos e o seu potencial e de participar na sociedade” (Lourenço et al., 2019, p. 20). Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 104 E, no que diz respeito à literacia matemática, consideram que é “a capacidade de um indivíduo formular, aplicar e interpretar a matemática em contextos diversos. Inclui raciocinar matematicamente e utilizar conceitos, processos, factos e ferramentas da matemática para descrever, explicar e prever fenóme nos” (Lourenço et al., 2019, p. 39). A IEA, responsável pelo TIMSS e pelo PIRLS, não aborda diretamente o conceito de literacia e localiza-a nas performances académicas. No caso do TIMSS, a literacia matemática e científica é encarada como a capacidade do indivíduo para usar e aplicar o conhecimento em cada uma destas áreas, mas, como já se referiu, essa capacidade está centrada nas competências curricula res e nas competências cognitivas conhecer, aplicar e raciocinar (IAVE, 2019). No entanto, não encontrámos qualquer definição de literacia nos diferentes relatórios do TIMSS nem qualquer preocupação em fazê-lo. Por exemplo, no Relatório de 1995, no volume referente a Conceção e Desenvolvimento, a primeira referência a literacia é feita na página 11, dizendo somente que o “TIMSS planeia relatar medidas de literacia em matemática e em ciências” (Martin & Kelly, 1997, p. 11); nas restantes vezes que a palavra “literacia” apa rece é para dizer se se trata de matemática ou ciências ou para a relacionar com a população em causa. 4.1. AS DEFINIÇÕES DE LITERACIA SEGUNDO O PISA, O TIMSS E O PIRLS A avaliação da literacia de leitura feita pelo PIRLS assenta numa noção abrangente do que é saber ler, uma noção que inclui a capacidade de refletir sobre o que se lê e de fazer uso disso para alcançar objetivos individuais e sociais. Segundo Campbell et al. (2000), a IEA define literacia de leitura como “a aptidão para compreender e utilizar as formas de linguagem escrita, que a sociedade tem necessidade e que são necessárias ao indivíduo” (p. 3). Ou seja, para o PIRLS, o aluno lê para aprender, mas, tal como no PISA, deve também participar na vida coletiva, chamada “comunida de de leitores” (Mullis et al., 2009, p. 11; Rémond, 2006, p. 72). Ora, em termos gerais, a OCDE (1999) assume, para os estudos do PISA, que literacia é a capacidade de “compreender, de utilizar e de analisar os textos escritos, mas também de refletir sobre o seu propósito, por forma a realizar os seus objetivos, de desenvolver os seus conhecimentos e o seu po tencial e assumir um papel ativo na sociedade” (p. 20). Portanto, apesar de a OCDE e a IEA apontarem para focos diferentes nas suas avaliações, a posição da OCDE sobre o que é literacia não difere muito da IEA. De facto, também para Rémond (2006), estas duas definições, elaboradas num intervalo de um ano, apontam numa perspetiva semelhante, pois ambas refletem “uma conce ção interativa da leitura, onde o leitor apela aos seus conhecimentos e às suas estratégias para construir a significação do texto e utilizar a escrita” (p. 72). 4.2. O CONTEÚDO DOS PROTOCOLOS E DO TRATAMENTO DE DADOS NO PISA, NO TIMSS E NO PIRLS Uma complexa metodologia condiciona a construção e a organização do material, elaborado em inglês, para formar uma “versão fonte”, servindo de base comum às traduções. Vários itens foram criados (141 para o PISA-2000), mas os alunos apenas veem uma parte do material produzido, sob o princípio de “cadernos rotativos”. Os protocolos do PIRLS recaem sobre textos longos e integrais, de onde são elaboradas várias questões. São constituídas diversas versões do teste. No PIRLS-2016, por exemplo, foram utilizados 16 cadernos Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 105 de teste, com diferentes combinações de itens que avaliam diferentemente os processos e as finalidades de leitura. Utilizando uma multiple matrix sampling test design (distribuição dos itens pelos cadernos de teste), assegura-se a “estimação da literacia de leitura para grupos grandes” (IAVE, 2017, p. 5). Devido às limitações da amostragem, os estudos como o PIRLS recorrem à estimação de valores plausíveis para o desempenho dos alunos. Considera-se a aptidão dos alunos em função das respostas aos itens do teste. O PISA, como outras avaliações da OCDE, incide sobre um grande número de exercícios, na medida em que procuram cruzar vários fatores para obter uma declinação dos itens, correspondendo às caraterísticas definidas (OCDE, 1999, 2003). No caso do TIMSS, e consoante o ano em causa (4º, 8º ou 12º), os domínios específicos avaliados em matemática e em ciências são: números, formas geométricas, medidas, álgebra, probabilidades, ciências da vida, ciências físicas e da terra, biologia, química, física, entre outros. Para além dos conteúdos, o quadro de referência define os processos cognitivos na resolução dos itens (aplicar, conhecer e raciocinar). Tabela 5 Nível de desempenho dos alunos no PIRLS, em %, 2011 e 2016 PIRLS 2016 Avançado (625) Elevado (550) Intermediário (475) Baixo (400) Portugal 2016 7% 38% 79% 97% 2011 9% 47% 84% 98% União Europeia 2016 12% 48% 82% 96% Fonte: IEA Tabela 6 Resultados do PIRLS em Portugal, 2011 e 2016 PIRLS – Resultados 2011 2016 Portugal 541 528 R i P ã Médi 548 529 Os relatórios internacionais publicados pela OCDE e pela IEA colo cam a ênfase na comparação de resultados entre os países e são representa dos como se traduzissem uma escala de competências que induz um ranking. * Em 2011e 2015, Portugal só participou com alunos do 4.º ano de escolaridade. Em 2016, realizou-se também o PIRLS Online (ePIRLS). * A participação dos países é variável durante as várias edições. 4.3. ALGUMAS COMPARAÇÕES DE RESULTADOS ENTRE O PISA, O TIMSS E O PIRLS Desde o primeiro ciclo do PISA, em 2000, Portugal tem registado uma melhoria significativa dos resultados obtidos nos três domínios analisados. Em 2018, os alunos obtiveram 492 pontos nas três literacias (leitura, matemática e científica), e, segundo Lourenço et al. (2019), as regiões de Coimbra, Leiria, Área Metropolitana do Porto e Médio Tejo são as que obtêm melhores resultados nos três domínios, com a região do Ave a sobressair também na leitura e na matemática. As regiões com resultados menos conseguidos nos três domínios foram a Região Autónoma dos Açores, o Baixo Alentejo e o Alto Alentejo. Estes resultados representam uma manutenção dos valores de 2015 ou uma descida não significativa, pois em 2015 os alunos obtiveram 498 pontos em leitura, 492 pontos em matemática e 501 pontos em ciências. As unidades territoriais com resultados mais conseguidos e menos conseguidos em 2018 também são diferentes das correspondentes em 2015. No relatório de Marôco (2016a), referente ao PISA-2015, é menciona do que são as unidades territoriais do litoral, em particular o Alentejo Litoral, que apresentam os melhores resultados médios dos alunos. Em contraparti da, são as unidades do interior norte (Alto Tâmega, Terras de Trás-os-Montes e Tâmega e Sousa) e do Baixo Alentejo onde se verifica que o desempenho dos alunos em leitura foi mais débil. A família, nas suas diferentes dimensões (económica, cultural e social), afeta o desempenho escolar dos seus filhos (Ferreira, Flores, & Casas-Novas, 2017). A OCDE criou um indicador, denominado “Estatuto Socioeconómico e Cultural” (ESCS), que agrega várias características do agregado familiar (nível de escolaridade, estatuto profissional, bens materiais, indicadores culturais). No estudo de Ferreira et al. (2017) refere-se que “desde 2003, Portugal tem vindo a melhorar a condição socioeconómica dos seus alunos” (p. 25). No en tanto, ela é ainda relativamente baixa quando comparada com a de outros paí ses (Espanha e Polónia, por exemplo). O desemprego ou subemprego a que as famílias foram expostas a partir de 2008, devido à crise económica, poderá ter contribuído para os efeitos negativos na performance dos alunos. Os autores referem que “Portugal tem feito uma trajetória decrescente, sendo que a cor relação entre ESCS e scores PISA tem vindo a reduzir-se ao longo do tempo, estando em 0,4 em 2015” (Ferreira et al., 2017, p. 46), isto é, em Portugal, a in fluência das condições socioeconómicas das famílias nos resultados do PISA tem vindo a reduzir-se. 4.2. O CONTEÚDO DOS PROTOCOLOS E DO TRATAMENTO DE DADOS NO PISA, NO TIMSS E NO PIRLS Raparigas - Pontuação Média 548 529 Rapazes - Pontuação Média 534 527 Raparigas (% de Alunos) 49 49 Rapazes (% de Alunos) 51 51 A Tabela 7 resume alguns aspetos dos questionários internacionais e a participação portuguesa. Fonte: Mullis, Martin, Foy e Drucker (2012); IAVE (2017) Tabela 7 Questionários internacionais e a participação de Portugal * A participação dos países é variável durante as várias edições. ** Em 2016, realizou-se também o PIRLS Online (ePIRLS). Tabela 7 Inquéritos internacionais e a participação de Portugal PISA PIRLS TIMSS TIMSS Advanced Pilotagem do Inquérito OCDE Organismo Intergovernamental IEA Cooperativa Internacional de Instituições de Investigação Data de Criação 2000 2001 1995 1995 Periodicidade Todos os 3 anos Todos os 5 anos Todos os 4 anos Irregular Número de Países/Regiões* 72 (34 da OCDE) 50 (26 da OCDE) 48 (26 da OCDE) 9 (7 da OCDE) Público-Alvo Alunos de 15 anos de idade que frequentam, pelo menos, o 7.º ano de escolaridade 4.º ano de escolaridade 4.º, 8.º anos de escolaridade 12.º ano de escolaridade Domínios Avaliados Literacia de Leitura Literacia Matemática Literacia Científica Literacia de Leitura Matemática e Ciências Matemática e Física Contexto dos Itens Competências úteis ao futuro cidadão Competências de leitura Saberes disciplinares Exercícios Libertados Poucos Poucos Muitos Participação de Portugal 2000, 2003, 2006, 2009, 2012, 2015, 2018 2011, 2016 1995, 2011, 2015*, 2019 2015 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 106 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 4.3. ALGUMAS COMPARAÇÕES DE RESULTADOS ENTRE O PISA, O TIMSS E O PIRLS Numa análise aos dados do PISA-2012, Ferrão (2015) afirma que “os alunos mais desfavorecidos socioeconomicamente [apresen tam] um imenso cenário de desigualdade de oportunidades, com início na es cola, a partir dos 7 anos de idade” (p. 113). Numa análise dos resultados nacionais do TIMSS-2015 (4º ano) por regiões da NUTS III, tomando como referência o estudo de Marôco (2016b), é possível verificar que quase um quarto dos alunos da região de Leiria alcan çaram resultados de nível Avançado (625 pontos ou mais na escala de ma temática). Em contraste, foi a região do Tâmega e Sousa, tal como aconteceu no PISA-2015, que obteve os dados mais fracos a nível nacional (500 pontos em matemática; 477 pontos em ciências). De uma forma global, as regiões do litoral centro e norte apresentam, no âmbito das duas literacias, resultados muito positivos. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal Figura 4 Figura 4 Médias dos três questionários, por domínios e por género, 2015-2016 Figura 3 Médias dos três questionários e domínios de avaliação, 2015-2016 Médias dos três questionários e domínios de avaliação, 2015-2016 q ç , Fontes: IAVE (2017); Marôco (2016b, 2016c). Fontes: IAVE (2017); Marôco (2016b, 2016c). Numa análise por género, também relativamente a todos os estudos referidos (cf. Figura 4), verifica-se que, em literacia matemática, de ciências e de física, os rapazes têm médias mais altas do que as raparigas e que estas têm médias mais elevadas na literacia de leitura. PISA, TIMSS e PIRLS em Portugal 107 No que respeita ao PIRLS, em 2016, Portugal, com 528 pontos, en contra-se no conjunto de participantes que registaram uma pontuação média significativamente superior ao ponto central da escala (500 pontos). No en tanto, face a 2011, esta pontuação representa uma descida significativa de 13 pontos (541, em 2011, desce para 528, em 2016). Na escala ordenada dos re sultados, entre 2011 e 2016, Portugal passou do 19º para o 30º lugar. A Figura 2 apresenta a distribuição dos resultados nacionais por regiões da NUTS III. Foi a região do Ave que obteve a melhor classificação no PIRLS-2016, com 544 pontos. É uma diferença de 16 pontos relativamente à média nacional (528). As regiões que registaram os desempenhos médios mais baixos foram: Alto Tâmega, Baixo Alentejo, Tâmega e Sousa, Beiras e Serra da Estrela, Algarve, Região Autónoma dos Açores e Douro. No entanto, todas elas registaram valo res acima de ou iguais a 500. Figura 2 Resultados do PIRLS (média e erro-padrão da média), por regiões da NUTS III, 2016 Figura 2 Resultados do PIRLS (média e erro-padrão da média), por regiões da NUTS III, 2016 Fonte: IAVE (2017). Fonte: IAVE (2017). Fonte: IAVE (2017). Comparando os resultados de todos os estudos referidos, verifica mos que Portugal apresenta resultados mais fracos em física e matemática no TIMSS Advanced-2015 (cf. Figura 3) do que nos restantes estudos. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 108 Figura 4 Figura 4 Figura 4 Médias dos três questionários, por domínios e por género, 2015-2016 Fontes: IAVE (2017); Marôco (2016b, 2016c). Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 109 Numa análise sobre as medidas e os impactos de política educativa no respetivo desempenho, em 2015, dos alunos do 4.º ano na área da mate mática (referente ao TIMSS) e dos alunos de 15 anos no que se refere à litera cia em ciências, Saraiva (2017) sublinha que Ao nível das ciências, e ao contrário do que sucedeu em matemáti ca, os relatórios nacionais do TIMSS (4.º ano de escolaridade) e do PISA (2015) revelam resultados discrepantes. Se no PISA a média dos alunos portugueses foi significativamente superior à obtida em anos anteriores e à média da OCDE, o mesmo não sucedeu no TIMSS …. Mas esta discrepância ficou quase “esquecida” no debate público, talvez porque as ciências permanecem afastadas dos “palcos” dos rankings nacionais, onde se mantém o protagonismo da matemática e do português. (p. 5) Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 4.4. ANÁLISE CORRELACIONAL ENTRE OS RESULTADOS DO PISA, DO TIMSS E DO PIRLS, POR REGIÕES DA NUTS III Com o intuito de se saber se haveria alguma relação entre os resultados dos estudos que temos vindo a mencionar, comparámos os RM, por regiões da NUTS III, dos seguintes estudos do PISA, TIMSS e PIRLS: PISA-2015 (15 anos – leitura, matemática e ciências); TIMSS-2015 (4.º ano – matemática e ciências); TIMSS Advanced-2015 (12.º ano – matemática e física); PIRLS-2016 e ePIRLS-2016 (ambos no 4.º ano – leitura). Fizemos uma análise correlacional entre esses RM, por regiões da NUTS III, calculando o coeficiente de correlação linear de Pearson entre os dados emparelhados de cada par desses estudos. Como há nove estudos/ domínios, calculámos as 36 correlações possíveis entre dois desses estudos. Verificámos que havia seis correlações estatisticamente significativas, todas positivas (ver Figura 5): a) no PISA-2015, entre a leitura, a matemática e as ciências, com valores entre 0,89 e 0,96 (em qualquer dos casos verifica-se um p<0,001); b) no TIMSS-2015, entre a matemática e as ciências, com 0,97 (p<0,001); c) no TIMSS Advanced-2015, entre a matemática e a física, com 0,48 (p=0,016); entre o PIRLS e o ePIRLS, referente à leitura, com 0,93 (p<0,001). Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 110 Figura 5 Correlações significativas positivas dentro de cada estudo g Correlações significativas positivas dentro de cada estudo Dessa análise, duas evidências se destacam: • Entre os RM de dois domínios do mesmo estudo, há sempre uma cor relação positiva, estatisticamente significativa, quaisquer que sejam os domínios em causa; • Entre os RM de dois questionários referentes ao mesmo domínio ou à mesma faixa etária, mas de estudos diferentes, não há qualquer correlação estatisticamente significativa (ver Tabela 8). A participação dos países é variável durante as várias edições. Em 2016, realizou-se também o PIRLS Online (ePIRLS) * l i i l d d l id d Tabela 8 Correlações não significativas entre RM de estudos diferentes ela 8 relações não significativas entre RM de estudos diferentes Correlações não significativas entre RM de estudos diferentes ela 8 relações não significativas entre RM de estudos diferentes Correlações não significativas entre RM de estudos diferentes ela 8 relações não significativas entre RM de estudos diferentes LITERACIAS CORRELAÇÕES PISA- 2015 TIMSS- 2015 TIMSS Advanced- 2015 PIRLS- 2016 ePIRLS- 2016 MATEMÁTICA PISA-2015 --- 31% 15% TIMSS-2015 31% --- 6% TIMS Advanced- 2015 15% 6% --- CIÊNCIAS TIMSS-2015 30% LEITURA PISA-2015 40% 30% PIRLS-2016 40% ePIRLS-2016 30% Verificar-se uma correlação muito forte entre os RM do PIRLS e do ePIRLS não é de estranhar, pois correspondem ao mesmo ano e à mesma área, mas o mesmo já não se pode dizer em relação a correlações muito fortes entre os RM de estudos referentes a domínios diferentes, como, por exemplo, den tro do PISA. A Figura 6 mostra os RM do PISA-2015, de leitura, matemática e PISA-2015 15 anos Leitura Matemática Ciências Coeficientes de correlação entre as três literacias variam entre 89% e 96% Figura 5 Correlações significativas positivas dentro de cada estudo Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 111 ciências, e do TIMSS Advanced-2015, de matemática, por regiões da NUTS III. Nela podemos verificar que os RM dos três domínios do PISA-2015 seguem linhas muito próximas, o que confirma a correlação quase perfeita que existe entre eles, e o desfasamento da linha do TIMSS Advanced-2105, da matemática, em relação às outras, o que mostra a não existência de correlação significativa entre qualquer dos RM do PISA-2015 e os do TIMSS Advanced-2015 referido. De facto, o coeficiente de correlação entre os RM do TIMSS Advanced-2015, da matemática, e do PISA-2015, da matemática, é de 0,15 (p=0,42). Correlações não significativas entre RM de estudos diferentes ela 8 relações não significativas entre RM de estudos diferentes Figura 6 Resultados do PISA-2015, de leitura, matemática e ciências, e do TIMSS Advanced, de matemática, por regiões da NUTS III Figura 6 Resultados do PISA-2015, de leitura, matemática e ciências, e do TIMSS Advanced, de matemática, por regiões da NUTS III Figura 6 Resultados do PISA-2015, de leitura, matemática e ciências, e do TIMSS Advanced, de matemática, por regiões da NUTS III g Resultados do PISA-2015, de leitura, matemática e ciências, e do TIMSS Advanced, de matemática, por regiões da NUTS III A evidência a) – entre os RM de dois domínios do mesmo estudo, há sempre uma correlação positiva, estatisticamente significativa, quaisquer que sejam os domínios em causa –, isto é, o facto de as correlações entre os RM de dois domínios do mesmo estudo serem sempre positivas e estatisticamen te significativas (em quatro das cinco correlações possíveis nesta situação o coeficiente de correlação é muito alto: ≥0,89, o que corresponde a p<0,001) diz-nos que, dentro de cada estudo, as regiões da NUTS III com bons RM num domínio também os têm no outro ou nos outros domínios do mesmo estudo. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 112 Isto aponta para que, dentro de cada estudo, o resultado obtido por um aluno aleatoriamente escolhido dentro dos que preencheram um dos questionários em causa dependa muito menos do domínio estudado do que da região da NUTS III a que o aluno pertence. Assumindo que as amostras de cada estudo são representativas de cada região e de Portugal no seu todo e que os ques tionários são válidos e fidedignos, isto indicia que obter bons resultados em qualquer dos questionários, de qualquer domínio, é muito mais dependente da região a que pertence o aluno do que do domínio em causa. Para aprofundar a evidência b) – entre os RM de dois questionários referentes ao mesmo domínio ou à mesma faixa etária, mas de estudos di ferentes, não há qualquer correlação estatisticamente significativa –, especi fiquemos os resultados obtidos. No que diz respeito à matemática, entre o PISA-2015 e o TIMSS-2015, entre este e o TIMSS Advanced-2015 e entre o último e o primeiro, os coeficientes de correlação obtidos foram, respetiva mente, de 0,31 (p=0,14), de 0,06 (p=0,78) e de 0,15 (p=0,48). 5. Este valor corresponde ao limite de uma correlação não significativa (p=0,05). Correlações não significativas entre RM de estudos diferentes ela 8 relações não significativas entre RM de estudos diferentes No que res peita ao domínio ciências, entre o TIMSS-2015 e o PISA-2015, obteve-se um coeficiente de correlação de 0,29 (p=0,15) e, em relação ao domínio leitura, o coeficiente de correlação entre o PIRLS-2016 e o PISA-2015 foi de 0,405 e entre o ePIRLS-2016 e o PISA-2015 foi de 0,30 (p=0,15). Todos estes valores, como dissemos, não têm significado estatístico. A não existência de correla ção estatisticamente significativa entre os RM de estudos diferentes, mesmo referentes ao mesmo domínio, aponta para que as diferenças de objeto de avaliação em cada questionário (competências para a vida no PISA e compe tências académicas no TIMSS e no PIRLS) possam ser fatores relevantes para os RM obtidos em cada questionário. O facto de também não haver correlação entre os RM dos diferentes questionários da IEA no que respeita ao mesmo domínio, isto é, entre os RM do PIRLS-2016 e do ePIRLS-2016 (4º ano) em comparação com os RM do PISA-2015 (15 anos), em relação à leitura, e entre os RM do TIMSS-2015 e do TIMSS Advanced-2015, em relação à matemática, poderá ser justificado pela diferença etária e de nível de escolaridade dos alu nos em causa, já que dificilmente se poderá dizer que os objetos de avaliação (currículos, recursos e ambientes familiares e escolares e sucesso dos alunos na escola) no 4º ano e no 12º ano de escolaridade sejam semelhantes. Ou seja, o fator objeto de avaliação em cada estudo parece ser decisivo para que não haja correlação significativa entre os RM de um domínio, mas de estudos diferentes. Porém, a não existência de correlação linear entre os RM de ques tionários aplicados a alunos do mesmo ano de escolaridade ou nível etário, como é o caso do TIMSS, do PIRLS e do ePIRLS (todos aplicados a alunos do 4º ano e em anos consecutivos, portanto da mesma geração), já não aponta para a mesma justificação. De facto, as quatro correlações calculáveis entre os RM de cada um dos dois domínios do TIMSS e os RM do PIRLS e do ePIRLS estão compreendidas entre 0,25 (p=0,26) e 0,34 (p=0,09) e são, como dissemos, todas estatisticamente não significativas. Sendo estes estudos da responsa bilidade da mesma organização, a IEA, portanto, com os mesmos princípios e objetivos genéricos, talvez só possa ser justificada pela diferença entre domí nios (leitura no PIRLS e ePIRLS e matemática e ciências no TIMSS). Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 4.5. RELAÇÃO ENTRE OS RESULTADOS DOS TRÊS ESTUDOS COM O PIB POR REGIÕES DA NUTS III Investigámos também se haveria alguma relação entre os RM de cada um destes estudos e o PIB/habitante de 2015 (dados obtidos na PORDATA – Base de Dados Portugal Contemporâneo)6, por regiões da NUTS III, dado que a maioria dos resultados estudados são deste ano. 6. Em https://www.pordata.pt/ 6. Em https://www.pordata.pt/ Relativamente às correlações lineares entre o PIB/habitante e os RM em cada domínio de cada questionário, constatámos que, das nove cor relações possíveis, a única estatisticamente significativa (0,47, o que corres ponde a p=0,017) verificou-se entre os valores do PIB/habitante e os RM do PISA-2015 de leitura, o que parece apontar para que as diferenças regionais a nível de PIB tenham pouca influência nos resultados destes questioná rios, conforme Marôco (2018) refere a propósito do PIRLS-2016. Contudo, se olharmos para os valores extremos do PIB/habitante, verificamos que há algumas coincidências entre estes e os RM de cada estudo. A região que apre senta o valor máximo de PIB/habitante – Alentejo Litoral – é também a região que, no PISA-2015, obteve melhores RM em cada um dos três domínios. Em qualquer dos outros questionários isso não sucede. Se alargarmos a análise à segunda região (Área Metropolitana de Lisboa) com maior PIB/habitante, verificamos que não é a região com melhores RM em qualquer dos questio nários TIMSS-2015, TIMSS Advanced-2015 e PIRLS-2016. Quanto à região com menor PIB/habitante – Tâmega e Sousa –, verificamos que é a região com RM mais baixo no PISA-2015, de matemática e de ciências, e no TIMSS-2015, de matemática e ciências. A segunda região com menor PIB/habitante (Alto Tâmega) é a região com RM mais baixo no PISA-2015, de leitura, no TIMSS Advanced-2015, de física, e no PIRLS-2016, de leitura. Somente o RM mais baixo no ePIRLS, de leitura, e no TIMSS Advanced-2015, de matemática, não é obtido por uma das duas regiões com menor PIB/habitante da NUTS III. Sintetizando, a região com PIB/habitante mais alto é a região com os RM mais altos em três dos nove estudos/domínios e as duas regiões com PIB/ habitante mais baixo são as regiões com RM mais baixos em sete dos nove estudos/domínios. 7. Note-se que, se não considerarmos a região do Alto Alentejo (a que tem PIB/habitante mais alto), a correlação entre o PIB/ habitante e os RM do PISA- 2015, de leitura, já não seria estatisticamente significativa, acompanhando, assim, o que sucede com os RM de todos os restantes estudos. Correlações não significativas entre RM de estudos diferentes ela 8 relações não significativas entre RM de estudos diferentes Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 113 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 5. CONCLUSÕES A análise dos resultados globais referentes a Portugal desde 2000 evidencia que estes têm vindo a melhorar no PISA, nos três domínios (leitura, matemática e ciências); que, no TIMSS, progrediram em matemática, mas que, em ciências, as médias oscilaram; e que no PIRLS, em leitura, pioraram. g g que estes têm vindo a melhorar no PISA, nos três domínios (leitura, matemática e ciências); que, no TIMSS, progrediram em matemática, mas que, em ciências, as médias oscilaram; e que no PIRLS, em leitura, pioraram. No entanto, os resultados obtidos pelos alunos não são iguais em todo o país, havendo diferenças entre as várias regiões da NUTS III. Na análise correlacional, por regiões da NUTS III, entre os RM em cada domínio de cada d á No entanto, os resultados obtidos pelos alunos não são iguais em todo o país, havendo diferenças entre as várias regiões da NUTS III. Na análise correlacional, por regiões da NUTS III, entre os RM em cada domínio de cada estudo, constatámos que: • Entre os RM de dois domínios do mesmo estudo, há sempre uma cor relação positiva estatisticamente significativa, quaisquer que sejam os domínios em causa; • Entre os RM de dois questionários referentes ao mesmo domínio ou à mesma faixa etária, mas de estudos diferentes, não há qualquer correlação estatisticamente significativa. Estes resultados fazem destacar dois fatores com relevâncias de sen idos opostos nos resultados das regiões da NUTS III: • 1) O objeto de avaliação (competências académicas nos estudos da IEA – PIRLS, ePIRLS, TIMSS e TIMSS Advanced, e competências para a vida nos estudos da OCDE – PISA) parece ter uma importância rele vante nos resultados dos questionários; • 2) O domínio (leitura, matemática, ciências e física) que se avalia pa rece não ser determinante nos resultados de cada estudo. A irrelevância do domínio diz-nos que as regiões têm bons ou maus resultados independentemente do domínio em causa, e a relevância do objeto de avaliação indica-nos que a competência na escola poderá estar pouco rela cionada com as competências para a vida. Olhando para a relação entre as questões socioeconómicas, se Portugal tem vindo a melhorar a condição socioeconómica dos seus alunos, ela é ainda baixa quando comparada com a de outros países europeus e reper cute-se nos resultados escolares dos alunos. 4.5. RELAÇÃO ENTRE OS RESULTADOS DOS TRÊS ESTUDOS COM O PIB POR REGIÕES DA NUTS III Ou seja, embora a maioria7 das correlações, por regiões, entre o PIB/habitante e os RM de cada domínio dos estudos citados seja não significativa, o facto de a maioria dos resultados mais altos e mais baixos em cada domínio de cada estudo serem obtidos nas regiões, respetivamente, com PIB/habitante mais alto e com PIB/habitante mais baixo parece mostrar que há alguma relação entre o PIB/habitante das regiões e os RM dos diferentes estudos/domínios e que essa relação é essencialmente nas regiões com valo res extremos de PIB/habitante. Se atendermos ao facto de, nos três domínios do PISA, os RM mais elevados serem na região de PIB/habitante mais alto e isso não se verificar em qualquer dos estudos TIMSS e PIRLS e ao facto de o PISA avaliar competências para a vida e os outros estudos competências académicas, podemos pôr a hipótese de isso apontar para que os filhos das famílias com mais rendimentos estejam mais preparados para a vida, embora não sejam necessariamente aqueles que têm mais sucesso escolar. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 114 PISA, TIMSS e PIRLS em Portugal Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 5. CONCLUSÕES A escola reproduz as desigual dades sociais, como concluíram, entre outros, Bourdieu e Passeron (1970), e, como sublinha Ferrão (2015), a desigualdade dos alunos mais desfavorecidos socioeconomicamente começa logo a partir dos 7 anos de idade. A compara ção que fizemos, por regiões da NUTS III, entre o PIB/habitante e os RM em cada domínio dos diferentes estudos mostra-nos uma relação direta entre o PIB/habitante e os RM dos estudos referidos nas regiões com PIB/habitan te extremos: a região com PIB/habitante mais alto é a região com RM mais elevados nos três estudos do PISA; as duas regiões com PIB/habitante mais baixo são as que têm RM mais baixos em sete dos nove estudos/domínios aqui citados, incluindo os três do PISA. No entanto, essa relação não se verifi ca nas restantes regiões, isto é, não há correlações significativas entre o PIB/ habitante e os RM em cada domínio de cada estudo, por regiões da NUTS III, se excluirmos as que têm valores extremos de PIB/habitante. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 115 Os resultados apresentados neste artigo e correspondentes conclu sões têm as suas limitações. A principal dessas limitações tem que ver com a assunção que fizemos sobre a representatividade das amostras de cada uma das unidades territoriais da NUTS III usadas em cada um dos estudos citados. De facto, o cuidado que as entidades competentes tiveram em usar amostras que representassem as diversas regiões da NUTS III é evidente nos relatórios nacionais sobre os diferentes estudos usados nas comparações correlacio nais realizadas neste artigo (cf. IAVE, 2017; Marôco, 2016a, 2016b, 2016c). Contudo, alguns erros amostrais associados aos RM das regiões da NUTS III são elevados e, portanto, como refere Marôco (2016a), os resultados que ob tivemos “deverão ser lidos com precaução” (p. 62). Pretendemos, com este artigo, dar alguns contributos para a educa ção em Portugal e também noutros países, no sentido de melhor se conhecer os estudos do PISA, do TIMSS e do PIRLS, em particular numa ótica compa rativa. As análises sobre o conceito de literacia, o conteúdo dos protocolos e do tratamento de dados usados em cada estudo já tinham sido desenvolvidas noutros artigos, mas não com o caráter comparativo que apresentámos e que se acrescenta ao saber já adquirido pela comunidade. 5. CONCLUSÕES As comparações entre os resultados globais dos ILSAs também são muito pouco frequentes e como, sobre Portugal, só conhecemos o estudo de Marôco (2020), publicado muito recentemente, entendemos que o nosso artigo acrescenta informação e outras comparações que permitem perceber melhor as diferenças que existem entre eles. Por fim, a análise correlacional entre os resultados do PISA, do TIMSS e do PIRLS, por regiões da NUTS III, e destes com o PIB/habitante, que apresen tamos e que cremos não ter sido tratada em artigo algum, consideramos que é uma abordagem nova sobre a avaliação de currículos e de competências para a vida. Cremos que abre uma nova janela por onde outros autores podem vis lumbrar novos caminhos de investigação, em particular sobre a relação entre os percursos académicos e os das competências para a vida e entre estas e as assimetrias socioeconómicas regionais das famílias. AGRADECIMENTOS Este trabalho foi financiado pela Fundação para a Ciência e a Tecnologia (FCT) [PTDC/CED-EDG/30084/2017]. AGRADECIMENTOS Este trabalho foi financiado pela Fundação para a Ciência e a Tecnologia (FCT) [PTDC/CED-EDG/30084/2017]. PISA, TIMSS e PIRLS em Portugal 116 Carvalho, L., & Costa, E. (2009). Production of OECD’s Programme for International Student Assessment (PISA). Rapport pour le projet Know&Pol. Université de Lisbonne, Faculté de Psychologie et de Sciences de l’Éducation. Conseil National d’Évaluation du Système Scolaire. (2016). Comparaison des évaluations PISA et TIMSS: Acquis des élèves: Comprendre les évaluations internationales – Rapport scientifique. CNESCO. 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Cham Springer. https://doi.org/10.1007/978-3-030-38969-7 Instituto de Avaliação Educativa. (2017). Resultados globais PIRLS 2016 – ePIRLS 2016 – Portugal. Literacia de leitura & literacia online. Lisboa, Portugal: IAVE. Harju-Luukkainen, H., McElvany, N., & Stang, J. (Eds.). (2020). Monitoring student achievement in the 21st century. European policy perspectives and assessment strategies. Cham Springer. https://doi.org/10.1007/978-3-030-38969-7 student achievement in the 21st century. European policy perspectives and assessment strategies. Cham Springer. https://doi.org/10.1007/978-3-030-38969-7 Instituto de Avaliação Educativa. (2017). Resultados globais PIRLS 2016 – ePIRLS 2016 – Portugal. Literacia de leitura & literacia online. Lisboa, Portugal: IAVE. Instituto de Avaliação Educativa. (2019). TIMSS8 – Itens de matemática e de ciências – I IAVE Instituto de Avaliação Educativa. (2017). Resultados globais PIRLS 2016 – Instituto de Avaliação Educativa. (2017). 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(2000). i Framework and specifications for PIRLS assessment 2001: Progress in international reading literacy study. Boston College. Framework and specifications for PIRLS assessment 2001: Progress in international reading literacy study. Boston College. Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 117 achievement at the fourth grade – Implications for early learning (pp. 109-178). TIMSS & PIRLS International Study Center, Boston College. Martin, M., & Kelly, D. (Org.). (1997). TIMSS – Technical report, vol. I. IEA. Mullis, I., Martin, M., Foy, P., & Drucker, K. (2012). PIRLS 2011 international results in reading. IEA. achievement at the fourth grade – Implications for early learning (pp. 109-178). 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A aprendizagem das ciências em Portugal: Uma leitura a partir dos resultados do TIMSS e do PISA. Mediações – Revista OnLine da Escola Superior de Educação do Instituto Politécnico de Setúbal, 5(2), 4-18. Saraiva, L. (2017). PISA, TIMSS e PIRLS em Portugal Lourenço, V., Duarte, A., Nunes, A., Amaral, A., Gonçalves, C., Mota, M., Lourenço, V., Duarte, A., Nunes, A., Amaral, A., Gonçalves, C., Mota, M., & Mendes, R. (2019). PISA 2018 – Portugal. Relatório nacional. IAVE. Disponível em: http://www.cnedu.pt/pt/noticias/internacional/1517-resultados-pisa-2018. Marôco, J. (2018). O bom leitor: Preditores da literacia de leitura dos alunos portugueses no PIRLS 2016. Revista Portuguesa de Educação, 31(2), 115-131. https://doi.org/10.21814/rpe.14105 Marôco, J. (2020). International large-scale assessments: Trends and effects on the Portuguese public education system. In H. Harju-Luukkainen, N. McElvany, & J. Stang (Eds.), Monitoring student achievement in the 21st century. 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Marôco, J., Lourenço, V., Mendes, R., & Gonçalves, C. (2016c). TIMSS Advanced 2015 – Portugal 1 – Desempenhos em matemática e em física. IAVE. Martin, M., Foy, P., Mullis, I., & O’Dwyer, L. (2013). Effective schools in reading, mathematics, and science at the fourth grade. In M. Marin & I. Mullis (Eds.), TIMSS and PIRLS 2011: Relationships among reading, mathematics, and science Portugal. Volume I: Desempenhos em matemática e em Ciências. IAVE. Marôco, J., Lourenço, V., Mendes, R., & Gonçalves, C. (2016c). TIMSS Advanced 2015 – Portugal 1 – Desempenhos em matemática e em física. IAVE. Marôco, J., Lourenço, V., Mendes, R., & Gonçalves, C. (2016c). TIMSS Advanced 2015 – Portugal 1 – Desempenhos em matemática e em física. IAVE. Martin, M., Foy, P., Mullis, I., & O’Dwyer, L. (2013). Effective schools in reading, mathematics, and science at the fourth grade. In M. Marin & I. Mullis (Eds.), TIMSS and PIRLS 2011: Relationships among reading, mathematics, and science Revista Portuguesa de Educação, 33(1), 94-120. PISA, TIMSS e PIRLS em Portugal Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal A aprendizagem das ciências em Portugal: Uma leitura a partir dos resultados do TIMSS e do PISA. Mediações – Revista OnLine da Escola Superior de Educação do Instituto Politécnico de Setúbal, 5(2), 4-18. Schleicher, A. (2018). World class: How to build a 21st-century school system, strong performers and successful reformers in education. OCDE Publishing. https://www.oecd.org/education/world-class-9789264300002-en.htm Schleicher, A. (2018). World class: How to build a 21st-century school system, strong performers and successful reformers in education. OCDE Publishing. https://www.oecd.org/education/world-class-9789264300002-en.htm Teodoro, A. (2010). Educação, globalização e neoliberalismo. Os novos modos de regulação transacional das políticas de educação. Lisboa, Portugal: Edições Universitárias Lusófonas. Teodoro, A. (2019). The end of isolationism: Examining the OCDE influence in Portuguese education policies, 1955-1974. Paedagogica Historica: International Journal of the History of Education, 1-14. https://doi.org/10.1080/00309 230.2019.1606022 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 PISA, TIMSS e PIRLS em Portugal 118 PISA, TIMSS e PIRLS em Portugal Informação dos autores: i Universidade Lusófona de Humanidades e Tecnologias, Centro de Estudos Interdisciplinares em Educação e Desenvolvimento (CeiED), Portugal. https://orcid.org/0000-0002-5093-8115 ii Universidade Lusófona do Porto, Centro de Estudos Interdisciplinares em Educação e Desenvolvimento (CeiED), Portugal. http://orcid.org/0000-0003-1466-1013 iii Instituto Politécnico do Porto, Escola Superior de Educação, Centro de Estudos Interdisciplinares em Educação e Desenvolvimento (CeiED), Portugal. https://orcid.org/0000-0001-5854-536X iv Universidade Lusófona de Humanidades e Tecnologias, Centro de Estudos Interdisciplinares em Educação e Desenvolvimento (CeiED), Portugal. https://orcid.org/0000-0001-7819-0498 Toda a correspondência relativa a este artigo deve ser enviada para: Vítor Rosa ULHT/CeiED Campo Grande, 376 1749-024 Lisboa, Portugal vitor.rosa@ulusofona.pt Recebido em 29 de julho de 2019 Aceite para publicação em 4 de junho de 2020 Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 119 PISA, TIMSS e PIRLS em Portugal Keywords: NUTS III Regions; PISA; TIMSS; PIRLS ABSTRACT In this article we present a comparative analysis of three international sur veys: PISA, carried out by OECD, and TIMSS and PIRLS, undertaken by IEA, considering their general objectives and the data correlation on Portugal (by NUTS III regions). The literature and documental review has shown how results can be reformulated or reinterpreted by the different social actors, and that they are seen by the political decision makers and organizations as a tool for school system comparison, revealing strengths and weakness es. The analysis of general results reveals that Portugal’s results has been improving in PISA, in the three domains (reading, mathematics and scienc es), that they have improved in TIMSS in mathematics, while the reading performance in PIRLS has declined. The comparative analysis by regions has shown that there is a difference in results by regions and that, while the domain factor does not seem relevant, the object of evaluation factor in each study seems decisive for the results of the surveys. This comparison has also revealed a direct link between the GDP/inhabitant and the results of the surveys in regions with extreme values of GDP/inhabitant, although this relation does not exist for most regions. Keywords: NUTS III Regions; PISA; TIMSS; PIRLS 120 PISA, TIMSS e PIRLS em Portugal Revista Portuguesa de Educação, 33(1), 94-120. http://doi.org/10.21814/rpe.18380 Palabras clave: Regiones NUTS III; PISA; TIMSS; PIRLS Palabras clave: Regiones NUTS III; PISA; TIMSS; PIRLS RESUMEN En este estudio presentamos un análisis comparativo de tres encuestas in ternacionales: PISA, realizada por la OCDE, y TIMSS y PIRLS, realizadas por la AIE, teniendo en cuenta sus objetivos generales y una correlación de los datos referentes a Portugal (por regiones de la NUTS III). La revisión biblio gráfica y documental mostró cómo los resultados pueden ser reformulados o reinterpretados por los diferentes actores sociales y que son vistos por los responsables políticos y las organizaciones como una herramienta de comparación de los sistemas escolares, revelando sus fortalezas y debilida des. El análisis de los resultados generales muestra que Portugal ha me jorado sus resultados en PISA, en los tres dominios (lectura, matemáticas y ciencias), que mejoró en TIMSS en matemáticas y empeoró en lectura en PIRLS. El análisis comparativo por regiones reveló que existen diferencias de resultados por regiones y que, si bien el factor dominio no es relevante, el factor objeto de evaluación en cada estudio parece ser un factor determi nante de los resultados de los cuestionarios. También reveló que existe una relación directa entre el PIB/habitante y los resultados de los cuestionarios en regiones con valores extremos de PIB/habitante, pero que esta relación no existe para la mayoría de las regiones. Palabras clave: Regiones NUTS III; PISA; TIMSS; PIRLS
https://openalex.org/W4256515532	https://www.qeios.com/read/JF5O24/pdf	English	null	Exposure Lot Number	Definitions	2,020	cc-by	60	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Exposure Lot Number National Cancer Institute National Cancer Institute Qeios ID: JF5O24 · https://doi.org/10.32388/JF5O24 Source National Cancer Institute. Exposure Lot Number. NCI Thesaurus. Code C83099. National Cancer Institute. Exposure Lot Number. NCI Thesaurus. Code C83099. The numerical identifier of the medication exposure lot. 1/1
https://openalex.org/W4385454583	https://rshare.library.torontomu.ca/articles/journal_contribution/Effect_of_electrode_physical_and_chemical_properties_on_lithium-ion_battery_performance/23811207/1/files/41776818.pdf	English	null	Effect of electrode physical and chemical properties on lithium-ion battery performance	null	2,023	cc-by	7,431	Victor Chabot1, Siamak Farhad1, Zhongwei Chen1,,†, Alan S. Fung2, Aiping Yu1and Feridun Hamdullahpur3 1Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada 2Department of Mechanical and Industrial Engineering, Ryerson University, 350 Victoria Street, Toronto, Ontario M5B 2K3, Canada 3Department of Mechanical and Mechatronics Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada INTERNATIONAL JOURNAL OF ENERGY RESEARCH Int. J. Energy Res. 2013; 37:1723–1736 Published online 11 September 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/er.3114 INTERNATIONAL JOURNAL OF ENERGY RESEARCH Int. J. Energy Res. 2013; 37:1723–1736 Published online 11 September 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/er.3114 INTERNATIONAL JOURNAL OF ENERGY RESEARCH gy Published online 11 September 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/er.3114 Published online 11 September 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/er.3114 ACCELERATED PUBLICATION Correspondence Correspondence Zhongwei Chen, Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada. †E il h h @ l Received 19 June 2013; Revised 8 August 2013; Accepted 12 August 2013 Copyright © 2013 John Wiley & Sons, Ltd. SUMMARY The effect of physical and chemical properties on the performance of both positive and negative electrodes is studied for lithium-ion (Li-ion) batteries. These properties include the lithium diffusivity in the active electrode material, the electrical conductivity of the electrode, and the reaction rate constant at electrode active sites. The speciﬁc energy and power of the cells are determined at various discharge rates for electrodes with different properties. In addition, this study is conducted across various cell design cases. The results reveal that at moderate discharge rates, lithium diffusivity in the active negative-electrode material has the highest impact on cell performance. The speciﬁc energy and power of the cell are improved ~11% by increasing the lithium diffusivity in the active negative-electrode material by one order of magnitude. Around 4% improvement in the cell performance is achieved by increasing the reaction rate constant at the active sites of either electrodes by one order of magnitude. Copyright © 2013 John Wiley & Sons, Ltd. KEY WORDS lithium-ion battery; electrodes; material properties; performance improvement; modeling 1. INTRODUCTION LiMn2O4, as an active positive-electrode material is being improved by substitution of nickel and aluminum within its spinel structure [6,7]. Development, altering, or doping materials may signiﬁcantly affect the physical and chemi- cal properties of an electrode. According to our literature survey that is summarized in Table I, some of the electrodes properties may vary several orders of magnitude by alteration or doping. The comprehensive survey accomplished by Park et al. in 2010 [8] also conﬁrms this statement. The value of electrode properties in Table I were determined based on com- plicated experimental methods, and the reader can refer to the cited papers in this table to learn about these methods. Although there are some experimental methods to determine the electrode properties, there is no answer for the question, ‘how much does the change of electrode physical and chemi- cal properties affect cell performance?’ In this study, we answer this question by conducting a comprehensive study on the effect of physical and chemical properties of both the positive and negative electrodes on Li-ion cell performance. The lithium-ion (Li-ion) battery is a high-capacity recharge- able electrical energy storage device with applications in portable electronics and growing applications in electric vehicles, military, and aerospace [1–3]. In this battery, lithium ions move from the negative electrode to the positive electrode and are stored in the active positive-electrode material during discharge. The process is reversed during charging. The lithium intercalation compounds act as active materials to store a large amount of electrical energy and are usually employed to fabricate both negative and positive electrodes of a Li-ion cell. At present, new materials with special properties to improve the cell performance are being developed for both electrodes [4,5]. The commercialized materials are also being altered or doped to enhance certain physical and chemical properties of the electrodes, such as lithium diffusivity, electrical conductivity, and reaction rate constants, to improve cell performance. For example, 1723 1723 V. Chabot et al. Energy storage Table I. The values suggested in literature for physical and chemical properties of electrodes for Lithium-ion cells. 1. INTRODUCTION Material Suggested in literature Lithium diffusivity in active electrode material (m2/s) Natural graphite (LixC6) 1014 to 109 (0.04 < x < 0.4) [9] 1014 to 1013 (x = 0) [10] 1013 to 1011 (0.1 < x < 0.95) [11] Graphitized MCMB (LixC6) 1014 to 1012 (0.15 < x < 0.8) [12] LixMn2O4 1015 to 1013 (x = 1) [13] 1015 to 1012 (0.5 < x < 1) [14] 1.23 × 1015 (x = 1) [15] 1012 (x = 1) [16] 2.7 × 1015 (x = 1) [17] LixCoO2 1014 to 1013 [8] 4 × 1015 to 3 × 1014 (0.45 < x < 0.7) [18] 8 × 1016 to 6 × 1013 (x = 0.5) [19] LixFePO4 2 × 1019 to 1.3 × 1018 (0.1 < x < 0.9) [20] Electrical Conductivity (S/m) Graphite 3 × 103 to 2.5 × 104 [21] 100 [22] LixMn2O4 103.5 to 103 (x ≈1) [14] 104 (x = 1) [8] LixCoO2 102 (x = 1) [8] 20 (x = 1) [23] LixFePO4 107 (x = 1) [8] 108 to 107 (x = 1) [24] Reaction rate constant Graphite 107 m/s [25] 4.92 × 1010 A/m2.5/mol1.5 [26] LiCoO2 5 × 107 m/s [25] 1.39 × 1010 A/m2.5/mol1.5 [26] Although this study can be performed by experimental methods, these methods are complicated and expensive. Thus, we conduct this study based on mathematical modeling. The result of this study is important because identifying the effect of electrode properties might enable the assembly of a cell with better electrode performance and tailored for the discharge rate of a speciﬁc application. material, electrical conductivity, and reaction rate constant at active sites of the electrode – on the speciﬁc energy and power of the cell at various discharge rates are quantiﬁed. To achieve generalized results, this study is conducted for multiple cell design cases including cells with various electrode thicknesses, volume fractions of active electrode material, and initial electrolyte salt concentrations. Computer simulation of Li-ion cells is an efﬁcient tool for guiding material development, pinpointing areas of future improvement, and optimizing cell performance [27,28]. From computer simulation, some electrode prop- erties may be shown to have negligible impact under certain cell design and operating conditions. In this study, the impacts of physical and chemical properties of electrodes – including the lithium diffusivity in active 2. MODELING AND COMPUTER SIMULATION In this study, the mathematical model developed by Newman’s group [29] was employed for computer simulation of the Li-ion cell. In this model, it is assumed that both Figure 1. Schematic of the structure of a Lithium-ion cell (not to scale). Figure 1. Schematic of the structure of a Lithium-ion cell (not to scale). Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er 1724 V. Chabot et al. Energy storage Table II. The modeling equations in general form of partial differential equation, and their corresponding boundary and initial conditions [29]. Table II. The modeling equations in general form of partial differential equation, and their corresponding boundary and initial conditions [29]. Table II. The modeling equations in general form of partial differential equation, and their corresponding boundary and initial conditions [29]. [ ] Transport equation Dependent variable Coefﬁcients of general form partial differential equation 1-D electron transfer in negative electrode ϕe ea = 0, da = 0, Γ ¼ Keff e;neg∇ϕe, f = Sneg jloc,neg B.C: (n. Γ)neg\|sep = 0, ϕeneg CCneg¼0 j I.C: ϕe = 0 1-D electron transfer in positive electrode ϕe ea = 0, da = 0, Γ ¼ Keff e;pos∇ϕe, f = Spos jloc,pos B.C: (n. 2. MODELING AND COMPUTER SIMULATION Γ)pos\|sep = 0, n:Γ ð Þpos CCpos¼icell¼QcellCrate j I.C: ϕe ¼ Eref;pos t¼0 ð Þ Eref;neg t¼0 ð Þ 1-D ion transfer through electrolyte in negative electrode ϕi ea = 0, da = 0, Γ ¼ Keff i;neg ∇ϕi 2RT F 1 tþ ð Þ ∇cLiþ cLiþ , f = Sneg jloc,neg B.C: n:Γ ð Þneg CCneg¼0 j , ϕineg\|sep = ϕisep\|neg I.C: ϕi = 0 1-D ion transfer through electrolyte in positive electrode ϕi ea = 0, da = 0, Γ ¼ Keff i;pos ∇ϕi 2RT F 1 tþ ð Þ ∇cLiþ cLiþ , f = Spos jloc,pos B.C: n:Γ ð Þpos CCpos¼0 j , ϕipos\|sep = ϕisep\|pos I.C: ϕi = 0 1-D ion transfer through electrolyte in separator ϕi ea = 0, da = 0, Γ ¼ Keff i;sep ∇ϕi 2RT F 1 tþ ð Þ ∇cLiþ cLiþ , f = 0 B.C: ϕisep\|neg = ϕineg\|sep, ϕisep\|pos = ϕipos\|sep I.C: ϕi = 0 1-D Li+ mass transfer through electrolyte in negative electrode cLiþ ea = 0, da = εneg, Γ ¼ Deff Liþ;neg∇cLiþ, f ¼ 1 F 1 tþ ð ÞSneg jloc;neg B.C: n:Γ ð Þneg CCneg¼0 j , cLiþneg sep¼cLiþsep neg j j I.C: cLiþ ¼ csalt;initial 1-D Li+ mass transfer through electrolyte in positive electrode cLiþ ea = 0, da = εpos, Γ ¼ Deff Liþ;pos∇cLiþ, f ¼ 1 F 1 tþ ð ÞSpos jloc;pos B.C: n:Γ ð Þpos CCpos¼0 j , cLiþpos sep¼cLiþsep pos j j I.C: cLiþ ¼ csalt;initial 1-D Li+ mass transfer through electrolyte in separator cLiþ ea = 0, da = εsep, Γ ¼ Deff Liþ;sep∇cLiþ, f = 0 B.C: cLiþsep neg¼cLiþneg sep j j , cLiþsep pos¼cLiþpos sep j j I.C: cLiþ ¼ csalt;initial 2-D Li diffusion in active material (solid particles) of negative electrode cLi ea = 0, da = r2, Γ ¼ 0 r2DLi;negactive material ∂cLi ∂r 8 < : 9 = ;, f = 0 B.C: n:Γ ð Þneg CCneg¼0 j , (n. Γ)neg\|sep = 0, (n. 2. MODELING AND COMPUTER SIMULATION Γ)r = 0 = 0, n:Γ ð Þr¼Rparticle;neg ¼ jloc;neg F I.C: cLi = cLi,0,neg 2-D Li diffusion in active material (solid particles) of positive electrode cLi ea = 0, da = r2, Γ ¼ 0 r2DLi;posactive material ∂cLi ∂r 8 < : 9 = ;, f = 0 B.C: n:Γ ð Þpos CCpos¼0 j , (n. Γ)pos\|sep = 0, (n. Γ)r = 0 = 0, n:Γ ð Þr¼Rparticle;pos ¼ jloc;pos F I.C: cLi = cLi,0,pos 2-D Li diffusion in active material (solid particles) of positive electrode Table III. Design and operating parameters of the base cell. Component Parameter Value Negative electrode (Osaka Gas MCMB 25–10 petroleum coke) Thickness of electrode 100 μm Thickness of current collector (copper) 25 μm Density of current collector 8930 kg/m3 Diameter of particles (active electrode material) 25 μm Volume fraction of active electrode material 0.47 Density of active electrode material 1900 kg/m3 Volume fraction of binder 0.146 Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er V. Chabot et al. Energy storage Table III. 2. MODELING AND COMPUTER SIMULATION (Continued) Component Parameter Value Density of binder 1780 kg/m3 Volume fraction of conductive additive material (carbon) 0.026 Density of conductive additive material 1900 kg/m3 Volume fraction of electrolyte (or electrode porosity) 0.357 Diffusivity of Li in active material 3.9 × 1014 m2/s Electrical conductivity 100 S/m Reaction rate constant at active sites of the electrode 1.88 × 106 A/m2.5/mol1.5 Maximum concentration of Li in the active electrode material 26390 mol/m3 Initial concentration of Li in the active electrode material 14870 mol/m3 Positive electrode (LiMn2O4) Thickness of electrode 174 μm Thickness of current collector (aluminum) 25 μm Density of current collector 2700 kg/m3 Diameter of particles (active electrode material) 16 μm Volume fraction of active electrode material 0.297 Density of active electrode material 4140 kg/m3 Volume fraction of binder 0.186 Density of binder 1780 kg/m3 Volume fraction of conductive additive material (carbon) 0.073 Density of conductive additive material (carbon) 1900 kg/m3 Volume fraction of electrolyte (or electrode porosity) 0.444 Diffusivity of Li in active material 1 × 1013 m2/s Electrical conductivity 3.8 S/m Reaction rate constant at active sites of the electrode 2.08 × 106 A/m2.5/mol1.5 Maximum concentration of Li in active electrode material 22860 mol/m3 Initial concentration of Li in active electrode material 3900 mol/m3 Separator and electrolyte Thickness of separator (polyoleﬁn membrane) 52 μm Volume fraction of electrolyte in separator (separator porosity) ~1 Initial salt concentration of electrolyte (LiPF6 in 2 : 1 EC:DMC) 2000 mol/m3 Salt diffusivity of electrolyte 7.50 × 1011 m2/s Cationic transport number in electrolyte 0.363 Density of electrolyte 1324 kg/m3 Total contact resistance between the cell components 97.345 × 104 (Ω m2) Cell temperature 298 K Cell cross-section active area 24 cm2 Component Positive electrode (LiMn2O4) Separator and electrolyte Positive electrode (LiMn2O4) Cell cross-section active area In reactions R1 and R2,Mneg andMpos represent the active materials of the negative and positive electrodes, respectively. negative and positive electrodes of the cell are made of uniform-sized solid spherical particles. These particles are the active material of the electrodes. The size of these particles is assumed to be unchanged during the cell charging and discharging processes. Both electrodes contain uniform composition of binder, conductive additive, and liquid electro- lyte. The volume fraction of liquid electrolyte in electrode and the electrode porosity are assumed to be identical. Reactions R1 and R2 take place in the negative and positive electrodes, respectively, and all side reactions in the cell are neglected. It is assumed that the effective electron and ion conduc- tivities and lithium ion diffusivity that take both porosity and tortuosity of the electrodes into account are estimated from the Bruggeman correlation [29]: Keff ¼ K ε1:5 (1) Deff ¼ D ε1:5 (2) (1) (2) (2) A quasi-two dimensional (2-D) model was considered for computer simulation of the cell. In this model, one dimensional mass and charge transports in both electrodes and electrolyte along x-axis and 2-D lithium diffusion in electrodes solid particles along r-axis and x-axis were taken into account (Figure 1). The cell was simulated by COMSOL Multiphysics® version 4.2 (COMSOL, Inc., Burlington, MA, USA). Lix1Mneg→ discharge Lix2Mneg þ x1 x2 ð Þ Liþ þ x1 x2 ð Þe (R1) (R1) Liy1Mpos þ y2 y1 ð ÞLiþ þ y2 y1 ð Þe→ discharge Liy2Mpos (R2) (R2) 1726 1726 Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er V. Chabot et al. Energy storage Figure 2. Effect of lithium diffusivity in the active positive-elec- trode material on the base-cell performance. Figure 2. Effect of lithium diffusivity in the active positive-elec- In the computer simulation by COMSOL, only the general form of partial differential equations shown in Eq. 3 was employed to build the system of transport equations: ea ∂2u ∂t2 þ da ∂u ∂t þ ∇Γ ¼ f (3) (3) In this equation, u is the dependent variable, and the parameters of ea, da, Γ, and f are the mass coefﬁcient, damping coefﬁcient, conservative ﬂux, and source term, respectively. The value of these parameters for all transport equations in simulation of Li-ion cell are listed in Table II. Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er Cell cross-section active area The active area per unit volume of the electrode and the local current generated per unit active area are determined from Eqs. 4 and 5, respectively [29]: Figure 2. Effect of lithium diffusivity in the active positive-elec- trode material on the base-cell performance. Figure 3. Ragone plots displaying the effect of lithium diffusivity in the active positive-electrode material on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). Figure 3. Ragone plots displaying the effect of lithium diffusivity in the active positive-electrode material on the performance of t cells with (a) thin electrodes (b) thick electrodes (c) low active material volume fraction in electrodes (d) high active material volum Figure 3. Ragone plots displaying the effect of lithium diffusivity in the active positive-electrode material on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). 1727 V. Chabot et al. Figure 4. Effect of lithium diffusivity in the active negative-elec- trode material on the base-cell performance. Sℓ¼ 3νℓ Rparticle;ℓ ; ℓ∈neg; pos f g (4) (4) jloc;ℓ¼ κℓc0:5 Liþc0:5 Li;ℓ; r¼Rparticle;ℓ ð Þ cLi;ℓ;max cLi;ℓ; r¼Rparticle;ℓ ð Þ 0:5 ( exp F ϕe ϕi Eref;ℓ 2RT 0 @ 1 A exp F ϕe ϕi Eref;ℓ 2RT 0 @ 1 A ) ; ℓ∈neg; pos f g (5) (5) To demonstrate the results of the computer simulation, the Ragone plot is employed. This plot is useful to interpret changes in both speciﬁc energy and power of the cell at various discharge rates (Crate). To build this plot, the Figure 4. Effect of lithium diffusivity in the active negative-elec- trode material on the base-cell performance. various discharge rates (Crate). To build this plot, the trode material on the base-cell performance. Figure 5. Cell cross-section active area Ragone plots displaying the effect of lithium diffusivity in the active negative-electrode material on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). inelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Figure 5. Ragone plots displaying the effect of lithium diffusivity in the active negative-electrode material on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). 1728 V. Chabot et al. V. Chabot et al. Energy storage Figure 6. Effect of electrical conductivity of the positive elec- trode on the base-cell performance. speciﬁc energy and power of the cell should be calculated from the following equations [29]: Ecell ¼ icell ð Þ ωcell ∫ tdischarge 0 vcell ð Þ dt ¼ Qcell Crate ð Þ ωcell ∫ tdischarge 0 ϕe;ccpos ϕe;ccneg Rcontact Qcell Crate dt (6) (6) Pcell ¼ Ecell tdischarge (7) (7) Where, tdischarge is the cell discharge time to reach a given cut-off voltage, and ωcell is the total mass per unit area of the cell, which is calculated from Eq. 8. Figure 6. Effect of electrical conductivity of the positive elec- trode on the base-cell performance. Figure 7. Ragone plots displaying the effect of electrical conductivity of the positive electrode on the performance of the cells with (a) hin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). Figure 7. Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er Cell cross-section active area Ragone plots displaying the effect of electrical conductivity of the positive electrode on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). 1729 1099114x, 2013, 14, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/er.3114 by University Of Toronto Mississauga, Wiley Online Library on [31/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online L Energy storage V. Chabot et al. Figure 8. Effect of electrical conductivity of the negative elec- trode on the base-cell performance. ωcell ¼ δneg ρMnegνMneg þ ρel εneg þ ρbinder;negνbinder;neg þρconductive material;negνconductive material;negÞ þδpos ρMposνMpos þ ρel εel;pos þ ρbinder;posνbinder;pos þρconductive material;posνconductive material;posÞ þδsep ρsep νsepþ ρel εel;sep þ ρccnegδccneg þ ρccposδccpos (8) (8) Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er 3. RESULTS AND DISCUSSION A comprehensive parametric study was conducted to investigate the effects of lithium diffusivity in the active material, electrical conductivity, and reaction rate constant at active sites of both electrodes on the speciﬁc energy and power of Li-ion cells. The cell with design and operating conditions listed in Table III was considered as the base Figure 8. Effect of electrical conductivity of the negative elec- trode on the base-cell performance. conditions listed in Table III was considered as the base trode on the base-cell performance. Figure 9. Ragone plots displaying the effect of electrical conductivity of the negative electrode on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). Figure 9. Ragone plots displaying the effect of electrical conductivity of the negative electrode on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). 1730 1099114x, 2013, 14, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/er.3114 by University Of Toronto Mississauga, Wiley Online Library on [31/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Com V. Chabot et al. Energy storage cell in the parametric study. In addition to the base cell, the parametric study was carried out for six different cell de- signs including a cell with thick electrodes (δneg = 200 μm), a cell with thin electrodes (δneg = 50 μm), a cell with high active material volume fraction in the electrodes (νMneg ¼ 0:67), a cell with low active material volume fraction in the electrodes (νMneg ¼ 0:37), a cell with high initial electrolyte salt concentration (csalt,initial = 3 mol/l), and a cell with low initial electrolyte salt concentration (csalt,initial = 0.5 mol/l). In this study, the ratios of the electrodes thickness (δneg/δpos) and the volume fraction of active electrodes material (νMneg=νMpos ) for different cell designs studied were kept constant: volume fractions. 3.2. Lithium diffusivity in the active negative-electrode material The effect of lithium diffusivity in the active material of negative electrode on the base-cell performance is illus- trated in Figure 4. As shown in this ﬁgure, diffusivity signiﬁcantly affects the cell performance. Selection of an active material with lithium diffusivity of less than 3.9 × 1014 m2/s leads to a signiﬁcant decrease in the cell performance. However, attempts to select an active material with diffusivity greater than 3.9 × 1013 would not yield better cell performance. If the lithium diffusivity in the active negative-electrode material of the base cell is boosted by one order of magnitude, speciﬁc energy and power of the cell are enhanced by 11%. From the modeling δneg δpos case study ¼ δneg δpos base cell (9) νMneg νMpos ! case study ¼ νMneg νMpos ! base cell (10) δneg δpos case study ¼ δneg δpos base cell (9) (9) νMneg νMpos ! case study ¼ νMneg νMpos ! base cell (10) (10) Online Library on [31/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by The volume fraction of electrolyte in the electrodes of the cells with high and low active material volume fractions is determined from Eq. 11: Figure 10. Effect of reaction rate constant at active sites of the (a) positive electrode and (b) negative electrode, on the base-cell performance. νel ð Þcase study ¼ νM ð Þbase cell þ νel ð Þbase cell νM ð Þcase study (11) (11) In the following section, the results of the parametric study are presented. 3. RESULTS AND DISCUSSION Active materials with lithium diffusivity less than 1 × 1014 m2/s are not recommended for the positive electrode of cells with thin electrodes. It seems that the electrolyte salt concentration has negligible impact on the effect of this diffusivity on the cell performance. Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er 3.1. Lithium diffusivity in the active positive-electrode material inelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Figure 2 depicts the effect of lithium diffusivity in active material of the positive electrode on the base-cell perfor- mance. The computer simulation results indicated that this diffusivity should be more than 1 × 1014 m2/s to achieve a high cell performance. Of course, attempts to improve this diffusivity would not yield better cell performance if its value is more than 1 × 1013 m2/s. From the modeling point of view, if the measured value for this diffusivity is estimated to be more than one order of magnitude lower, it could signiﬁcantly affect the modeling result. At moderate discharge rates, there will be a 22% error in prediction of both speciﬁc power and energy when diffu- sivity is estimated to be two orders of magnitude lower than in the base cell. In order to have a reliable computer simulation result, it is recommended that special attention is paid to measure the diffusivity for new active positive- electrode materials if its value is less than 1 × 1014 m2/s. In addition to the base cell, the effect of this diffusivity on the cell performance was studied for all six different cell designs introduced in the last section. The results of this study are shown in Figure 3. The results revealed that the effect of lithium diffusivity is less signiﬁcant for the cells with thick electrodes or high active electrode material Figure 10. Effect of reaction rate constant at active sites of the (a) positive electrode and (b) negative electrode, on the base-cell performance. 1731 1099114x, 2013, 14, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/er.3114 by University Of Toronto Mississauga, Wiley Online Library on [31/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for ru V. Chabot et al. Energy storage concentration has negligible impact on the effect of this diffusivity on the cell performance. point of view, inaccurate measurement of this parameter may cause large error in the computer simulation results. At moderate discharge rates, there will be a 40% error in both speciﬁc power and energy of the base cell once diffusivity is estimated to be one order of magnitude lower than in the base cell. Thus, special attention should be paid to selecting the lithium diffusivity mea- surement technique [30]. Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er 3.3. Electrical conductivity of the positive electrode The effect of electrical conductivity of the positive elec- trode on the base-cell performance is demonstrated in Figure 6. The results of the computer simulation indicated that the reduction in electrical conductivity to less than 0.38 S/m leads to a signiﬁcant drop in the cell performance, especially at moderate discharge rates. This states the importance of using a sufﬁcient amount of conductive additive materials in positive electrode to eliminate perfor- mance limitations because of the electrical conductivity. Of course, adding more conductive material to the positive The effect of lithium diffusivity in the active material of the negative electrode on the performance of the cells with various design parameters is shown in Figure 5. The results indicated that the effect of this diffusivity is more signiﬁ- cant for the cells with thin electrodes or low active material volume fractions. For these cell designs, the active material with lithium diffusivities less than 3.9 × 1013 m2/s is not recommended. It seems that the electrolyte salt Of Toronto Mississauga, Wiley Online Library on [31/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of Figure 11. Ragone plots displaying the effect of reaction rate constant at active sites of the positive electrode on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active materia volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). inelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Figure 11. Ragone plots displaying the effect of reaction rate constant at active sites of the positive electrode on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material volume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). 1732 Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er 1099114x, 2013, 14, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/er.3114 by University Of Toronto Mississauga, Wiley Online Li V. Chabot et al. Energy storage Figure 12. 3.3. Electrical conductivity of the positive electrode Ragone plots displaying the effect of reaction rate constant at active sites of the negative electrode on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material vol- ume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). aded from https://onlinelibrary.wiley.com/doi/10.1002/er.3114 by University Of Toronto Mississauga, Wiley Online Library on [31/07/2023]. See the Terms and Conditions (https://onlineli wiley.com/doi/10.1002/er.3114 by University Of Toronto Mississauga, Wiley Online Library on [31/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-cond Figure 12. Ragone plots displaying the effect of reaction rate constant at active sites of the negative electrode on the performance of the cells with (a) thin electrodes, (b) thick electrodes, (c) low active material volume fraction in electrodes, (d) high active material vol- ume fraction in electrodes, (e) low initial electrolyte salt concentration, and (f) high initial electrolyte salt concentration (y-axis in all graphs is speciﬁc energy (Wh/kg), and x-axis is average speciﬁc power (W/kg)). without any signiﬁcant impact on the cell-speciﬁc power and energy. electrode of the base cell will not cause any observable improvement in the cell performance because this elec- trode already has sufﬁciently conductive materials. Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er 4. SUMMARY AND CONCLUSIONS the cells with low and high initial electrolyte salt con- centrations. Therefore, the conductive additive material in fabrication of negative electrodes can be minimized. Of course, it cannot be neglected because this additive material increases the contact surface area between the electrode particles that are point-to-point connected to each other. The increased contact surface area between the particles facilitates the electron migration in the electrode. From the cell modeling point of view, it is not necessary to measure the electrical conductivity of new negative electrodes with high accuracy because it has a negligible effect on the computer simulation results. The impact of lithium diffusivity in active material, electrical conductivity, and reaction rate constant at active sites of both positive and negative electrodes on the speciﬁc energy and power of Li-ion cells was quantiﬁed. A quasi-2-D model was employed for computer simulation of Li-ion cells. The results revealed that lithium diffusivity in the active negative-electrode material has the highest impact on the cell performance. This is followed by the reaction rate constant at active sites of the negative electrode and then positive electrode, lithium diffusivity in the active positive-electrode material, electrical conduc- tivity of the positive electrode, and ﬁnally, the electrical conductivity of the negative electrode. If the lithium diffu- sivity in the active negative-electrode material is boosted by one order of magnitude, the cell-speciﬁc energy and power can be enhanced around 11%. The speciﬁc energy and power can be also enhanced around 4% by improving the active material reaction rate constant in either positive or negative electrodes. It was also revealed that the effect of lithium diffusivity is more signiﬁcant for the cells with thin electrodes or low active material volume fractions. This statement is valid for the reaction rate constant at active sites of the negative electrode. The effect of electri- cal conductivity of the positive electrode on the cell perfor- mance is more signiﬁcant for the cell designs with thick electrodes, low active material volume fractions, and low initial electrolyte salt concentrations. 3.5. Reaction rate constant at electrodes active sites The effect of reaction rate constant at the active sites of the positive and negative electrodes on the base-cell perfor- mance is shown in Figure 10. Even at slow discharge rates, an improvement in the performance of this cell is observed if the reaction rate constant for either positive or negative electrodes is increased to around 2 × 105 A/m2.5/mol1.5. However, no further performance improvement is seen if the reaction rate constant is beyond this value. The speciﬁc power and energy of the base cell can be increased up to 3% or 4% by improving the active material reaction rate constant in either positive or negative electrodes, respectively. From the modeling point of view, underestimation of the reaction rate constant of an electrode by one order of magnitude leads to a 7.5% error in prediction of both speciﬁc energy and power of the cell at mod- erate discharge rates. This error is reduced to around 4.6% for slower discharge rates if the negative-electrode reaction rate constant is underestimated by one order of magnitude. 3.4. Electrical conductivity of the negative electrode The effect of electrical conductivity of the positive electrode on the performance of the cells with various design parameters is shown in Figure 7. The results indi- cated that the effect of electrical conductivity is more signiﬁcant for the cells with thick electrodes (because of a longer electron migration path), low active material volume fractions, or low initial electrolyte salt concentra- tion. For these cell designs, electrical conductivity less than 3.8 S/m is not recommended. For the cell designs with thin electrodes, high active material volume fractions, or high initial electrolyte salt concentration, the conductive addi- tive material in the positive electrode may be reduced The effect of electrical conductivity of the negative elec- trode on the base-cell performance is shown in Figure 8. The computer simulation results conﬁrmed that varying electrical conductivity of the negative electrode from 1 to 104 S/m has no signiﬁcant impact on performance of the base cell at all the discharge rates studied. Fur- ther, Figure 9 illustrates that this statement is valid for the cell designs with thin and thick electrodes, the cells with low and high active material volume fractions, and 1733 V. Chabot et al. Energy storage Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er REFERENCES 1. Karimi G, Li X. Thermal management of lithium-ion batteries for electric vehicles. International Journal of Energy Research 2013; 37:13. 2. Awarke A, Jaeger M, Oezdemir O, Pischinger S. Thermal analysis of a Li-ion battery module under realistic EV operating conditions. International Jour- nal of Energy Research 2013; 37:617. 3. Feng ZC, Zhang Y. Thermal runaway due to symmetry breaking in parallel-connected battery cells. International Journal of Energy Research 2013. doi:10.1002/er.3086. 4. Cai R, Yuan T, Ran R, Liu X, Shao Z. Preparation and re-examination of Li4Ti4.85Al0.15O12 as anode material of lithium-ion battery. 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NOMENCLATURE DOI: 10.1002/er t = time (s) T = temperature (K) t+ = cationic transport number () u = dependent variable in general form of parti differential equation vcell = cell voltage (V) x = spatial coordinate along the cell thickness Greek letters Γ = conservative ﬂux () δ = thickness (m) ε = porosity () ℓ = negative or positive electrode M = denotes the active electrode material κ = reaction rate constant (A/m2.5/mol1.5) ν = volume fraction () ρ = density (kg/m3) ϕ = potential (V) ωcell = mass per unit area of the cell (kg/m2) Subscripts 0 = initial \| = interface cc = current collector e = electron el = electrolyte i = ion neg = negative electrode pos = positive electrode salt = salt in electrolyte sep = separator Superscripts eff = effective Abbreviations 1-D = one dimensional 2-D = two dimensional B.C = boundary conditions I.C = initial conditions ACKNOWLEDGEMENTS The authors gratefully acknowledge the support pro Energy storage V. Chabot et al. NOMENCLATURE = concentration (mol/m3) = discharge rate (/h) = diffusivity (m2/s) = damping coefﬁcient = mass coefﬁcient = cell-speciﬁc energy (Wh/kg) = electromotive force (V) = source term = Faraday’s constant (C/mol) = current applied to the cell (A/m2) = local current generated per unit active area (A/m2) = electrical conductivity (S/m) = lithium ion = normal vector to the cell surface = cell-speciﬁc power (W/kg) = cell capacity (Ah/m2) = spatial coordinate along the electrode particles radius = universal gas constant (J/mol/K) = residual contact resistance between cell components (Ω) = average radius of solid particles (active material) in electrode (m) = electrode active area per unit volume (/m) The effect of reaction rate constant at the active sites of the positive electrode on the performance of the cells with various design parameters is shown in Figure 11. The computer simulation results indicated that the effect of this reaction rate constant on the cell performance is slightly more signiﬁcant for the cells with thin electrodes or high initial electrolyte salt concentrations. No signiﬁcant varia- tion in the effect of this reaction rate constant was observed for the cells with high and low volume fractions of the active material. The effect of reaction rate constant at active sites of the negative electrode on the performance of the cells with various design parameters is illustrated in Figure 12. The results pointed out that the effect of this reaction rate constant on the cell performance is more signiﬁcant for the cells with thin electrodes or low active material volume fraction of electrodes. No signiﬁcant variation in the effect of the reaction rate constant for the cells with high and low initial electrolyte salt concentrations was observed. = average radius of solid particles (active material) in electrode (m) = electrode active area per unit volume (/m) 1734 Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. ACKNOWLEDGEMENTS 14. Marzec J. Conduction mechanism in operating a LiMn2O4 positive electrode. Solid State Ionics 2002; 146:225. The authors gratefully acknowledge the support pro- vided by General Motors, the University of Waterloo, Ryerson University, and Natural Sciences and Engi- neering Research Council of Canada (NSERC). 15. Okumura T, Fukutsuka T, Uchimoto Y, Sakai N, Yamaji K, Yokokawa H. Determination of lithium 1735 Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er V. Chabot et al. Energy storage ion diffusion in lithium–manganese-oxide-spinel thin ﬁlms by secondary-ion mass spectrometry. 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Effect of porosity on the capacity fade of a lithium-ion battery. Journal of the Electrochemical Society 2004; 151:A1104. 18. Jang Y-I, Neudecker BJ, Dudney NJ. Lithium diffu- sion in LixCoO2 (0.45<x<0.7) intercalation cathodes. Electrochemical and Solid-State Letters 2001; 4:A74. 19. Xie J, Imanishi N, Matsumura T, Hirano A, Takeda Y, Yamamoto O. Orientation dependence of Li-ion diffusion kinetics in LiCoO2 thin ﬁlms prepared by RF magnetron sputtering. Solid State Ionics 2008; 179:362. 27. Abdul-Quadir Y, Laurila T, Karppinen J, Paulasto- Kröckel M. Thermal simulation of high-power Li-ion battery with LiMn1/3Ni1/3Co1/3O2 cathode on cell and module levels. International Journal of Energy Research 2013. doi:10.1002/er.3049. 20. Prosini PP, Lisi M, Zane D, Pasquali M. Determina- tion of the chemical diffusion coefﬁcient of lithium in LiFePO4. Solid State Ionics 2002; 148:45. 28. Sakti A, Michalek JJ, Chun S-E, Whitacre JF. A validation study of lithium-ion cell constant c-rate discharge simulation with battery design studio®. International Journal of Energy Research 2012. doi:10.1002/er.2999. 21. Hess M, Lebraud E, Levasseur A. Int. J. Energy Res. 2013; 37:1723–1736 © 2013 John Wiley & Sons, Ltd. DOI: 10.1002/er ACKNOWLEDGEMENTS Graphite multilayer thin ﬁlms: a new negative-electrode material for Li-ion microbatteries synthesis and characterization. Journal of Power Sources 1997; 68:204. 29. Fuller TF, Doyle M, Newman J. Simulation and opti- mization of the dual lithium ion insertion cell. Journal of the Electrochemical Society 1994; 141:1. 22. Doyle M. Comparison of modeling predictions with experimental data from plastic lithium ion cells. Jour- nal of the Electrochemical Society 1996; 143:1890. 30. Han BC, Van der Ven A, Morgan D, Ceder G. Electro- chemical modeling of intercalation processes with phase ﬁeld models. Electrochimica Acta 2004; 49:4691. 23. 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https://openalex.org/W2962768968	https://europepmc.org/articles/pmc6723480?pdf=render	English	null	Modelling of Molasses Fermentation for Bioethanol Production: A Comparative Investigation of Monod and Andrews Models Accuracy Assessment	Biomolecules	2,019	cc-by	7,493	Received: 27 February 2019; Accepted: 15 April 2019; Published: 26 July 2019 Abstract: Modelling has recently become a key tool to promote the bioethanol industry and to optimise the fermentation process to be easily integrated into the industrial sector. In this context, this study aims at investigating the applicability of two mathematical models (Andrews and Monod) for molasses fermentation. The kinetics parameters for Monod and Andrews were estimated from experimental data using Matlab and OriginLab software. The models were simulated and compared with another set of experimental data that was not used for parameters’ estimation. The results of modelling showed that µmax = 0.179 1/h and Ks = 11.37 g.L−1 for the Monod model, whereas µmax = 0.508 1/h, Ks = 47.53 g.L−1 and Ki = 181.01 g.L−1 for the Andrews model, which are too close to the values reported in previous studies. The validation of both models showed that the Monod model is more suitable for batch fermentation modelling at a low concentration, where the highest R squared was observed at S0 = 75 g.L−1 with an R squared equal to 0.99956, 0.99954, and 0.99859 for the biomass, substrate, and product concentrations, respectively. In contrast, the Andrews model was more accurate at a high initial substrate concentration and the model data showed a good agreement compared to the experimental data of batch fermentation at S0 = 225 g.L−1, which was reﬂected in a high R squared with values 0.99795, 0.99903, and 0.99962 for the biomass, substrate, and product concentrations respectively. Keywords: bioethanol; modelling; fermentation; molasses; Monod; Andrews Article Modelling of Molasses Fermentation for Bioethanol Production: A Comparative Investigation of Monod and Andrews Models Accuracy Assessment Hamid Zentou 1,* , Zurina Zainal Abidin 1,* , Robiah Yunus 1, Dayang Radiah Awang Biak 1, Mustapha Zouanti 2 and Abdelkader Hassani 1 Hamid Zentou 1,* , Zurina Zainal Abidin 1,* , Robiah Yunus 1, Dayang Radiah Awang Biak 1, Mustapha Zouanti 2 and Abdelkader Hassani 1 1 Department of Chemical and Environmental Engineering, Universiti Putra Malaysia, Serdang 43400, Malaysia 2 f f l l h l l 1 Department of Chemical and Environmental Engineering, Universiti Putra Malaysia, Serdang 43400, Malaysia 2 Department of Process Engineering, University of Djilali Bounaama, Khemis Miliana 44001, Algeria * Correspondence: hamidzen@gmail.com (H.Z.); zurina@upm.edu.my (Z.Z.A.) 2 Department of Process Engineering, University of Djilali Bounaama, Khemis Miliana 44001, Algeria * Correspondence: hamidzen@gmail.com (H.Z.); zurina@upm.edu.my (Z.Z.A.) 2 Department of Process Engineering, University of Djilali Bounaama, Khemis Miliana 44001, Algeria * Correspondence: hamidzen@gmail.com (H.Z.); zurina@upm.edu.my (Z.Z.A.) Biomolecules 2019, 9, 308; doi:10.3390/biom9080308 biomolecules biomolecules biomolecules 1. Introduction In the last century, an exponential increase in energy consumption and demand has been noted owing to industrial development, population and economic growth, and modernisation. Researchers have taken this seriously to fulﬁl future energy demand [1]. Currently, fossil fuel derived from non-sustainable energy sources is being used as the main source of power [2]. However, the high consumption of fossil fuels has led to certain economic and environmental implications owing to a depletion of fossil fuel reserves and global warming [3,4]. Renewable energy as biofuel, wind energy, solar energy, and hydroelectric energy can be alternate sources of sustainable power generation in order to replace conventional fossil fuel and to limit its implications. Renewable energy usage has increased in recent years, but it is not really widespread as it represents only 4.4% of the primary energy consumption [5]. The new renewable energy resources need to be developed, promoted, and supported for meeting the future needs of energy [6]. Among these renewable energy resources, biofuels such as bioethanol or biodiesel, which are predominantly produced from biomass, are the Biomolecules 2019, 9, 308; doi:10.3390/biom9080308 www.mdpi.com/journal/biomolecules www.mdpi.com/journal/biomolecules 2 of 11 Biomolecules 2019, 9, 308 most promising alternate fuels owing to its advantages in terms of energy sustainability, reduction in the greenhouse eﬀect, and rural areas development [7]. Starch and sugars are currently the main bioethanol production feedstocks, but there has been considerable debate about their sustainability in the recent years due to the competitivity between this sector and nutrition security, especially in the developing countries [8]. Thus, scientists have laid an emphasis on looking for new feedstocks for bioethanol production without implications on the agriculture sector and food security. In this regard, molasses has received more attention as it seems to be a promising feedstock for bioethanol production with a high yield and low cost and without competition to food crops [9]. Several researchers have highlighted the production of bioethanol using the alcoholic fermentation of molasses by Saccharomyces cerevisiae [9–12]. With the growing interest in the industrial application of batch alcoholic fermentation for bioethanol production, diﬀerent kinetic models have been developed to describe microbial growth, product formation, and substrate consumption to serve the bioethanol industry. 1. Introduction Owing to its help in the process control, a reduction in production costs, and an increase in the product quality, mathematical modelling may be regarded as a tool to optimise the fermentation process in order to meet the industry needs [13]. The mathematical modelling of fermentation processes can be classiﬁed into two main categories: structured and unstructured models. In unstructured models, the biomass is regarded as a chemical compound in a solution with an average formula, whereas it is regarded as a number of biochemical compounds in structured models taking into consideration the change in the internal composition of the organism [14]. Batch bioprocesses, in particular, are hard to model owing to the time-varying characteristics of biological systems, which often result in process nonlinearities. The multiplicity of reactions, the adaptability and evolution of organisms over short periods of time, and the continuous shift in environmental conditions are features that characterise batch processes. In this regard, a large number of studies have been conducted on the modelling of batch alcoholic fermentation kinetics. Batch reactors are ﬁrstly used to identify the main phenomena (limitation, inhibition, cell death, and maintenance, among others) that govern the fermentation kinetics by performing speciﬁc experiments for this purpose [15]. The literature related to the modelling of fermentation processes is quite extensive. However, the model presented by Monod (1950) seems to enjoy the widest acceptance model [16]. Table 1 reviews some recent articles highlighting the modelling of alcoholic fermentation using diﬀerent feedstocks based on the Monod model and Andrews model. Table 1. The fermentation kinetics parameters of the Monod model using diﬀerent feedstocks. Substrate Model µmax (h−1) Ks (g.L−1) Ki (g.L−1) Reference Sorghum leaves Monod 0.176 10.11 —- [17] Oil palm frond juice Monod 0.150 10.21 —- [18] Sweet sorghum juice Monod 0.313 47.51 —- [19] Banana peels Monod 1.500 25.00 —- [20] Glucose Monod 0.084 213.60 —- [21] Glucose Monod 0.650 11.39 —- [22] Citrus waste pulp Monod 0.350 10.69 —- [23] Glucose Monod 0.133 3.70 —- [24] Beet molasses Monod 0.355 6.65 —- [23] Soft drinks mixture Andrews 0.606 65.53 0.029 [25] Sucrose Andrews 0.103 30.24 109.8 [26] Sugar cane juice Andrews 0.500 0.006 139.7 [15] Glucose Andrews 0.088 700 3.730 [27] µmax—the maximum speciﬁc growth rate; Ks—the Monod constant; Ki—the substrate inhibition constant. Table 1. The fermentation kinetics parameters of the Monod model using diﬀerent feedstocks. 2.2. Fermentation Anaerobic fermentation was carried out in a batch bioreactor with a 1-L volume containing 250 mL of culture media with diﬀerent initial sugar concentrations (equivalent 5–25 g.L−1 glucose for the Monod model and equivalent of 50–200 g.L−1 glucose for the Andrews model), where the dilution rate was calculated in accordance with the chemical composition of molasses reported by Zentou et al. (2017) [9]. Yeast was added to the prepared fermentation medium with a concentration of 1 g.L−1 (calculated as fresh baker’s yeast). During the fermentation process, the pH value was adjusted at pH = 4.5 by the automatic addition of 0.1 M NaOH and the stirring speed was maintained at 250 rpm. The fermentation temperature was kept at 30 ◦C using a water jacket. The fermentation was carried out under micro-aeration conditions (1 vvm) for 2 h and turned later to anaerobic during the rest time of fermentation. A sample of 5 mL was taken at a predetermined time (0, 2, 4, 6, 8, 10, 12, 18, 24, 30, 36, 48, and 72 h) in order to determine the concentration of sugars, ethanol, and biomass. 2.1. Microorganism and Fermentation Medium Three loops of active dry Saccharomyces cerevisiae yeast from Saf-Levure (Lesaﬀre, Marcq, France) were dissolved in 50 mL of distilled water, which were then added directly into 200 mL of culture media containing diluted molasses (50 g.L−1 glucose), ammonium sulphate (0.7 g.L−1), and ammonium phosphate (0.4 g.L−1). The fermentation medium was incubated at 35 ◦C besides shaking with 250 rpm for 6 h under aerobic conditions. All the used chemicals and materials were sterilized in an autoclave at 121 ◦C for 20 min before the experiment. 1. Introduction The current study aims at developing and validating the Monod and Andrews mathematical models for predicting the dynamics of biomass, substrate, and ethanol for the batch fermentation of molasses using Saccharomyces cerevisiae besides assessing the accuracy of these models when compared with the experimental data diﬀerent from the experimental data set used for the model development. 3 of 11 Biomolecules 2019, 9, 308 2.3. Analytical Methods Yeast growth was evaluated by spectrophotometric measurements at 620 nm in Shimadzu UV-1280 spectrophotometer (Shimadzu Scientiﬁc Instruments, Inc., Kyoto, Japan) and calibrated against the cell dry weight measurements. The concentrations of glucose, fructose, sucrose, and ethanol were measured by high performance liquid chromatography (HPLC) using a Biored Aminex HPX 87H column (Bio-Rad Laboratories, Inc., California, USA) as described in NREL (National Renewable Energy Laboratory, USA) methods [28]. In order to simplify the calculations, the concentration of other sugars (Fructose and sucrose) was converted into the equivalent concentration of glucose. 2.4. Models Theory 2.4.1. Growth Models In the Monod model, an emphasis has been laid to the exponential or logarithmic phase in modelling the microbial cells growth as the formation of the product is found maximum and directly proportional to the microbial cell growth. The growth rate of a batch culture under the exponential phase is generally believed to follow the ﬁrst order kinetic model, i.e., the growth rate is proportional to the microbial mass in the system. Mathematically, dX dt = µX (1) (1) where dX dt denotes the bacterial growth rate (g.L−1.h−1), X (g.L−1) characterises the bacterial cell concentration, and µ (h−1) symbolises the proportional constant known as the speciﬁc growth rate. where dX dt denotes the bacterial growth rate (g.L−1.h−1), X (g.L−1) characterises the bacterial cell concentration, and µ (h−1) symbolises the proportional constant known as the speciﬁc growth rate. F E ti (1) th iﬁ th t (h−1) b itt f ll From Equation (1), the speciﬁc growth rate µ (h−1) can be written as follows: µ = ln(x) −ln(x0) t −t0 (2) (2) Biomolecules 2019, 9, 308 4 of 11 The Monod equation describes the dependence of a microorganism’s growth rate on the concentration of a limiting substrate as follows: µ = µmax S KS + S (3) (3) where µmax is the maximum speciﬁc growth rate (h−1), S is the concentration of the growth limiting substrate (g.L−1), and Ks is the half velocity constant, i.e., the substrate concentration at half of the maximum growth rate (g.L−1). Combining Equations (1) and (3) yields: where µmax is the maximum speciﬁc growth rate (h−1), S is the concentration of the growth limiting substrate (g.L−1), and Ks is the half velocity constant, i.e., the substrate concentration at half of the maximum growth rate (g.L−1). Combining Equations (1) and (3) yields: dX dt = µmaxS KS + S X (4) (4) The viable cell concentrations both in the carriers and the broth were determined and used for µ determination using Equation (2). The µ values were used to evaluate µmax and Ks by Lineweaver–Burk plots derived from Equation (3). In the Andrews model, the growth of S. 2.4.1. Growth Models cerevisiae can be described in relation to the predicted inhibition caused by the excess of the substrate as follows: µ = µmax S KS + S + S2 Ki (5) (5) The experimental growth data was ﬁtted to determine the mathematical parameters (µmax, Ks, and Ki) using Origin 2018a software based on the SubstrateInhib function, where µmax (h−1) is the maximum speciﬁc growth, Ks (g.L−1) is the half velocity constant, and Ki (g.L−1) is the substrate inhibition coeﬃcient. 3. Results and Discussion 3.1. Calculation of Kinetics Parameters 2.4.2. Substrate and Product Models Once µmax, Ks and Ki are calculated and µ is determined, the mathematical model for the substrate consumption and ethanol production can be expressed as follows: dS dt = − 1 Yx/s dX dt (6) dS dt = − 1 Yx/s dX dt (6) dP dt = Yp/s ds dt (7) (6) dP dt = Yp/s ds dt (7) (7) re Yx/s and Yp/s are the substrate- and product-speciﬁc yield coeﬃcients, respectively. where Yx/s and Yp/s are the substrate- and product-speciﬁc yield coeﬃcients, respectively. 2.5. Models Simulations and Validation 2.5. Models Simulations and Validation The models were solved and simulated by using the fourth-order Runge–Kutta method ODE 45 with the MATLAB R2014a software (Version 8.3). The models’ performance was statistically estimated using the coeﬃcient of determination (R2) where the simulation data were validated and compared with the experimental data set that was not used for parameters estimation. 3.1. Calculation of Kinetics Parameters In order to estimate the value of µmax and Ks in the Monod model, a set of batch fermentation using diﬀerent substrate (5–25 g) concentrations was carried out. Figure 1 represents the variation of speciﬁc growth coeﬃcient µ as a function of substrate concentration. omolecules 2019, 9, 308 5 of μ , sing different substrate (5–25 g) concentrations was carried out. Figure 1 represents the variation pecific growth coefficient μ as a function of substrate concentration. Figure 1. The variation of the specific growth rate μ as a function of the substrate concentration S (5– Figure 1. The variation of the speciﬁc growth rate µ as a function of the substrate concentration S (5–25 g.L−1). 5 of 11 ation of Biomolecules 2019, 9, 308 using different subst specific growth coeff iomolecules 2019, 9, 308 5 o g ( g) g p pecific growth coefficient μ as a function of substrate concentration. Figure 1. The variation of the specific growth rate μ as a function of the substrate concentration S (5– Figure 1. The variation of the speciﬁc growth rate µ as a function of the substrate concentration S (5–25 g.L−1). Figure 1. The variation of the specific growth rate μ as a function of the substrate concentration S (5– 25 L 1) Figure 1. The variation of the speciﬁc growth rate µ as a function of the substrate concentration S (5–25 g.L−1). 25 g.L ). It is clear that the specific growth increases with the increase in the substrate concentration and that it achieved the maximum value for S = 20 g.L−1 and stabilized after that despite the augmentation It is clear that the speciﬁc growth increases with the increase in the substrate concentration and that it achieved the maximum value for S = 20 g.L−1 and stabilized after that despite the augmentation of the initial sugar concentration. g p g of the initial sugar concentration. 3.1. Calculation of Kinetics Parameters 6 of 11 6 of 11 6 of 11 Biomolecules 2019, 9, 308 Biomolecules 2019, 9, x FO Biomolecules 2019, 9, x FO Figure 2. A Lineweaver–Burk plot estimating the μmax and Ks values in a batch ethanol fermentation. Figure 2. A Lineweaver–Burk plot estimating the µmax and Ks values in a batch ethanol fermentation. Figure 2. A Lineweaver–Burk plot estimating the μmax and Ks values in a batch ethanol fermentation. Figure 2. A Lineweaver–Burk plot estimating the μmax and Ks values in a batch ethanol fermentation Figure 2. A Lineweaver–Burk plot estimating the µmax and Ks values in a batch ethanol fermentation. Figure 2. A Lineweaver–Burk plot estimating the μmax and Ks values in a batch ethanol fermentation. For the Andrews model, the mathematical model coefficients (μmax, Ks, and Ki) have been estimated for a set of batch fermentation at different substrate concentrations (100, 200, and 300 g.L−1) i i i 2018 ft b d th S b t t I hib f ti For the Andrews model, the mathematical model coeﬃcients (µmax, Ks, and Ki) have been estimated for a set of batch fermentation at diﬀerent substrate concentrations (100, 200, and 300 g.L−1) using origin 2018a software based on the SubstrateInhib function. For the Andrews model, the mathematical model coefficients (μmax, Ks, and Ki) have been estimated for a set of batch fermentation at different substrate concentrations (100, 200, and 300 g.L−1) using origin 2018a software based on the SubstrateInhib function. using origin 2018a software based on the SubstrateInhib function. Figure 3 represents the variations of the specific growth rate μ as a function of the initial substrate concentration S (50–300 g.L−1). The graph shows that the specific growth rate μ increased from 0.225 to 0.250 h−1 with the increase in the glucose concentration in the range of 50–100 g.L−1. However, a remarkable decline in the specific growth rate μ from 0.250 to 0.185 h−1 was noted at the range of 100–300 g.L−1 of the glucose concentration which is contrary to the reported theory in the Monod model. It confirms that the continuity of the specific growth rate increases with the increase in the glucose concentration Figure 3 represents the variations of the speciﬁc growth rate µ as a function of the initial substrate concentration S (50–300 g.L−1). 3.1. Calculation of Kinetics Parameters g µmax and Ks were calculated based on Equation (3), which could be simpliﬁed to Equation (8 d on Equation (3), which could be simplified to Equation (8): 1 𝐾 1 1 1 µ = KS µmax 1 S + 1 µmax (8) ) (8) 1 μ = 𝐾ௌ μ௠௔௫ 1 𝑆 + 1 μ௠௔௫ (8) Figure 2 shows the Lineweaver–Burk plot estimating the μmax and Ks values in a batch ethanol fermentation where the values of μmax and Ks were found to be 0.179 h−1 and 11.37 g.L−1 respectively, which were too close to the values reported in previous studies [17,18] using sweet sorghum leaves and oil palm jus. However, these values were different from the values reported in other studies [19– 23]. μmax was 0.179 h−1, which is half of the value reported by Raposo et al. [23] for molasses fermentation, whereas Ks was almost double as compared to the same study and it amounted to 11.37 g.L−1. This difference may be due to the diversity of the used mediums and their composition, biomass concentration, and strain nature besides the change in operating conditions such as pH, temperature, d h h ff h h h h f h d f f d l Figure 2 shows the Lineweaver–Burk plot estimating the µmax and Ks values in a batch ethanol fermentation where the values of µmax and Ks were found to be 0.179 h−1 and 11.37 g.L−1 respectively, which were too close to the values reported in previous studies [17,18] using sweet sorghum leaves and oil palm jus. However, these values were diﬀerent from the values reported in other studies [19–23]. µmax was 0.179 h−1, which is half of the value reported by Raposo et al. [23] for molasses fermentation, whereas Ks was almost double as compared to the same study and it amounted to 11.37 g.L−1. This diﬀerence may be due to the diversity of the used mediums and their composition, biomass concentration, and strain nature besides the change in operating conditions such as pH, temperature, and aeration which may aﬀect the growth mechanisms. This conﬁrms the need for a speciﬁc model for each feedstock fermentation separately, since it is not practical to generalize a standard model to be applicable to all sets of fermentation. 3.1. Calculation of Kinetics Parameters The graph shows that the speciﬁc growth rate µ increased from 0.225 to 0.250 h−1 with the increase in the glucose concentration in the range of 50–100 g.L−1. However, a remarkable decline in the speciﬁc growth rate µ from 0.250 to 0.185 h−1 was noted at the range of 100–300 g.L−1 of the glucose concentration which is contrary to the reported theory in the Monod model. It conﬁrms that the continuity of the speciﬁc growth rate increases with the increase in the glucose concentration. g g Figure 3 represents the variations of the specific growth rate μ as a function of the initial substrate concentration S (50–300 g.L−1). The graph shows that the specific growth rate μ increased from 0.225 to 0.250 h−1 with the increase in the glucose concentration in the range of 50–100 g.L−1. However, a remarkable decline in the specific growth rate μ from 0.250 to 0.185 h−1 was noted at the range of 100–300 g.L−1 of the glucose concentration which is contrary to the reported theory in the Monod model. It confirms that the continuity of the specific growth rate increases with the increase in the glucose concentration. g Figure 3. The variation of the specific growth rate μ as a function of the substrate concentration S Figure 3. The variation of the specific growth rate μ as a function of the substrate concentration S (50 300 g L-1) Figure 3. The variation of the speciﬁc growth rate µ as a function of the substrate concentration S (50–300 g.L−1). Figure 3. The variation of the specific growth rate μ as a function of the substrate concentration S Figure 3. The variation of the specific growth rate μ as a function of the substrate concentration S (50–300 g.L-1). Figure 3. The variation of the speciﬁc growth rate µ as a function of the substrate concentration S (50–300 g.L−1). (50–300 g.L-1). The Andrews kinetics parameters were estimated using the origin 2018b Software and are presented in Table 2, where μmax, Ks, and Ki were 0.508 h−1, 47.53 (g.L−1), and 181.02 (g.L−1), i l d i d i h hi h (R2 0 9907) The Andrews kinetics parameters were estimated using the origin 2018b Software and are presented in Table 2, where μmax, Ks, and Ki were 0.508 h−1, 47.53 (g.L−1), and 181.02 (g.L−1), respectively, and were estimated with a high accuracy (R2 = 0.9907). 3.3. Model Validation To validate the Monod and Andrews models, the simulation data of the model was compared with the experimental data obtained from the batch fermentation with diﬀerent initial substrate concentrations (75, 150, and 225 g.L−1). A common way to assess the reliability of the model is to use statistical indicators such as the coeﬃcient of determination (R2). Although there is much discussion in the literature about the validity of using R2 to validate a nonlinear model, this statistic can provide a fair ﬁrst indication of how much of the variance in the experimental data is explained by the model. However, a key limitation of R2 is that this statistic cannot determine whether the parameter estimates and predictions are biased, which is why the residual plots must be assessed. Despite this limitation, the value of R2 has been widely accepted to validate the mathematical models of bioprocesses [27–31]. Figure 4 represents the simulation data using Matlab 2014a for the Andrews and Monod models at diﬀerent substrate concentrations (75, 150, and 225 g.L−1) as compared to the experimental data of a series of batch fermentation at the same concentrations (75, 150, and 225 g.L−1). The values of substrate concentration used for the validation of the models are diﬀerent from the concentrations which were used for the modelling which gives the validation more reliability. Figure 4 showed a variance in both models’ accuracy when they were compared to the experimental data. This variance seemed to be dependent to the initial substrate concentration. However, a statistical analysis is needed for more validation. To validate the Monod and Andrews models, the simulation data of the model was compared with the experimental data obtained from the batch fermentation with diﬀerent initial substrate concentrations (75, 150, and 225 g.L−1). A common way to assess the reliability of the model is to use statistical indicators such as the coeﬃcient of determination (R2). Although there is much discussion in the literature about the validity of using R2 to validate a nonlinear model, this statistic can provide a fair ﬁrst indication of how much of the variance in the experimental data is explained by the model. However, a key limitation of R2 is that this statistic cannot determine whether the parameter estimates and predictions are biased, which is why the residual plots must be assessed. 3.2. Calculation of Yield Coeﬃcients The formulation of Equations (9) and (10) are based on the parallel conversion stoichiometry equations. The cell mass yield coeﬃcient (Yx/s) and the product yield coeﬃcient (Yp/s) can be calculated during the growth phase as follows: Yx/s = ∆X ∆s (9) Yp/s = ∆P ∆s (10) (9) (10) Table 3 represents the values of Yx/s and Yp/s respectively in the range between 5 and 25 g.L−1. No signiﬁcant change in both yield coeﬃcients was noted in this range, and the average values of Yx/s and Yp/s were 0.518 and 0.365, respectively. The same was noted for the Andrews model where the average values of Yx/s and Yp/s were 0.286 and 0.431 respectively. Table 3. The variation of the yield coeﬃcients at diﬀerent initial substrate concentrations for the Monod model. Table 3. The variation of the yield coeﬃcients at diﬀerent initial substrate concentrations for the Monod model. Initial Substrate Concentration (g.L−1) Yx/s Yp/s 5 0.267 0.384 10 0.282 0.397 15 0.290 0.446 20 0.278 0.435 25 0.283 0.439 Average 0.280 ± 0.0084 0.420 ± 0.0028 l Validation Table 2. The estimated kinetics parameters ﬁtted to the Andrews model. Table 2. The estimated kinetics parameters ﬁtted to the Andrews model. µ=µmax∗S/KS+S+( S2 ki ) Reduced Chi Squared 7.90971 × 10−6 Adjusted R-Squared 0.99071 µmax (h−1) 0.5086 ± 0.04 Ks (g.L−1) 47.53789 ± 9.27 Ki (g.L−1) 181.01639 ± 29.14 3.2. Calculation of Yield Coeﬃcients 3.2. Calculation of Yield Coeﬃcients 3.1. Calculation of Kinetics Parameters The Andrews kinetics parameters were estimated using the origin 2018b Software and are presented in Table 2, where µmax, Ks, and Ki were 0.508 h−1, 47.53 (g.L−1), and 181.02 (g.L−1), respectively, and were estimated with a high accuracy (R2 = 0.9907). 7 of 11 Biomolecules 2019, 9, 308 3.3. Model Validation Despite this limitation, the value of R2 has been widely accepted to validate the mathematical models of bioprocesses [27–31]. Figure 4 represents the simulation data using Matlab 2014a for the Andrews and Monod models at diﬀerent substrate concentrations (75, 150, and 225 g.L−1) as compared to the experimental data of a series of batch fermentation at the same concentrations (75, 150, and 225 g.L−1). The values of substrate concentration used for the validation of the models are diﬀerent from the concentrations which were used for the modelling which gives the validation more reliability. Figure 4 showed a variance in both models’ accuracy when they were compared to the experimental data. This variance seemed to be dependent to the initial substrate concentration. However, a statistical analysis is needed for more validation. Biomolecules 2019, 9, 308 seemed to be depe 8 of 11 s is needed for more validation. Figure 4. A comparison between the models’ data and the experimental data for x0 = 1 g.L-1 at different Figure 4. A comparison between the models’ data and the experimental data for x0 = 1 g.L−1 at diﬀerent substrate concentrations: (a) S0 = 75 g.L−1, (b) S0 = 150 g.L−1, and (c) S0 = 225 g.L−1. Figure 4. A comparison between the models’ data and the experimental data for x0 = 1 g.L-1 at different Figure 4. A comparison between the models’ data and the experimental data for x0 = 1 g.L−1 at diﬀerent substrate concentrations: (a) S0 = 75 g.L−1, (b) S0 = 150 g.L−1, and (c) S0 = 225 g.L−1. substrate concentrations: (a) S0 = 75 g.L-1, (b) S0 = 150 g.L-1, and (c) S0 = 225 g.L-1. Figure 5 represents the variance of R2 of both models for biomass, substrate, and product concentrations at different initial substrate concentrations (75, 150, and 225 g.L−1). The results represented in Figure 5 show the dependency of both models’ accuracy on the initial substrate concentration. The Monod model showed a good agreement with the experimental data at a low concentration where the maximum R2 values for the biomass, substrate, and ethanol concentrations were achieved at S0 = 50 g.L−1. 3.3. Model Validation The decline in R2 of the Monod model was observed with the increase in the initial substrate concentration from R2 biomass = 0.99956, R2 substrate = 0.99954, and R2 product = 0.99859 to R2 biomass = 0.92462, R2 substrate = 0.921547, and R2 product = 0.916246 for an increase in the initial substrate concentration from 75 to 225 g.L−1. In contrast, the Andrews model showed a good performance at high concentration (225 g.L−1) with R2 equal to 0.99795, 0.99903, and 0.99962 for the biomass, substrate, and product concentrations, respectively. However, the Andrews model was less accurate at a low concentration, with R2 equal to 0.8743932, 0.891686, and 0.90020 for the biomass, substrate, and product concentrations, respectively at S0 = 75 g.L−1. It was also noted that any change in the R2 biomass led to a change in the R2 substrate and the R2 product in both models, which is understood due to the association of the product and substrate expressions with the cell growth expression in both models. Figure 5 represents the variance of R2 of both models for biomass, substrate, and product concentrations at diﬀerent initial substrate concentrations (75, 150, and 225 g.L−1). The results represented in Figure 5 show the dependency of both models’ accuracy on the initial substrate concentration. The Monod model showed a good agreement with the experimental data at a low concentration where the maximum R2 values for the biomass, substrate, and ethanol concentrations were achieved at S0 = 50 g.L−1. The decline in R2 of the Monod model was observed with the increase in the initial substrate concentration from R2 biomass = 0.99956, R2 substrate = 0.99954, and R2 product = 0.99859 to R2 biomass = 0.92462, R2 substrate = 0.921547, and R2 product = 0.916246 for an increase in the initial substrate concentration from 75 to 225 g.L−1. In contrast, the Andrews model showed a good performance at high concentration (225 g.L−1) with R2 equal to 0.99795, 0.99903, and 0.99962 for the biomass, substrate, and product concentrations, respectively. However, the Andrews model was less accurate at a low concentration, with R2 equal to 0.8743932, 0.891686, and 0.90020 for the biomass, substrate, and product concentrations, respectively at S0 = 75 g.L−1. 3.3. Model Validation It was also noted that any change in the R2 biomass led to a change in the R2 substrate and the R2 product in both models, which is understood due to the association of the product and substrate expressions with the cell growth expression in both models. 9 of 11 9 of 11 Biomolecules 2019, 9, 308 Biomolecules 2019 9 x FOR Figure 5. R2 of both models for x0 = 1 g.L-1 at different initial substrate concentrations: (X) R2 for the biomass concentration, (S) R2 for the substrate concentration, and (P) R2 for the ethanol concentration. Figure 5. R2 of both models for x0 = 1 g.L−1 at diﬀerent initial substrate concentrations: (X) R2 for the biomass concentration, (S) R2 for the substrate concentration, and (P) R2 for the ethanol concentration. Figure 5. R2 of both models for x0 = 1 g.L-1 at different initial substrate concentrations: (X) R2 for the biomass concentration, (S) R2 for the substrate concentration, and (P) R2 for the ethanol concentration. Figure 5. R2 of both models for x0 = 1 g.L−1 at diﬀerent initial substrate concentrations: (X) R2 for the biomass concentration, (S) R2 for the substrate concentration, and (P) R2 for the ethanol concentration. Figure 5. R2 of both models for x0 = 1 g.L-1 at different initial substrate concentrations: (X) R2 for the biomass concentration, (S) R2 for the substrate concentration, and (P) R2 for the ethanol concentration. Figure 5. R2 of both models for x0 = 1 g.L−1 at diﬀerent initial substrate concentrations: (X) R2 for the biomass concentration, (S) R2 for the substrate concentration, and (P) R2 for the ethanol concentration. The low accuracy of the Monod model in a high substrate concentration is due to the fact that the classical Monod equation does not consider the inhibitory effect of a high substrate on cell growth. On the other hand, the Andrews model has successfully presented the experimental dataset at a high concentration; however, it does not show the same performance at a low concentration. This can be explained by the estimation of the Andrews model coefficients at high substrate concentrations in order to investigate the inhibitory effect of the substrate which leads to a low accuracy at low initial substrate concentrations as compared to the Monod model. These results confirm the limitation of these models to widely describe the fermentation process. 3.3. Model Validation However, these models can be more practical under specific conditions. Despite the efforts devoted to developing complex models to be more accurate and more practical, a universal model for different fermentation cultures does not exist. Although increasing the model complexity often results in an improved curve fitting, the most appropriate model should be selected on the basis of statistical considerations. Unfortunately, there is evidence that complex equations have often been constructed in an attempt to explain a set of experimental data that exhibited so much scattering that it was impossible to discriminate between the different models. Although both the numerical and analytical methods played a critical role for the advancement of these models, it will still be difficult to find the necessary balance between avoiding an unnecessary complexity and ensuring an adequate reality [32] The low accuracy of the Monod model in a high substrate concentration is due to the fact that the classical Monod equation does not consider the inhibitory eﬀect of a high substrate on cell growth. On the other hand, the Andrews model has successfully presented the experimental dataset at a high concentration; however, it does not show the same performance at a low concentration. This can be explained by the estimation of the Andrews model coeﬃcients at high substrate concentrations in order to investigate the inhibitory eﬀect of the substrate which leads to a low accuracy at low initial substrate concentrations as compared to the Monod model. These results conﬁrm the limitation of these models to widely describe the fermentation process. However, these models can be more practical under speciﬁc conditions. Despite the eﬀorts devoted to developing complex models to be more accurate and more practical, a universal model for diﬀerent fermentation cultures does not exist. Although increasing the model complexity often results in an improved curve ﬁtting, the most appropriate model should be selected on the basis of statistical considerations. Unfortunately, there is evidence that complex equations have often been constructed in an attempt to explain a set of experimental data that exhibited so much scattering that it was impossible to discriminate between the diﬀerent models. Although both the numerical and analytical methods played a critical role for the advancement of these models, it will still be diﬃcult to ﬁnd the necessary balance between avoiding an unnecessary complexity and ensuring an adequate reality [32]. 4 Conclusion 4. Conclusions 4. Conclusion A mathematical model which predicts, simulates, and controls the alcoholic fermentation development would be a valuable technical tool to promote the bioethanol industry and to increase A mathematical model which predicts, simulates, and controls the alcoholic fermentation development would be a valuable technical tool to promote the bioethanol industry and to increase 10 of 11 Biomolecules 2019, 9, 308 its economical competitivity. The Monod model is the most used basic model to simulate alcoholic fermentation. However, diﬀerent modiﬁed models have been developed based on the Monod model to ameliorate the model reliability and acceptance. In the present study, a comparative investigation was conducted to evaluate the performance and reliability of the Monod and Andrews models for batch alcoholic fermentation. However, the results conﬁrm that it is so diﬃcult to deﬁne the most suitable model at diﬀerent microbial growth conditions. However, the suitability of a model can only be determined for a speciﬁc range. p g It was found that the Andrews model showed a better agreement with the experimental data at a high initial substrate concentration due to the consideration of the substrate inhibitory eﬀect. On the other hand, the Andrews model was less accurate at a low initial substrate concentration compared to the Monod model which showed more suitability to represent the experimental data at a low concentration. It is clear that the complexity of a model does not always serve the objective to develop the accuracy of a model but can only be used under certain conditions when the simple models fail to give a real representation of the experimental data. Author Contributions: Conceptualization, H.Z. and Z.Z.A.; methodology, H.Z., Z.Z.A., R.Y., and D.R.A.B; software, M.Z.; validation, H.Z. and M.Z.; investigation, H.Z., A.H.; writing—original draft preparation, H.Z; writing—review and editing, Z.Z.A., A.H.; supervision, Z.Z.A., R.Y., and D.R.A.B; project administration, Z.Z.A.; funding acquisition, Z.Z.A. Author Contributions: Conceptualization, H.Z. and Z.Z.A.; methodology, H.Z., Z.Z.A., R.Y., and D.R.A.B; software, M.Z.; validation, H.Z. and M.Z.; investigation, H.Z., A.H.; writing—original draft preparation, H.Z; writing—review and editing, Z.Z.A., A.H.; supervision, Z.Z.A., R.Y., and D.R.A.B; project administration, Z.Z.A.; funding acquisition, Z.Z.A. Funding: This work was supported by the Malaysian Ministry of Higher Education for sponsoring the work under Fundamental Research Grant Scheme (FRGS/2/2013/TK05/UPM/01/3), Universiti Putra Malaysia for Grant Putra (GP-IPS/2016/9502500), and Aerospace Malaysian Innovative Centre (AMIC) under grant number: 6300801. Acknowledgments: The authors would like to thank Mr. 4 Conclusion 4. Conclusions Rachid Ben Abdulrahman, Head of the Laboratory Department in Rassila Sucre Reﬁnery, for his cooperation and support. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Hasanuzzaman, M.; Rahim, N.A.; Hosenuzzaman, M.; Saidur, R.; Mahbubul, I.M.; Rashid, M.M. Energy savings in the combustion based process heating in industrial sector. Renew. Sustain. Energy Rev. 2012, 16, 4527–4536. [CrossRef] 1. 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https://openalex.org/W2914949082	https://europepmc.org/articles/pmc6353128?pdf=render	English	null	Demand and supply factors of iron-folic acid supplementation and its association with anaemia in North Indian pregnant women	PloS one	2,019	cc-by	6,789	Demand and supply factors of iron-folic acid supplementation and its association with anaemia in North Indian pregnant women 1 Division of Nutrition, St. John’s Research Institute, Bangalore, India, 2 Department of Physiology, St. John’s Medical College, Bangalore, India, 3 Department of Biostatistics, St. John’s Medical College, Bangalore, India 1 Division of Nutrition, St. John’s Research Institute, Bangalore, India, 2 Department of Physiology, St. John’s Medical College, Bangalore, India, 3 Department of Biostatistics, St. John’s Medical College, Bangalore, India * tinku.sarah@sjri.res.in * tinku.sarah@sjri.res.in Editor: Jacobus P. van Wouwe, TNO, NETHERLANDS Received: April 2, 2018 Accepted: December 29, 2018 Published: January 30, 2019 Copyright: © 2019 Varghese 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. RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Varghese JS, Swaminathan S, Kurpad AV, Thomas T (2019) Demand and supply factors of iron-folic acid supplementation and its association with anaemia in North Indian pregnant women. PLoS ONE 14(1): e0210634. https://doi.org/ 10.1371/journal.pone.0210634 Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Anaemia prevalence in pregnant women of India declined from 57.9% to 50.3% from National Family Health Survey (NFHS)-3 to NFHS-4. However, over the course of that decade, the uptake of iron and folic acid (IFA) supplementation for 100 days of pregnancy improved by only 15%. To understand demand side risk factors of anaemia specifically related to IFA intake, an in-depth survey was conducted on pregnant women (n = 436) in 50 villages and wards of Sirohi district of Rajasthan, India. At the demand side, consistent IFA consumption in the previous trimester was inversely and strongly associated with anaemia (OR: 0.26, 95% CI: 0.12, 0.55). Reasons for inconsistent consumption included not register- ing to antenatal clinic, not receiving IFA tablets from the health worker and perceived lack of need. At the supply side, an analysis of IFA stock data at various levels of the health care (n = 168) providers from primary to tertiary levels showed that 14 out of 52 villages surveyed did not have access to IFA tablets. The closest availability of an IFA tablet for 16 villages, was more than 5 km away. To improve the uptake of IFA supplementation and thereby reduce iron deficiency anaemia in pregnant women, a constant supply of IFA at the village or sub-centre level, where frontline workers can promote uptake, should be ensured. Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply significantly in the last 10 years between the nationally representative surveys, NFHS-3 con- ducted in 2005–2006 and NFHS-4 conducted between 2015–2016 among women aged 15–49 years. (Fig 1). Plausible explanations for this continuing burden of anaemia, in spite of an apparently improved, but still not satisfactory compliance to the NIPI program as reported by NFHS sur- veys, are attributed to dietary and environmental reasons. The dietary reasons include an even lower than estimated iron density of diet, inhibition of iron supplement absorption due to diet components such as phytate and polyphenols [6,7] and a predominantly vegetarian diet lead- ing to low intake compared to highly bioavailable heme iron [8–10]. The presence of the “leaky gut” syndrome [11,12] or environmental enteric dysfunction (EED) driven by water, sanitation and hygiene (WASH), hookworm infestation [13], and genetic causes [14,15] are other possible reasons. An equally plausible set of reasons for the continuing prevalence of anaemia relates to the poor coverage of the IFA supplementation. This is expected to reach the pregnant women either through the antenatal services (ANC) or through frontline health workers. Although, the NFHS [3] surveys report an improvement in the uptake of IFA from 15.2% to 30.3% at a national level from round 3 to 4, it is far from being sufficient. There are both demand-side and supply-side problems contributing to the situation. There are important demand-side considerations, such as side effects, poor antenatal care utilization and forgetfulness, which are well known [16–18] and are held responsible for low compliance. However, there are little data on the supply-side deficiencies in an uncontrolled setting, such as relationship between antenatal care provision, health worker motivation [19] and availability of IFA stocks across different public health facilities [20,21], though periodical surveys such as the National Family Health Surveys tend to report some, but not all of these issues. An important logistic compo- nent relates to the distance to health facilities [22–24], that in turn determines health seeking behaviour. This has not been studied in any systematic way, to determine its impact. This paper studies the risk factors associated with anaemia and the coverage of the flagship IFA sup- plementation program (NIPI), among pregnant women in Sirohi district, Rajasthan, and maps the distance to the nearest iron supplements for women in the villages surveyed. Fig 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Introduction Anaemia is characterised by low haemoglobin concentrations and could lead to adverse health outcomes such as maternal and peri-natal mortality and low birth weight [1,2]. In India, par- ticularly among pregnant women, anaemia is a major public health problem, with the recently concluded nationally representative NFHS-4 [3] reporting a prevalence of 50.3%, with not much variation between rural (54.3%) and urban (50.9%) populations. Data Availability Statement: Data are available from GitHub: https://github.com/jvargh7/how-far- is-the-ifa. Iron deficiency is probably the most common cause of anaemia in India [4]. For this reason, under the National Iron Plus Initiative (NIPI, [5]), the government of India provides daily doses of IFA to pregnant women for a period of 100 days during their pregnancy. However, despite revisions to the NIPI programme with an increase in provision of elemental iron from 60 mg to 100 mg, the prevalence of anaemia in pregnant women has not come down Funding: This work was supported by Tata Trusts (RAPID MCN Study), www.tatatrusts.org. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 1 / 13 Survey design A multi-stage cluster sampling of Primary Sampling Units (PSU) which were villages (for rural areas) and wards (for urban areas) as per the Census of India [25] was employed. The sampling was stratified by block, rural-urban status and the PSUs were selected by probability propor- tional to size sampling. The definition of a village in rural areas corresponded to geographically defined villages, that relate to meaningful social and administrative divisions; in urban areas, communities are based on well-defined wards and are considered to be demographically homogeneous [26]. The eligible households were randomly sampled in the next stage of sam- pling for the demand survey. For the supply side component of the survey, the facilities cater- ing to the sampled villages were surveyed. Geographical Information System (GIS) mapping of each village/ward was done. A village representative provided information on distance of vil- lage from different health facilities such as Sub Centre, Primary Health Centre (PHC), Com- munity Health Centre (CHC), Sub Divisional Hospital, District Hospital and Private Clinics, and health workers such as Accredited Social Health Activists (ASHA) and Anganwadi Work- ers (AWW). ASHA workers report to an Auxiliary Nurse Midwife (AN) located at the Sub Centre. In Sirohi, six Sub Centres report administratively to either a PHC or a CHC, which then report to the District Hospital. Village level information on items such as local agricul- tural production, waste disposal, industrial emissions etc. were also collected from the Pan- chayat head or any senior literate and knowledgeable member of a village or ward. Ethical approval Ethical approval was obtained from the institutional ethics committee of the St John’s Medical College and informed written consent was obtained from each of the survey respondents both from the demand and the supply side. Permission from the state health department and the office of the district collector were procured prior to commencement of the survey. Findings from National Family Health Survey-4 related to anaemia in pregnant women. (A) Anaemia among Pregnant women and (B) percentage of women consuming 100 or more IFA tablets in India by district (NFHS- 4). The maps were created from shapefiles available freely from datameet.org using tmap package (https://cran.r- project.org/web/packages/tmap/index.html) in R. Fig 1. Findings from National Family Health Survey-4 related to anaemia in pregnant women. (A) Anaemia among Pregnant women and (B) percentage of women consuming 100 or more IFA tablets in India by district (NFHS- 4). The maps were created from shapefiles available freely from datameet.org using tmap package (https://cran.r- project.org/web/packages/tmap/index.html) in R. https://doi.org/10.1371/journal.pone.0210634.g001 https://doi.org/10.1371/journal.pone.0210634.g001 2 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply Methods An in-depth district level survey was conducted to collect data pertaining to both the demand- side from the perspective of the individual and the supply-side by examining facility level data. To study associated risk factors of anaemia at an individual level resulting from inadequate die- tary intake and low compliance towards IFA tablets, data from survey, conducted in pregnant women in Sirohi District in Rajasthan state between March to June 2016, were used. This survey had collected detailed information on 5,324 households from 52 villages and wards, on maternal and child health in Sirohi, and aimed to identify the major risk factors of anaemia in children (0 to 59 months), adolescent girls (10 to 19 years), pregnant and lactating women, and stunting in children and adolescent girls. The survey also included a supply-side component which aimed to understand the functioning of the health system (from village to district level) in addressing malnutrition. All data other than the biomarkers were collected using questionnaires which were developed in consultation with experts and referring to literature on the current evidence available on nutrition specific and nutrition sensitive factors associated with malnutrition, anae- mia and stunting, surveys conducted in India and elsewhere, and health system guidelines. The questionnaires were pre-tested and translated to local language before being used in the field. Household information (Demand) Household information on possessions, food security and details on education, age, occupa- tion and marital status of members were collected from the head or a responsible adult mem- ber in the household. Data pertaining to medical information and diet of pregnant women 3 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply collected in the survey was considered for this analysis. Trained field staff conducted inter- views and anthropometric measurements of height and weight in all eligible individuals. Hae- moglobin (Hb) was measured from capillary blood samples using Hemocue 301 analyzer in all sampled pregnant women. Pregnant women with Hb levels below 110g/l [27] were classified as anaemic for this analysis. The survey covered 454 pregnant women in Sirohi district, Rajas- than. Out of these, data on 436 subjects who had Hb measurements were considered for this analysis. There was no altitude adjustment of Hb required. Reported symptoms related to anaemia such as fatigue, were collected. IFA demand was measured in terms of receipt and consumption of IFA tablets for each trimester up to the trimester when a pregnant woman was recruited. Diet diversity was measured in terms of the food group consumed and had 11 cate- gories of food groups. Additionally, frequency of consumption of vitamin A and vitamin C rich foods on a monthly basis was also collected. GIS data (Latitude, Longitude and Altitude) for households was measured using Samsung Tab 4—T231 (Samsung Electronics), an Android device commonly used in surveys. Facility information (Supply) To get the perspective from the supply side, health care personnel from 168 facilities (70 Accred- ited Social Health Activists or ASHAs, 52 Anganwadi workers, 30 Sub Centres, 20 Primary Health Centres, 6 Community Health Centres) in Sirohi catering to the villages/wards that were sampled were interviewed. In addition, specific questions on IFA supply was recorded at these centres, both by recording responses from health care providers as well as through observations by field workers of drug kits provided for ASHAs and AWW, and through stock checks in the pharmacy for Sub Centres and Primary Health Centres. GIS data for frontline workers (FLWs) in a village was assumed to be centroid of surveyed households in the village. GIS data for facili- ties was assumed to be location of village/town where the facility was housed, as this informa- tion for facilities was not collected. The distance to the nearest IFA tablet was the distance to the closest health facility which had IFA stocks had the sampled village in its catchment area. Statistical analysis Descriptive statistics were reported as numbers and percentage for categorical data and mean ± standard deviation for continuous normally distributed data. In the individual dataset, the factors associated with anaemia were analysed using chi square test, independent sample t test and Mann-Whitney U Test as appropriate. The significant factors from these analyses were used in bivariate and multiple variable logistic regression of anaemia. The factors considered for analysis in individual data sets were dietary diversity, health service utilization, morbidity, usage of intoxicant substances like paan (betel leaf, arecanut) and tobacco, water, sanitation and hygiene practices. Mobile usage was considered as a proxy for woman’s economic status. The factors were considered in the multiple variable analysis with a cut off value of p < 0.2 in bivari- ate logistic regression. The final model in the multiple variable analysis was identified using a hierarchical iterative process. Confounding and interaction effects were also explored. All anal- yses were performed using R software (https://cran.r-project.org/bin/windows/base/old/3.3.3/) and survey weighted logistic regression was carried with R survey package [28]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 https://doi.org/10.1371/journal.pone.0210634.t001 Results The survey covered a sample of 454 pregnant women of which haemoglobin was measured for 436 (4% non-respondents). There were 110, 187 and 139 pregnant women in their first, second PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 4 / 13 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply and third trimesters respectively out of the 436, at the time of survey. A detailed description of the study population is provided in Table 1. While 66% of the women were anaemic, 35% were moderately or severely anaemic. General fatigue was the most reported anaemia related symptom among the anaemic women (59%) fol- lowed by giddiness, dizziness or headache. Among 299 anaemic women, 37 (12%) did not report any symptom and out of these 17 (6%) were mildly anaemic. Among 436 pregnant women, 303 (69%) reported registering for antenatal care, although the ANC card was avail- able with only 209 (48%) of the respondents. Of the 227 with whom the card was not available, 66 (29%) were in their third trimester and 88 (39%) in the second trimester. About 59% of Table 1. Socio-demographic and economic characteristics of pregnant women in Sirohi district, India. N (%) Number of Pregnant women interviewed 454 Rural 346 (76) Urban 108 (24) Number of Pregnant women for whom Hb was measured 436 (96) Rural 334 (97) Urban 102 (94) Trimester First 110 (25) Second 187 (43) Third 139 (32) Age of household head# 39.6 ± 13.9 Educational level of household head (%) Illiterate 47.9 Primary (1st to 5th grade) 16.5 Type of family Nuclear 258 (59) Extended 47 (11) Joint 131 (30) Main source of income Agriculture 55 (13) Non-agriculture 265 (61) Both 116 (27) Household size# 5.6 ± 2.2 Socio-demographic characteristics of pregnant women Age (yrs.) # 24.9 ± 4.6 Literate 169 (39) Work for income 111 (25) Read state language 140 (32) Write state language 133 (31) Currently married 429 (98) Age at marriage > = 18 years 274 (63) Multiparous 203 (47) Use mosquito net 228 (52) Suffer from malaria in last three months 16 (4) Use mobile phone 266 (61) #- Mean ± SD https://doi.org/10.1371/journal.pone.0210634.t001 able 1. Socio-demographic and economic characteristics of pregnant women in Sirohi district, India. Results 5 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply pregnant women chose to register at a government facility. Those who registered elsewhere (10%) cited quality of care, higher trust of non-government facilities and distance to govern- ment health centre as major reasons for not registering at a government centre. More informa- tion on utilization is available in Table 2. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 https://doi.org/10.1371/journal.pone.0210634.t002 Factors associated with anaemia in pregnant women The factors associated with anaemia in pregnancy in the bivariate and multiple variable logistic regression analyses are presented in Table 3. The final logistic regression model showed IFA supplementation to be negatively associated with anaemia such that there was 74% lower odds of anaemia with consistent consumption of IFA or folic acid supplement in previous trimester (OR: 0.26, 95% CI: 0.12 to 0.55, p = 0.001). In addition, month of pregnancy (OR: 1.21, p = 0.012), mobile usage (OR: 0.41, p = 0.010), usage of intoxicant substances (OR: 2.27, Table 2. Antenatal care and morbidity of pregnant women in Sirohi district, India (N = 436). Antenatal services received by pregnant women N (%) Received antenatal check-up 208 (48) Physical Examination 65 (15) Measurement of weight 62 (14) BP measured 59 (13) Received TT vaccination 48 (11) Consumed IFA in previous month’s trimester^ 155 (36) Folic Acid received in 1st trimester as per ANC card# 4 (4) Folic Acid consumed in 1st trimester (previous month’s trimester = 1)^# 30 (20) IFA received in 2nd or 3rd trimester as per ANC card 47 (14) IFA consumed in 2nd/3rd trimester (previous month’s trimester = 2 or 3; N = 270) 125 (46) Ever Received deworming tablet as per ANC card during pregnancy 11 (2) Received professional breastfeeding counselling as per ANC card 14 (4) Hb examined at health centre as per ANC card 71 (29) Prevalence of Anaemia 299 (69) Mild anaemia 146 (34) Moderate anaemia 140 (32) Severe anaemia 13 (3) Morbidity (last one month) Diarrhoea 64 (15) Vomiting 204 (47) Fever 117 (27) Symptoms related to anaemia General fatigue 259 (59) Breathlessness on routine and somewhat strenuous work 156 (36) Giddiness, dizziness, headache 197 (45) Appetite loss 125 (29) Weight loss 57 (13) Blurring of vision 149 (34) Sudden swelling of feet 68 (16) ^—calculation does not include those women (N = 13) in their first month of pregnancy; #—Folic Acid is provided in Trimester 1; —IFA is provided in Trimester 2 and 3 htt //d i /10 1371/j l 0210634 t002 2. Antenatal care and morbidity of pregnant women in Sirohi district, India (N = 436). 6 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 https://doi.org/10.1371/journal.pone.0210634.t003 Factors associated with IFA consumption in pregnant women Since consistent consumption of iron and folic acid or folic acid tablets is associated with anae- mia, the timely receipt of IFA supplements by pregnant women in Sirohi is critical and is examined in Fig 2. Among those who did not receive IFA, ‘no provision’ from the health worker was the most common reason. Among those who received supplements and did not consume them, low palatability, unrealized need, constipation and forgetfulness were the major reasons. ANC registration was significantly associated with consumption of IFA or folic acid supple- ments by pregnant women in the previous month’s trimester (χ2 = 69.7, p<0.01, N = 423). Majority of those who did not consume did not receive IFA (88%). Table 4 provides a sum- mary of ANC registration, IFA supplementation status and health worker visit. There was no association between health worker visit and receiving IFA (χ 2 = 2.8, p>0.05, N = 326) in Tri- mester 2 and 3. A health worker was more likely to visit those pregnant women who were reg- istered under ANC (χ 2 = 29.7, p<0.01, N = 436). Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply Table 3. Individual and household characteristics associated with anaemia outcome in pregnant women of Sirohi district, India. Unadjusted Adjusted Individual characteristics Odds Ratio P-value Odds Ratio P-value Month of Pregnancy 1.13 0.07 1.211 0.012 Previous month’s trimester 1.58 0.05 Writing state language 0.49 0.09 Mobile usage 0.29 <0.001 0.409 0.010 Low BMI 2.04 0.04 Usage of tobacco 3.20 0.03 2.268 0.075 Past history of seizures 2.45 0.06 Awareness of health and nutrition programs 0.95 0.08 Receipt of blood transfusion in last 1 year 9.74 0.03 Dietary consumption vegetables and pulses 0.62 0.02 Guava 1.33 0.09 Jackfruit 0.63 0.02 Papaya 1.69 0.05 Pumpkin 0.02 0.04 Avoid meat 9.10 0.03 Avoid poultry 23.31 0.01 Avoid fish 4.55 0.09 Avoid eggs 4.61 0.10 ICDSa Consuming ICDS Supplementary Nutrition alone 2.82 0.05 Consuming ICDS Supplementary Nutrition completely 2.49 0.10 Antenatal Care Consistent IFA/FA consumed (every day in last month’s trimester) 0.29 <0.001 0.258 0.001 Never Received IFA tablets 1.64 0.06 ANC Check-up in 3rd Trimester 0.89 0.04 Mobile Registration for ANC- Registered vs Not registered 4.17 0.01 WASH General use of soap 0.19 0.05 Brushing teeth at least once a day 0.35 0.06 Health seeking behaviour index 0.86 0.07 Household Characteristics Altitude 0.99 0.07 Total Household Members 0.91 0.05 Number of women in household 0.70 0.04 0.724 0.079 Relationship to head of household 6.01 0.06 Non-Hindu religion 6.13 0.05 Wealth Index 0.88 0.02 Wealth Quintile 0.83 <0.001 NREGA beneficiary 2.42 0.05 Expected number of pregnant women in village 0.994 0.575 aICDS- Integrated Child Development Services scheme. Odds ratio from survey weighted logistic regression analysis. Adjusted odds ratio after including all variables PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 7 / 13 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply p = 0.075), number of women in household (OR: 0.72, p = 0.079) were also significantly associ- ated with anaemia (Table 3). PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Availability of IFA to pregnant women Fig 3 represents the location of different facilities, administrative relationships between the facilities and the status of their IFA stock spatially. Table 5 contains information on health facilities with IFA supple- ments and their proximity to villages. availability of IFA, for the remaining villages the nearest tablet was at geodesic distance of 3.6 km [IQR: 1.2, 8.5] while the reported distance was 5 km [IQR: 0.5, 10]. For 15 villages, the nearest IFA tablets were more than 5 km away (geodesic) from the source. Fig 3 represents the location of different facilities, administrative relationships between the facilities and the status of their IFA stock spatially. Table 5 contains information on health facilities with IFA supple- ments and their proximity to villages. Fig 3 provides administrative linkages between different health facilities and the status of their IFA stocks. We randomly shifted the location of the village to North, South, East or West by 5km for confidentiality purposes. “1” refers to an ideal case where all facilities from village level onwards have IFA stocks. “2” refers to a non-ideal case where none of the facilities, except at the district level have IFA stocks. Approximately 50% of villages do not have IFA stocks below or at Sub-Centre level. Both geodesic (r = -0.21, p = 0.15) and reported distance (r = -0.18, p = 0.23) to the health facility with IFA supplements were negatively (but not significantly) correlated with the pro- portion of women receiving IFA supplementation in the village. Availability of IFA to pregnant women Fourteen of 52 surveyed villages in Sirohi did not have access to an IFA tablet except at the CHC or District Hospital level. Nineteen villages had access at the ASHA or Anganwadi level, while 9 and 10 villages had access at the sub-centre and PHC level respectively. Distance between two geographic locations, also known as the geodesic, was calculated as the shortest distance between two points on an ellipsoid, namely WGS84 [29]. Reported distance to each type of facility, per information from the village representative, was also recorded. There are 4 villages which had IFA availability only at the District Hospital level (as per referral structure) which was 31.5 km (median reported distance) from the villages. On the other end, among the remaining villages 17 had IFA tablet available with either the ASHA or Anganwadi worker within the village at geodesic distance 0.6 km [IQR: 0,5.5], while the reported median distance was 0 km [IQR: 0, 7.2]. Excluding the farthest and nearest Fig 2. Reporting of consumption and receipt of iron and folic acid supplements. Values are number of subjects. https://doi.org/10.1371/journal.pone.0210634.g002 Fig 2. Reporting of consumption and receipt of iron and folic acid supplements. Values are number of subjects. https://doi.org/10.1371/journal.pone.0210634.g002 https://doi.org/10.1371/journal.pone.0210634.g002 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply Table 4. Antenatal care registration and health worker visits in surveyed population (N = 436). Health worker visits regularly Health worker does not visit ANC Registered 135 168 Did not receive IFA (Trimester 1) 19 (14%) 20 (12%) Received IFA (Trimester 2 and 3) 24 (18%) 23 (14%) Did not receive IFA (Trimester 2 and 3) 92 (68%) 125 (74%) ANC Not Registered 23 110 Did not receive IFA (Trimester 1) 12 (52%) 59 (54%) Received IFA (Trimester 2 and 3) 0 (0%) 0 (0%) Did not receive IFA (Trimester 2 and 3) 11 (48%) 51 (46%) Eligible for folic acid; IFA- Iron and Folic Acid https://doi.org/10.1371/journal.pone.0210634.t004 Table 4. Antenatal care registration and health worker visits in surveyed population (N = 436). availability of IFA, for the remaining villages the nearest tablet was at geodesic distance of 3.6 km [IQR: 1.2, 8.5] while the reported distance was 5 km [IQR: 0.5, 10]. For 15 villages, the nearest IFA tablets were more than 5 km away (geodesic) from the source. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Discussion The burden of anaemia was high at 66% in pregnant women of Sirohi district and consistent consumption of IFA/folic acid supplements during the previous trimester of pregnancy was the single most strongly inversely associated factor for anaemia. In addition low mobile usage, low number of women in household, use of tobacco and paan, late month of pregnancy increased the odds of anaemia. Of these, mobile usage (possibly a proxy for latent unmeasured variables such as woman’s empowerment and access to information in addition to woman’s economic status), substance use and IFA/folic acid intake are factors amenable to intervention. Poor consumption of IFA supplements is a key risk factor contributing to anaemia among pregnant women in Sirohi. Though anaemia is a complex and multi-faceted problem which involves interaction of various nutrients, iron supplementation could alleviate a considerable proportion [30] of anaemia in India. As observed in Sirohi, a low intake of IFA supplements resulting from a combined effect of low stocks and poor demand drives anaemia among preg- nant women. There was no significant difference of IFA receipt between women those who were visited by a health worker and those who were not. A possible explanation for this could be low stocks of IFA with frontline workers and IFA supply only from centralized locations such as Sub-centres or Anganwadi centres and not at homes. It could also mean that the health worker responsible for distributing IFA tablets was not the one who visited pregnant women at their homes. However, other studies have shown that antenatal care utilization can be PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 9 / 13 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply Fig 3. Geographical distribution of health facilities, inter-facility relationships and availability of IFA supplementation. The polygon was filtered from the shape file and modified using ggplot2 package (https://cran.r-project.org/web/packages/ggplot2/index.html) in R. https://doi.org/10.1371/journal.pone.0210634.g003 Fig 3. Geographical distribution of health facilities, inter-facility relationships and availability of IFA supplementation. The polygon was filtered from the shape file and modified using ggplot2 package (https://cran.r-project.org/web/packages/ggplot2/index.html) in R. https://doi.org/10.1371/journal.pone.0210634.g003 https://doi.org/10.1371/journal.pone.0210634.g003 improved from health worker visits [31]. The present study also shows that health worker visits are more likely for those women who are registered for antenatal care. It is important to note that more than one-fourth of the villages surveyed did not have access to IFA supplements within a 5 km radius. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 https://doi.org/10.1371/journal.pone.0210634.t005 Discussion As per Fig 2, most women did not consume IFA supplements not because of perceived side-effects but because they were not provided the same. NFHS-4 reports only 25.9% of pregnant women in rural areas consume 100 IFA tablets Table 5. Distance from different levels of public health facilities where IFA is available to surveyed villages. IFA Available with N Geodesic distance (km) Reported distance (km) Asha 10 0 (Within village) 0 (Within village) Anganwadi 7 0 (Within village) 0 (Within village) Sub Centre 8 1.06 (IQR: 0.5 to 2.5) 0.5 (IQR: 0 to 3.5) PHC 10 7.9 (IQR: 4.4 to 8.9) 10 (IQR: 7.3 to 11.8) CHC 9 2.2 (IQR: 1.2 to 8.7) 2.5 (IQR: 0 to 10) District Hospital 4 22.6 (IQR: 16.2 to 24.3) 31.5 (IQR: 22.25 to 38.75) IQR: Inter Quartile Range https://doi.org/10.1371/journal.pone.0210634.t005 Table 5. Distance from different levels of public health facilities where IFA is available to surveyed villages. 10 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 10 / PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply during pregnancy. Other studies in South Asia have reported that geographic proximity to health service is important in its utilization, especially for antenatal care, chronic diseases and infectious diseases [22,23]. To ensure stocks are in place in all health facilities, the local govern- ment could re-evaluate the supply chain management information system in place as well as provide for other measures such as buffer stocks and pharmacy budgets for health facilities. If so, health workers could plan the effective generation of demand in the community. For this, at least two actions are required; that the supply of IFA should be proactive and constant at the village level or the sub-centre, and that the distance to the nearest tablet should be at least at the Sub Centre level if not at the village level ensuring that visiting health workers (ANMs, ASHAs) have stock and would be able to supply pregnant women with IFA tablets. Anaemia as a result of use of substances such as paan (betel leaf and/or betel nut) or tobacco is well-known [32,33]. Almost 40 (9%) pregnant women consumed paan masala or chewable tobacco on a routine basis. Smokeless tobacco [32] during pregnancy is observed to decrease haemoglobin levels, similar to cigarette smoking. Conclusion The prevalence of anaemia among pregnant women in Sirohi was higher than the national prevalence. Anaemia in pregnant women was associated with inconsistent consumption of IFA/FA. IFA not supplied by health worker was identified as demand side factor associated with inconsistent IFA consumption. With more than half the sample of pregnant women not being visited by health worker during pregnancy, it is critical that IFA is available at the nearest health facility. However, it was observed that about 50% of the villages did not have IFA avail- able at the nearest health facility. While anaemia among pregnant Indian women continues to be a priority issue for the government, focussed interventions such as regular haemoglobin testing and ensuring successful implementation of the National Iron Plus Initiative nuanced for the local context and access to IFA can go a long way in meeting anaemia reduction targets in India. Discussion In addition to reducing maternal red blood cell count, the habit can also lead to decreased serum Vitamin B12 and maternal red blood cell folate. The increasing odds of anaemia with month of pregnancy is a well-known phenome- non, often referred to as the physiological anaemia of pregnancy [34]. This analysis is not without limitations. Firstly, we surveyed only 52 PSUs (villages and wards) of which, reported distance to health facilities were available only for 48. However, the PSUs were sampled based on a probability proportional to size (PPS) design and ought to be representative of the district. Secondly, access to different health facilities were not considered when estimating the distance to the nearest IFA supplement. It is possible [35,36] that a health facility is close to a village, but not accessible due to socio-cultural reasons. Thirdly, distance and receipt of IFA supplementation were not significantly associated at a village level. As a result, the optimal location of the health facility for optimal provision of IFA cannot be defined. Fourth, since the road networks in the rural part of the district is poor, geodesic might not convey the true picture of distance. We tried to overcome this limitation by using the reported distance to the health facility as reported by the village representative along with the geodesic. Finally, it is possible that the stock-out of IFA which our team observed (we did not assess folic acid stocks) prevailed only for the duration of the survey period. This limitation is common to all cross-sectional studies. Author Contributions Conceptualization: Sumathi Swaminathan, Anura V. Kurpad, Tinku Thomas. Data curation: Jithin Sam Varghese. Formal analysis: Jithin Sam Varghese, Tinku Thomas. Funding acquisition: Anura V. Kurpad. Investigation: Jithin Sam Varghese. Methodology: Sumathi Swaminathan, Anura V. Kurpad, Tinku Thomas. Project administration: Sumathi Swaminathan, Tinku Thomas. Software: Jithin Sam Varghese. Supervision: Sumathi Swaminathan, Tinku Thomas. Visualization: Jithin Sam Varghese. Writing – original draft: Jithin Sam Varghese. Writing – review & editing: Sumathi Swaminathan, Anura V. Kurpad, Tinku Thomas. Conceptualization: Sumathi Swaminathan, Anura V. Kurpad, Tinku Thomas. Methodology: Sumathi Swaminathan, Anura V. Kurpad, Tinku Thomas. Project administration: Sumathi Swaminathan, Tinku Thomas. Supervision: Sumathi Swaminathan, Tinku Thomas. Supervision: Sumathi Swaminathan, Tinku Thomas. Visualization: Jithin Sam Varghese. Writing – original draft: Jithin Sam Varghese. Writing – original draft: Jithin Sam Varghese. Writing – review & editing: Sumathi Swaminathan, Anura V. Kurpad, Tinku Thomas. Writing – review & editing: Sumathi Swaminathan, Anura V. Kurpad, Tinku Thomas. Acknowledgments The authors would like to acknowledge Divya Mishra, Dr. Nidhi Jaikrishna, Shruthi Cyriac, Sumedha Minocha, Dr. Swetha Rajeswari, Dr. Khushbu Gandhi and Vijay Singh for assisting 11 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210634 January 30, 2019 Poor compliance to the IFA supplement in anaemic North Indian pregnant women: Demand vs supply in field coordination and data pre-processing support. The authors would also like to acknowl- edge Dr. Prabal V. Singh, Dr. Navin Kumar. Dr. Nayan Kumar and Pooja Singh of Oxford Pol- icy Management Ltd. for assisting in the data collection process. in field coordination and data pre-processing support. The authors would also like to acknowl- edge Dr. Prabal V. Singh, Dr. Navin Kumar. Dr. Nayan Kumar and Pooja Singh of Oxford Pol- icy Management Ltd. for assisting in the data collection process. References 1. World Health Organization. The global prevalence of anaemia in 2011. 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https://openalex.org/W3030560551	https://europepmc.org/articles/pmc7312192?pdf=render	English	null	Estimated Cardiorespiratory Fitness Attenuates the Impacts of Sarcopenia and Obesity on Non-Alcoholic Fatty Liver in Korean Adults	International journal of environmental research and public health/International journal of environmental research and public health	2,020	cc-by	7,488	Received: 23 April 2020; Accepted: 29 May 2020; Published: 31 May 2020 Received: 23 April 2020; Accepted: 29 May 2020; Published: 31 May 2020 Abstract: This population-based, cross-sectional study examined the preventive role of non-exercise-based estimation of cardiorespiratory ﬁtness (eCRF) against the impacts of sarcopenia and obesity on the non-alcoholic fatty liver (NAFL) in Korean adults. Data were obtained from the 2008–2011 Korea National Health and Nutrition Examination Surveys IV and V (n = 14,015 Koreans aged ≥18 years, 64% women). eCRF was calculated with the age- and sex-speciﬁc algorithms, and classiﬁed as lower (lowest 25%), middle (middle 50%) and upper (highest 25%). Individuals were classiﬁed as optimal (i.e., the absence of both sarcopenia and obesity), sarcopenia (i.e., the presence of sarcopenia), obesity (i.e., the presence of obesity) or sarcopenic obesity (i.e., the coexistence of sarcopenia and obesity). Limited to the sarcopenia phenotype, the adjusted odds ratio (OR) of NAFL was 2.2 (95% conﬁdence interval, CI, 1.5–3.1) for the lower eCRF, 1.6 (95% CI, 1.3–2.1) for the middle eCRF and 2.1 (95% CI, 1.4–3.1) for the upper eCRF, compared to the optimal phenotype. Limited to the obesity phenotype, the adjusted OR of NAFL was 2.9 (95% CI, 2.0–4.2) for the lower eCRF, 3.5 (95% CI, 2.7–4.6) for the middle eCRF and 1.8 (95% CI, 1.2–2.8) for the upper eCRF, compared to the optimal phenotype. Limited to the sarcopenic obesity phenotype, the adjusted OR of NAFL was 5.9 (95% CI, 4.3–8.2) for the lower eCRF, 4.2 (95% CI, 3.2–5.5) for the middle eCRF and 2.5 (95% CI, 1.5–4.1) for the upper eCRF, compared to the optimal phenotype. The current ﬁndings suggest that high eCRF attenuates the individual and synergistic impacts of sarcopenia and obesity on NAFL in Korean adults. Keywords: fatty liver disease; physical ﬁtness; physical activity; sarcopenia; obesity International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2020, 17, 3902; doi:10.3390/ijerph17113902 www.mdpi.com/journal/ijerph 1. Introduction Non-alcoholic fatty liver disease (NAFLD) encompasses two subtypes of non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). The global prevalence of NAFLD is estimated to be approximately 25% in the general population [1], although it varies from 15% to 70% depending on age, sex, ethnicity and diagnostic method [2,3]. The prevalence of NAFLD is projected to rise, due to an increase in Westernized aﬄuent and sedentary lifestyles [4]. NAFL represents an excessive accumulation of hepatic fat in the absence of any signiﬁcant inﬂammation or ﬁbrosis. Pathologically, NAFL is closely associated with metabolic complications, such as hyperinsulinemia or dyslipidemia, resulting in such adverse health outcomes as type 2 diabetes [5] and cardiovascular diseases (CVDs) [6]. Obesity and cardiorespiratory ﬁtness (CRF), which reﬂects the ability of the respiratory and circulatory systems to deliver oxygen to working skeletal muscles during exercise, have been well established as independent predictors of metabolic syndrome [7], diabetes [8] and CVDs [9]. The two lifestyle risk factors are also associated with NAFL [10,11]. In addition, sarcopenia is known as another novel risk factor of NAFL in Western [12,13] and Asian populations [14–16], collectively suggesting Int. J. Environ. Res. Public Health 2020, 17, 3902; doi:10.3390/ijerph17113902 www.mdpi.com/journal/ijerph www.mdpi.com/journal/ijerph 2 of 12 Int. J. Environ. Res. Public Health 2020, 17, 3902 that an interaction among adipose tissue, skeletal muscle and the cardiorespiratory system may play an important role in the pathophysiology of NAFL [17]. In Korea, the prevalence of NAFL is steadily rising in those middle aged and older, particularly in men with central obesity [18], and in individuals with sarcopenia [16] or poor CRF [19]. Poor CRF increases the risk of both sarcopenia and visceral obesity in apparently healthy Korean adults [19]. Therefore, examining the individual and synergistic impacts of lifestyle risk factors on NAFL will provide critical information and help develop better therapeutic strategies for patients with NAFL. p g p An objective measurement of CRF, which requires laboratory-based assessments with gas analysis, is often limited due to such requirements as specialized equipment and trained personnel, as well as being time-consuming, especially in a large population-based study. Fortunately, CRF can be estimated with sex- and age-speciﬁc algorithms, developed on the basis of routinely-obtained health indictors, with acceptable accuracy [20]. 1. Introduction To the best of our knowledge, however, no previous study has investigated whether or not eCRF is a moderator in determining the association of sarcopenia and/or obesity with NAFL in the Korean population. This study aimed to investigate the protective role of eCRF against the synergistic impacts of sarcopenia and obesity on NAFL in Korean adults. 2 2 1 Definition of Obesity and Sarcopenia 2.2.1. Deﬁnition of Obesity and Sarcopenia y p Asians, including Koreans, have a higher risk of central obesity than Western populations, due to a higher chance of central fat deposition, despite having lower BMI [22]. More importantly, BMI fails to account for changes in body composition with aging, specifically the age-related decline in muscle mass, strength and function [23]. Consequently, WC is known as a better indicator of visceral adiposity, and is more closely associated with insulin resistance (IR) and its related metabolic disorders such as NALFD [24,25], especially in Koreans [26,27]. In this study, therefore, the same Korean-specific WC threshold for metabolic syndrome was used to determine central obesity: WC ≥ 90 cm in men or ≥85 cm in women [28] Asians, including Koreans, have a higher risk of central obesity than Western populations, due to a higher chance of central fat deposition, despite having lower BMI [22]. More importantly, BMI fails to account for changes in body composition with aging, speciﬁcally the age-related decline in muscle mass, strength and function [23]. Consequently, WC is known as a better indicator of visceral adiposity, and is more closely associated with insulin resistance (IR) and its related metabolic disorders such as NALFD [24,25], especially in Koreans [26,27]. In this study, therefore, the same Korean-speciﬁc WC threshold for metabolic syndrome was used to determine central obesity: WC ≥90 cm in men or ≥85 cm in women [28]. 90 cm in men or ≥ 85 cm in women [28]. Appendicular skeletal muscle mass (ASM) was defined as the sum of the muscle mass in the arms and legs, measured with dual energy X-ray absorptiometry (DEXA) (DISCOVERY-W fan-beam densitometer, Hologic, Inc., Bedford, MA, USA). Sarcopenic index was calculated with ASM (kg) divided by body weight (kg), and the index values that were < 1 standard deviation below the mean of the sex-specific healthy reference group (i.e., aged 20–39 years) from the same KNHANES IV and V database were considered sarcopenic [29]. Appendicular skeletal muscle mass (ASM) was deﬁned as the sum of the muscle mass in the arms and legs, measured with dual energy X-ray absorptiometry (DEXA) (DISCOVERY-W fan-beam densitometer, Hologic, Inc., Bedford, MA, USA). 2 2 1 Definition of Obesity and Sarcopenia 2.2.1. Deﬁnition of Obesity and Sarcopenia Sarcopenic index was calculated with ASM (kg) divided by body weight (kg), and the index values that were < 1 standard deviation below the mean of the sex-speciﬁc healthy reference group (i.e., aged 20–39 years) from the same KNHANES IV and V database were considered sarcopenic [29]. V database were considered sarcopenic [29]. Based on the sarcopenia and obesity assessments, individuals were classified as optimal (i.e., the absence of body sarcopenia and obesity), sarcopenia (i.e., the presence of sarcopenia), obesity (i.e., the presence of obesity) or sarcopenic obesity (i.e., the coexistence of sarcopenia and obesity). Based on the sarcopenia and obesity assessments, individuals were classified as optimal (i.e., the absence of body sarcopenia and obesity), sarcopenia (i.e., the presence of sarcopenia), obesity (i.e., the presence of obesity) or sarcopenic obesity (i.e., the coexistence of sarcopenia and obesity). 2.1. Data Source and Study Participants The data used in this study were obtained from the Korea National Health and Nutrition Examination Surveys (KNHANES) IV and V, conducted in the Republic of Korea from 2007 to 2011 [20]. In brief, KNHANES is a continuous survey of South Korea. Each survey consists of independent sets of non-institutionalized civilians aged 1 year and older from the South Korean population. All participants were randomly assigned from 600 randomly selected districts of cities and provinces in South Korea. KNHANES IV comprises survey 1 in 2007 (n = 4594), survey 2 in 2008 (n = 9744) and survey 3 in 2009 (n = 10,533). KNHANES V comprises survey 1 in 2010 (n = 8958) and survey 2 in 2011 (n = 8518). All statistics of this survey have been calculated using sample weights assigned to sample participants. A detailed description of the sample weights is available elsewhere [21]. For the current study, we initially selected 28,071 adults aged 18 years and older from KNHANES IV (survey 2 and survey 3) and V (survey 1 and survey 2). We then excluded individuals who had no data available regarding height and weight (n = 20), resting heart rate (n = 1287), waist circumference (WC) (n = 78), appendicular skeletal muscle mass (n = 9360), physical activity (n = 47), marital status (n = 260) or education (n = 35) at baseline. Individuals who met the following criteria were additionally excluded: (1) alcohol abuse (n = 2445), deﬁned as a weekly alcohol consumption > 140 g/week for men and > 70 g/week for women; 2) positive serologic markers for hepatitis B (n = 393) or hepatitis C virus (n = 41); and (3) the presence of liver cirrhosis (n = 62) or liver cancer (n = 28). Consequently, a total of 14,015 adults (8917 women) were included in the ﬁnal data analyses (Figure 1). The Institutional Review Board of the Korea Centre for Disease Control and Prevention in accordance with the Declaration of Helsinki reviewed and approved the KNHANES (Nos. 2008-04EXP-01-C, 2009-01CON-03-2C, 2010-02CON-21-C, 2011-02CON-06C). 3 of 12 Int. J. Environ. Res. Public Health 2020, 17, 3902 Figure 1. Flow chart of eligible participants in the study. Figure 1. Flow chart of eligible participants in the study. Figure 1. Flow chart of eligible participants in the study. Figure 1. Flow chart of eligible participants in the study. 2.2. Study Variables 2.2. Study Variables 2.2.3. Deﬁnition of Hepatic Steatosis Hepatic steatosis was determined using the following two indices of suspected hepati Hepatic steatosis was determined using the following two indices of suspected hepatic steatosis. NAFLD li f t (NAFLD LFS) [31] Hepatic steatosis was determined using the following two indices of suspected hepatic steatosis. NAFLD liver fat score (NAFLD-LFS) [31] Hepatic steatosis was determined using the following two indices of suspected hepatic steatosis NAFLD liver fat score (NAFLD-LFS) [31] NAFLD liver fat score (NAFLD-LFS) [31] NAFLD liver fat score (NAFLD-LFS) [31] NAFLD-LFS = −2.89 + 1.18 (metabolic syndrome; yes = 1, no = 0) + 0.45 (type 2 diabetes; yes = 2, no = 0) + 0.15 (insulin) + (0.04 × alanine aminotransferase) −0.94 × (aspartate aminotransferase/alanine aminotransferase), with fasting serum concentrations of insulin (µU/ml), alanine aminotransferase (U/L) and aspartate aminotransferase (U/L). Metabolic syndrome (Mets) was determined according to a revised National Cholesterol Education Program deﬁnition [32] with the adoption of a Korean-speciﬁc WC threshold [28]. Hepatic steatosis index (HSI) [33]. p HSI = 8 (alanine aminotransferase/aspartate aminotransferase ratio) + body mass index (+ 2 if diabetes, + 2 if female), with the cut-oﬀpoint of 30 for steatosis was used. 2.3. Statistical Analyses All variables were checked for normality, both visually and through the Kolmogorov–Smirnov test, and subjected to an appropriate transformation, if necessary, prior to statistical analyses. Descriptive statistics were presented as means and standard deviations for continuous variables, and as percentages for categorical variables. Parametric and non-parametric statistics were also used to compare continuous and categorical variables, respectively, among sarcopenia and obesity-based phenotypes (i.e., optimal, sarcopenia, obesity and sarcopenic obesity) stratiﬁed by eCRF categories (i.e., lower, middle and upper). Odd ratios (ORs) and 95% conﬁdence intervals (95% CIs) of sarcopenia and obesity-based phenotypes for NAFL were calculated in each eCRF category using multiple logistic regression analyses before and after adjustments for all measured covariates. Alpha was set at 0.05. All statistical analyses were performed using the SPSS-PC statistical software (version 23.0, SPSS Inc., Chicago, IL, USA). 2.2.4. Clinical and Laboratory Measurements Participants were required to complete self-administered questionnaires regarding smoking, alcohol habits, physical activity, income, marital status, education and past medical history. Height, weight, WC, systolic blood pressure (SBP), diastolic blood pressure (DBP) and resting heart rate (RHR) were assessed with standardized procedures by trained persons. Body mass index (BMI) was calculated as weight divided by height (kg/m2). Fasting venous blood sampling was performed after overnight fasting to determine concentrations of glucose, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDLC), insulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and platelet. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated; HOMA-IR = fasting insulin (uU/L) × fasting glucose (nmol/L)/22.5. A detailed description of the clinical and laboratory measurements is available elsewhere [22]. 2.2.2. Calculation of eCRF 2.2.2. Calculation of eCRF eCRF was calculated in units of metabolic equivalents (METs) [20]; eCRF (METs) = 2.77 (sex) − 0.10 (age) − 0.17 (body mass index) − 0.03 (resting heart rate) + 1.00 (physical activity score) + 18.07. Physical activity score was assessed with the Johnson Space Centre Physical Activity Rating scale, which was developed to provide a self-rating in the range of 0 (avoid physical activities whenever possible) to 7 (heavy physical activities done regularly for more than 3 hours per week) [30]. eCRF eCRF was calculated in units of metabolic equivalents (METs) [20]; eCRF (METs) = 2.77 (sex) − 0.10 (age) −0.17 (body mass index) −0.03 (resting heart rate) + 1.00 (physical activity score) + 18.07. Physical activity score was assessed with the Johnson Space Centre Physical Activity Rating scale, which was developed to provide a self-rating in the range of 0 (avoid physical activities whenever possible) to 7 (heavy physical activities done regularly for more than 3 hours per week) [30]. eCRF was 4 of 12 Int. J. Environ. Res. Public Health 2020, 17, 3902 then classiﬁed into lower (lowest 25%), middle (middle 50%) and upper (highest 25%), on the basis of sex- and age-speciﬁc tertiles of individual distributions. 2.2.3. Deﬁnition of Hepatic Steatosis 3. Results Table 1 represents the descriptive statistics of study participants. In general, men had more favorable proﬁles of marital status and education, and higher rates of physical inactivity, smoking, heavy alcohol consumption, hypertension, diabetes and metabolic syndrome, in conjunction with a higher rate of glucose-lowering medications and a lower rate of lipid-lowering medications than women. Men had poorer blood chemistry proﬁles than women, as shown in FBG, TG, AST, ALT, NAFLD-LFS, HSI and HDLC. 5 of 12 Int. J. Environ. Res. Public Health 2020, 17, 3902 Table 1. Description of study participants. Table 1. Description of study participants. Table 1. Description of study participants. 3. Results Women (n = 8917) Men (n = 5098) Total (n = 14,015) Body fatness and ﬁtness Age (year) 50.3 ± 16.2 49.7 ± 16.4 50.1 ± 16.3 BMI (kg/m2) 23.4 ± 3.4 23.9 ± 3.1 23.6 ± 3.3 Body fat (%) 33.0 ± 5.5 22.1 ± 5.5 29.1 ± 7.6 WC (cm) 78.8 ± 9.9 84.0 ± 9.0 80.7 ± 9.9 SMI (%) 26.9 ± 2.7 34.0 ± 3.0 29.5 ± 4.4 RHR (beats/min) 71.6 ± 9.3 70.0 ± 9.4 71.0 ± 9.3 eCRF (METs) 8.2 ± 2.2 11.2 ± 2.2 9.3 ± 2.7 Socio-economic status Income (10,000 won/month) 345.2 ± 823.7 375.1 ± 1229.8 356.1 ± 990.9 Marital status, n (%) Married 6308 (70.7) 4055 (79.5) 10,363 (73.9) Widow/divorced 1742 (19.6) 232 (4.6) 1974 (14.1) Unmarried 867 (9.7) 811 (15.9) 1678 (12.0) Education, n (%) Elementary 3101 (34.8) 923 (18.1) 4024 (28.7) Middle/high 3672 (41.2) 2426 (47.6) 6098 (43.5) College 2144 (24.0) 1749 (34.3) 3893 (27.8) Health conditions Smoking, n (%) 728 (8.2) 3856 (75.6) 4584 (32.7) Alcohol, n (%) 2049 (23.0) 373 (7.3) 2422 (17.3) Inactivity, n (%) 5540 (62.1) 2831 (55.5) 8371 (59.7) Hypertension, n (%) 2602 (29.2) 1939 (38.0) 4541 (32.4) Diabetes, n (%) 766 (9.0) 548 (11.1) 1314 (9.8) Metabolic syndrome, n (%) 2056 (24.2) 1380 (28.0) 3436 (25.6) Menopause, n (%) 4069 (45.6) 0 (0.0) 4069 (29.0) GLM, n (%) 568 (6.4) 381 (7.5) 949 (6.8) LLM, n (%) 499 (5.6) 198 (3.9) 697 (5.0) Blood markers FBG (mg/dL) 96.2 ± 21.3 99.7 ± 26.2 97.5 ± 23.3 Insulin (uIU/L) 10.0 ± 5.6 9.9 ± 5.4 10.0 ± 5.5 HOMA-IR 2.4 ± 2.3 2.5 ± 1.7 2.5 ± 2.1 TC (mg/dL) 189.0 ± 36.0 186.7 ± 34.8 188.2 ± 35.6 HDL-C (mg/dL) 49.8 ± 10.9 44.5 ± 10.2 47.8 ± 11.0 TG (mg/dL) 115.0 ± 76.6 147.9 ± 104.6 127.1 ± 89.3 AST (IU/L) 20.3 ± 8.1 23.7 ± 12.2 21.5 ± 10.0 ALT (IU/L) 17.7 ± 12.7 26.2 ± 22.0 20.8 ± 17.2 Platelet (109/L) 264.2 ± 59.7 243.3 ± 54.1 256.5 ± 58.6 Indices of hepatic steatosis NAFLD-LFS −1.44 ± 1.44 −1.02 ± 1.47 −1.29 ± 1.47 HSI 32.3 ± 4.8 32.7 ± 5.4 32.4 ± 5.0 BMI, body mass index; WC, waist circumference; SMI, skeletal muscle index; RHR, resting heart rate; eCRF, non-exercise-based estimation of cardiorespiratory ﬁtness; GLM, glucose-lowering medications; LLM, lipid-lowering medications; FBG, fasting blood glucose; HOMA-IR, homeostasis model assessment of insulin resistance; TC, total cholesterol; TG, triglycerides; AST, aspartate aminotransferase; ALT, alanine aminotransferase; NAFLD-LFS, non-alcoholic fatty liver disease-liver fatty score; HSI, hepatic steatosis index. 3. Results The impacts of sarcopenia and obesity on the prevalence of NAFL stratiﬁed by eCRF levels. Table 3 represents the ORs and 95% CIs of sarcopenia and obesity-based phenotypes for NAFL, stratiﬁed by eCRF levels. Limited to the sarcopenia phenotype, the adjusted OR of NAFL was 2.2 (95% CI, 1.5–3.1) for the lower eCRF, 1.6 (95% CI, 1.3–2.1) for the middle eCRF and 2.1 (95% CI, 1.4–3.1) for the upper, compared to the optimal phenotype. Limited to the obesity phenotype, the adjusted OR of NAFL was 2.9 (95% CI, 2.0–4.2) for the lower eCRF, 3.5 (95% CI, 2.7–4.6) for the middle eCRF and 1.8 (95% CI, 1.2–2.8) for the upper eCRF, compared to the optimal phenotype. Limited to the sarcopenic obesity phenotype, the adjusted OR of NAFL was 5.9 (95% CI, 4.3–8.2) for the lower eCRF, 4.2 (95% CI, 3.2–5.5) for the middle eCRF and 2.5 (95% CI, 1.5–4.1) for the upper eCRF, compared to the optimal phenotype. Together, the ﬁndings of the current study suggest that high eCRF attenuates the individual and synergistic impacts of sarcopenia and obesity on NAFL. Table 3. Odds ratio (OR) of sarcopenia and obesity-based phenotypes for NAFL, stratiﬁed by eCRF levels (data are presented as OR and 95% conﬁdence interval). Phenotypes Lower 25% eCRF Middle 50% eCRF Upper 5% eCRF SAR OB Crude OR Adjusted OR Crude OR Adjusted OR Crude OR Adjusted OR - - 1 (referent) 1 (referent) 1 (referent) 1 (referent) 1 (referent) 1 (referent) + - 2.2 (1.7–2.9) 2.1 (1.5–3.1) 2.1 (1.7–2.5) 1.6 (1.3–2.1) 2.2 (1.6–3.0) ** 2.1 (1.4–3.1) * - + 9.0 (7.1–11.6) 2.9 (2.0–4.2) 11.8 (9.8–11.4) 3.5 (2.7–4.6) 9.4 (7.0–12.5) ** 1.8 (1.2–2.8) * + + 13.0 (10.4–16.2) 5.9 (4.3–8.2) 13.7 (11.4–16.4) 4.2 (3.2–5.5) 11.6 (8.3–16.1) 2.5 (1.5–4.1) eCRF, non-exercise-based estimation of cardiorespiratory ﬁtness; SAR, sarcopenia; OB, obesity. Minus (-) and plus (+) indicate the absence of SAR or OB and the presence of SAR and/or OB, respectively. Adjusted for income, marital status, education, resting heart rate, smoking, alcohol consumption, hypertension and menopause. * p < 0.05, ** p < 0.001. Table 3. Odds ratio (OR) of sarcopenia and obesity-based phenotypes for NAFL, stratiﬁed by eCRF levels (data are presented as OR and 95% conﬁdence interval). eCRF, non-exercise-based estimation of cardiorespiratory ﬁtness; SAR, sarcopenia; OB, obesity. 3. Results Compared to optimal anthropometrics (i e age BMI percent body fat WC and SMI Women (n = 8917) Men (n = 5098) Total (n = 14,015) BMI, body mass index; WC, waist circumference; SMI, skeletal muscle index; RHR, resting heart rate; eCRF, non-exercise-based estimation of cardiorespiratory ﬁtness; GLM, glucose-lowering medications; LLM, lipid-lowering medications; FBG, fasting blood glucose; HOMA-IR, homeostasis model assessment of insulin resistance; TC, total cholesterol; TG, triglycerides; AST, aspartate aminotransferase; ALT, alanine aminotransferase; NAFLD-LFS, non-alcoholic fatty liver disease-liver fatty score; HSI, hepatic steatosis index. BMI, body mass index; WC, waist circumference; SMI, skeletal muscle index; RHR, resting heart rate; eCRF, non-exercise-based estimation of cardiorespiratory ﬁtness; GLM, glucose-lowering medications; LLM, lipid-lowering medications; FBG, fasting blood glucose; HOMA-IR, homeostasis model assessment of insulin resistance; TC, total cholesterol; TG, triglycerides; AST, aspartate aminotransferase; ALT, alanine aminotransferase; NAFLD-LFS, non-alcoholic fatty liver disease-liver fatty score; HSI, hepatic steatosis index. Compared to optimal, anthropometrics (i.e., age, BMI, percent body fat, WC and SMI), SES (i.e., marital status and education) and health conditions (i.e., smoking, alcohol consumption, inactivity, hypertension, diabetes, metabolic syndrome, menopause, glucose and lipid-lowering medications, and markers of NAFL) became worse in the order of sarcopenia, obesity and sarcopenic obesity, in the lower, middle and upper eCRF categories. Compared to optimal, blood chemistry proﬁles (i.e., FBG, insulin, HOMA-IR, TC, HDL-C, TG, AST and ALT) also became worse in the order Int. J. Environ. Res. Public Health 2020, 17, 3902 6 of 12 of sarcopenia, obesity and sarcopenic obesity, in the lower, middle and upper eCRF categories (refer to Tables S1–S3 in the Supplementary Materials). of sarcopenia, obesity and sarcopenic obesity, in the lower, middle and upper eCRF categories (refer to Tables S1–S3 in the Supplementary Materials). Table 2 represents the impacts of sarcopenia and obesity on the prevalence of NAFL, stratiﬁed by eCRF levels. Signiﬁcant inverse, linear trends in the prevalence of NAFL were found according to incremental eCRF categories (from lower to upper) in all the sarcopenia and obesity-based phenotypes. Table 2. The impacts of sarcopenia and obesity on the prevalence of NAFL stratiﬁed by eCRF levels. Optimal Sarcopenia Obesity Sarcopenic Obesity Lower eCRF 177 (14.9%) 127 (27.9%) 281 (61.2%) 524 (69.4%) Middle eCRF 418 (11.2%) 183 (20.7%) 410 (59.8%) 463 (63.3%) Upper eCRF 217 (9.3%) 68 (18.3%) 121 (49.0%) 94 (54.3%) p < 0.001 p = 0.001 p = 0.005 p < 0.001 eCRF: non-exercise-based estimation of cardiorespiratory ﬁtness. Table 2. 3. Results Minus (-) and plus (+) indicate the absence of SAR or OB and the presence of SAR and/or OB, respectively. Adjusted for income, marital status, education, resting heart rate, smoking, alcohol consumption, hypertension and menopause. * p < 0.05, ** p < 0.001. 4. Discussion In this population-based study, we examined the impacts of sarcopenia and obesity on NAFL, stratiﬁed by eCRF levels, in Korean adults, and found that NAFL was positively associated with sarcopenia and/or obesity, and inversely associated with eCRF. To the best of our knowledge, this is the ﬁrst study to report that high eCRF attenuates the individual and synergistic impacts of sarcopenia and obesity on NAFL in Korean adults. The ﬁndings of the current study are in accordance with previous studies reporting obesity and sarcopenia as important risk factors for NAFL. In an average 4.5-year follow-up cohort study, involving 77,425 metabolically healthy adults at baseline, Chang et al. [34] showed that overweight and obese individuals had higher hazard ratios of incident NAFLD (HR = 2.15, 95% CI = 2.06–2.26 and OR = 3.55, 95% CI = 3.37–3.74, respectively) compared with normal-weight individuals. By analyzing the data obtained from 29,994 Korean adults aged 18 years and older who had a routine health examination, Kwon et al. [35] showed that obese individuals had a higher prevalence of NAFLD (50.1% vs. 12.6%) compared to non-obese individuals. Yang et al. [36] examined the association between NAFLD and Int. J. Environ. Res. Public Health 2020, 17, 3902 7 of 12 obesity-related metabolic syndrome in a sample of 614 Korean participants recruited from a local community, and found that the risk of Mets was linearly increased according to incremental NAFLD severity (from mild to severe). Together, these ﬁndings of the current and previous studies suggest that obesity, especially central obesity, increases the odds for NAFL directly and/or indirectly by inducing metabolic disorders, such as metabolic syndrome and IR [37]. In addition to obesity, the ﬁndings of the current study are also in accordance with the ﬁndings from Korean cohort studies reporting sarcopenia as another novel risk factor of NAFL. In a total of 4398 Korean adults who had no baseline NAFLD, Lee et al. [38] examined the association between ASM and the 7-year follow-up incident of NAFLD, and found that decreased ASM was positively associated with incident of NAFLD independent of obesity. By analyzing a 7-year follow-up study, involving 12,624 Korean adults aged 20 years and older, Kim et al. [14] also showed that baseline skeletal muscle index (SMI) was inversely associated with incident NAFLD, and positively correlated with the resolution of baseline NAFLD. The association between sarcopenia and NAFL has been reported from population-based, cross-sectional studies. 4. Discussion By analyzing data obtained from KNHANES IV and V, Lee et al. [16] examined the association between sarcopenia and NAFLD in a representative sample of Korean adults, and found that decreased SMI was independently associated with an increased risk of NAFL and NASH in Korean adults. In a recent meta-analysis involving 19 articles, Cai et al. [39] showed that patients with NAFLD had lower SMI than normal controls. In that study, sarcopenic individuals were found to have a signiﬁcantly higher risk of developing NAFL (OR = 1.33), NASH (OR = 2.42) and hepatic ﬁbrosis (OR = 1.56) when compared with their non-sarcopenic counterparts. Those previous ﬁndings emphasize the clinical importance of increases in skeletal muscle mass as a therapeutic strategy against NAFL. Evidence has been accumulated to support lifestyle intervention as a therapeutic strategy for patients with NAFL. In a review paper, Glass et al. [40] showed that a combination of aerobic and resistance exercise, targeting both CRF and myokines, resulted in the greatest beneﬁts for NAFLD. In a lifestyle intervention with diet modiﬁcation and increase in physical activity, for example, CRF at baseline was the strongest predictor of reductions in fatty liver, independent of changes in body fatness [41]. Similarly, a 16 weeks of supervised exercise training resulted in improvements in both CRF and NAFL independent of weight loss, and the exercise training-induced improvements in CRF and NAFL were reversed after 1 year of exercise cessation [42], implying that CRF and NAFL are causally related to each other. Consequently, an intervention study will be necessary to elucidate the nature of the link between CRF and NAFL, as well as the underlying mechanism(s). Several explanations are possible for the high eCRF-related attenuation of the prevalence of NAFL, associated with sarcopenia and central obesity, observed in the current study. First, sarcopenia and obesity are two of the major risk factors that are frequently encountered in patients with NAFL. IR is considered as a common factor linking sarcopenia and obesity to hepatic steatosis, via perturbations in the release of myokines and adipokines, respectively [17]. On the other hand, CRF is positively associated with whole-body insulin sensitivity in apparently healthy men and women [39], as well as overweight/obese subjects [10]. Therefore, it seems reasonable to speculate that high CRF-related insulin sensitivity may attenuate the individual and synergistic impact of sarcopenia and obesity on the risk of hepatic steatosis. 4. Discussion Second, inﬂammation may be another explanation for the link between hepatic steatosis, sarcopenia and obesity [43,44]. As such, high CRF-related anti-inﬂammatory responses may attenuate the individual and synergistic impacts of sarcopenia and obesity on NAFL [11]. Third, metabolic syndrome, characterized by hyperglyceridemia, hyperlipidemia and hypertension, may contribute to the development of NAFL [45]. On the other hand, promotion of CRF via regular exercise hasbeenwellestablishedasaneffectivetherapeuticstrategyforpatientswithmetaboliccomplications[46,47]. Together, the findings of the current study suggest that sarcopenia and obesity are individually and synergistically associated with whole-body insulin resistance/diabetes, low grade inflammation and Int. J. Environ. Res. Public Health 2020, 17, 3902 8 of 12 metabolic syndrome, contributing to the development of NAFL, while high CRF may alleviate the individual and synergistic impacts of sarcopenia and obesity on NAFL by suppressing/minimizing those metabolic complications. metabolic syndrome, contributing to the development of NAFL, while high CRF may alleviate the individual and synergistic impacts of sarcopenia and obesity on NAFL by suppressing/minimizing those metabolic complications. p This study has limitations. First, the cross-sectional nature of this study does not allow causal inference regarding the potential role of eCRF in determining the relationships of sarcopenia and obesity with NAFL. Second, a further study will be necessary to investigate the cellular and molecular mechanism(s) through which CRF alleviates the impact of sarcopenic obesity on the risk of hepatic steatosis. Third, most previous studies involving Korean populations did not include CRF as an exposure, perhaps due to practical the diﬃculties/limitations related to its measurement. Alternatively, we included eCRF as an important exposure, so that the nature of the relationship of sarcopenic obesity with NAFLD may be better delineated or unveiled [18]. However, the accuracy of the eCRF used in this study remains to be validated in Korean populations. Fourth, BMI and waist-to-hip ratio (WHR) were used as obesity measures, and these are also closely associated with metabolic disorders [46]. Consequently, the impact of the coexistence of sarcopenia and obesity on NAFL should be further addressed in a future study using anthropometric measures of obesity. Fifth, assessment of alcohol consumption based on a self-reported questionnaire may not eliminate the possibility of underestimating the alcohol intake. Lastly, although using HSI and NAFLD-LFS as biomarkers of NAFL was previously validated against ultrasound or 1H-MRS [31], their accuracy in quantifying steatosis was also questioned, and contrasted with liver biopsy as the reference standard [48]. 4. Discussion Therefore, the accuracy of HSI and NAFLD-LFS should be validated against liver biopsy, ultrasound or 1H-MRS as reference standard in a large cohort study using Korean populations. Despite the limitations, the current ﬁndings of this population-based, cross-sectional study indicate the individual and synergistic impacts of sarcopenia and obesity on NAFL, and expand the current knowledge and literature regarding this issue, proving the possible role of eCRF as an important mediator in determining the relationship between sarcopenic obesity and NAFL in Korean adults. 5. Conclusions This study examined the impacts of sarcopenia and obesity on NAFL in relation to eCRF levels in Korean adults, and found that high eCRF attenuates the individual and synergistic impacts of sarcopenia and obesity on NALF. From a clinical perspective, the ﬁndings of our study suggest that the importance of interventions targeting both poor CRF and sarcopenic obesity should be emphasized in the management and prevention of NAFL. Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/17/11/3902/s1, Table S1: Descriptive statistics of sarcopenic obesity-based phenotypes in lower eCRF category, Table S2: Descriptive statistics of sarcopenic obesity-based phenotypes in middle eCRF category, Table S3: Descriptive statistics of sarcopenic obesity-based phenotypes in upper eCRF category. Author Contributions: I.L. and H.K. contributed to the conception and design, data analyses, data interpretation, and writing of the manuscript. J.K. contributed to data interpretation and preparing and writing of the manuscript. All authors read and approved the ﬁnal manuscript. Funding: This work was supported by the National Research Foundation Grant funded by the Korean Government (NRF-2019R1I1A1A01043771). Acknowledgments: We gratefully thank the Korea Centers for Disease Control and Prevention (KCDC) for allowing use of their data for this study. Conﬂicts of Interest: The authors declare no conﬂicts of interest to disclose. t: The authors declare no conﬂicts of interest to disclo Abbreviations CRF cardiorespiratory ﬁtness eCRF estimated cardiorespiratory ﬁtness WC waist circumference WHR waist-to-hip ratio BMI body mass index References 1. Araújo, A.R.; Rosso, N.; Bedogni, G.; Tiribelli, C.; Bellentani, S. Global epidemiology of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: What we need in the future. Liver Int. 2018, 38, 47–51. [CrossRef] [PubMed] 1. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2565605627	https://rebeca.socine.org.br/1/article/download/144/219	Spanish; Castilian	null	Producción, mercados y políticas públicas cinematográficas en América Latina	Rebeca	2,016	cc-by	9,509	N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 1 Consultor del Instituto de Estadísticas de la Unesco. Autor de dos libros, capítulos en compilaciones y decenas de artículos y papers sobre cine, audiovisual e industrias culturales, publicados en Estados Unidos, España, Italia, México, Brasil, Argentina y Colombia (como “Emerging Markets and the Digitalization of the Film Industry”, Institute of Statistics, UNESCO, Montreal, 2013, entre otros). Conformó, junto a Octavio Getino, el Observatorio del Cine y el Audiovisual Latinoamericano (Ocal-FNCL) y el Observatorio del Mercosur Audiovisual (OMA-Recam). Referente latinoamericano del Observatorio Europeo del Audiovisual. E-mail: roquegonzalez@gmail.com Producción, mercados y políticas públicas cinematográficas en América Latina Film production, market and public policies in Latin America Producción, mercados y políticas públicas cinematográficas en América Latina Resumo Durante os últimos 20 anos –depois do período neoliberal (baseado nos postulados do Consenso do Washington) - as políticas públicas de cinema na América Latina estão enfocadas quase exclusivamente na produção. Neste contexto, duas tentativas para fazer políticas públicas cinematográficas latino-americanas tiveram diferentes abordagens e resultados. Palavras-chave: Políticas públicas cinematográficas; produção; distribução; exhibição Resumen Desde hace 20 años —luego del período neoliberal (basado en los postulados del Consenso de Washington) — las políticas públicas de cinematografía en América Latina se encuentran enfocadas casi exclusivamente en la producción. La comercialización, la convergencia audiovisual y la digitalización están prácticamente ausentes en estas políticas. En este contexto, dos intentos de regionalizar políticas públicas cinematográficas tuvieron distintos abordajes y resultados. Palabras clave: Políticas públicas cinematográficas; producción; distribución; exhibición Film production, market and public policies in Latin America Roque González1 E-mail: roquegonzalez@gmail.com 209 Revista Brasileira de Estudos de Cinema e Audiovisual Abstract For the last 20 years--since the neoliberal period of the 1990s (based on Washington Consensus)--public policies on cinematography in Latin America have focused predominantly on production. In this context, two (intentos) to make public film policies in Latin America had different (abordajes) and (results). Keywords: Film policies; production; distribution; exhibition. 210 Revista Brasileira de Estudos de Cinema e Audiovisual El presente trabajo busca analizar la producción y los mercados cinemato gráficos en América Latina en los últimos años, en el contexto de las políticas públicas de fomento a esta actividad. Durante la última década dichas políticas fueron retomando fuerza -en casos como el argentino, mexicano y brasileño- o comenzaron a surgir -por ejemplo, en Colombia, Chile, Uruguay y Ecuador, entre otros países-. Por otra parte, éstas políticas buscaron expandirse a nivel regional —por ejemplo, en los casos de la Conferencia de Autoridades Cinematográficas de Iberoamérica (Caci) y de la Reunión Especializada de Autoridades de Cine y Audiovisual del Mercosur (Recam). La presente investigación se enmarcará en el mencionado campo de la Economía Política de la Comunicación y la Cultura (EPCC), y más específica mente, en la Economía Política del Cine. La EPCC estudia el rol del poder en la producción, en la distribución — especialmente, en este punto (PENDAKUR, 1990; BALIO, 1993; WASKO, 2011)— y en el intercambio de la comunicación mediada, analizando las rela ciones sociales, las estructuras del poder, el proceso por el cual los mensajes se transforman en mercancías y las relaciones entre producción material y producción intelectual. A nivel más básico, la EPCC estudia la manera en que la comunicación y la cultura formen parte del proceso de acumulación de capital, de la estratificación y las desigualdades de clases y de las relaciones entre los centros de poder político y los centros de poder económico (Guback, 1980; Pendakur, 1990; Wasko, 2003; Getino, 2005; Sánchez Ruiz, 2012). Abstract Según la economía política del cine, las películas son mercancías produ cidas y distribuidas dentro de una estructura industrial capitalista (Guback, 1980; Wasko, 2003, 2011), concentrada, con una asimétrica división interna cional del trabajo audiovisual (Miller y Yúdice, 2004, se refieren a la “nueva di visión internacional del trabajo cultural”), en el marco de relaciones de poder dentro del sistema cultural y político, con el agregado de que el cine es a la vez producto y servicio intangible (Pendakur, 1990), importante no sólo por su valor de cambio sino por su valor cultural (Pendakur, 2003; Getino 2005) —en cuanto elemento trascendente para la constitución de la identidad y la cultura de un pueblo. 211 Revista Brasileira de Estudos de Cinema e Audiovisual El presente trabajo parte del criterio de que el cine en América Latina du rante la última década se caracterizó por el regreso del apoyo estatal al cine (“neofomentismo”) —luego del desmantelamiento de las ayudas públicas a este sector hacia fines de la década de 1980 y comienzos de la de ‘90, enfa tizando en la producción. A su vez, esta reflexión busca mostrar que el sector cinematográfico en América Latina sólo puede existir por el fomento estatal, debido a que tienen nula recuperación de los costos a través de la taquilla en sus respectivos mer cados nacionales —y menos aún en los mercados internacionales, en donde prácticamente no circula. A esta situación se suma la posición oligopólica en el mercado de la distri bución y la exhibición por parte de las majors de Hollywood —tanto en Esta dos Unidos como en el resto del mundo (salvo contadas excepciones, como China e India). A continuación se profundizará en estos temas, aclarando que las variadas estadísticas y números de producción y mercado fueron realizados por el au tor de este artículo, basándose en datos oficiales, información de empresas del sector y de medios periodísticos (salvo cuando se especifique la fuente). 2 Elaboración propia basada en datos de las agencias nacionales de cine de Argentina, Brasil, México, Colombia, Chile, Perú, Uruguay, Venezuela, Bolivia, Ecuador, de las consultoras Rentrak, Ultracine y medios especializados. Producción y mercados Desde hace algunos años en América Latina se producen entre 300 y 350 largometrajes anuales (con picos de 400, en 2008), un número notablemente superior al de las décadas recientes: durante los años ochenta se produjo un promedio de 230 largometrajes anuales, y durante los noventa, ese promedio bajó a 91 producciones al año (GONZÁLEZ, 2012: 95). Durante el último decenio, la producción cinematográfica en varios países de la región ha alcanzado picos históricos. El fomento estatal a la producción cinematográfica existente en casi toda América Latina ha permitido el aumen to en la producción de películas como nunca antes en la región; inclusive en países como México, Brasil y Argentina el número de filmes realizados alcanza valores que sólo se habían conseguido décadas atrás. 212 América Latina produjo en total alrededor de 4500 largometrajes en el pe ríodo 2000-20132. La decidida acción del fomento estatal en la mayoría de los países latinoamericanos se vio reflejada en la cantidad de largometrajes pro ducidos. Sin embargo, a pesar de estos incrementos, en ningún país latinoa mericano se termina de configurar una “industria”, sino sólo un ecosistema fragmentado en el que conviven algunas pocas empresas profesionales con innumerables pequeños emprendimientos sin sustentabilidad, atomizados e inconexos, con el peso puesto —tanto desde el sector público como en el privado— en la producción más no en la comercialización y exhibición de los filmes nacionales. Por su parte, la coproducción internacional se convirtió, en las últimas dé cadas, en una importante estrategia para hacer frente a obstáculos como el difícil financiamiento y las dificultades para que los filmes circulen más allá de las fronteras nacionales. Para varios países de la región la coproducción fue fundamental para revitalizar y fortalecer su sector cinematográfico, como en los casos de Cuba, Bolivia y Uruguay. 3 Elaboración propia basada en datos de las agencias nacionales de cine de Argentina, Brasil, México, Colombia, Chile, Perú, Uruguay, Venezuela, Bolivia, Ecuador, de las consultoras Rentrak, Ultracine, de distribuidoras, exhibidoras y medios especializados. 4 Elaboración propia basada en datos de las agencias nacionales de cine de Argentina, Brasil, México, Colombia, Chile, Perú, Uruguay, Venezuela, Bolivia, Ecuador, de las consultoras Rentrak, Ultracine, de distribuidoras, exhibidoras y medios especializados. 5 “K” equivale, en este caso, a 2 elevado a 10 (sistema binario), es decir, 1024 píxeles de resolución. Se denominan 2K y 4K a imágenes de 2048 x 1080 píxeles y 4096 x 2160 píxeles, respectivamente, destinadas a proyectores y archivos de cine digital. Concentración en la exhibición Durante la década del 2000 se consolidó la concentración elitista del mer cado cinematográfico, que prepondera las ciudades y las zonas más ricas, a la vez que a las clases más pudientes, para consolidar el incremento sostenido en las taquillas con menos diversidad en las pantallas y de espaldas a las ma sas que sustentan la base de la pirámide social. En efecto: entre las décadas del ochenta y el noventa cerraron casi la mi tad de las salas de cine que había en América Latina (GETINO, 2005: 82). La mayoría de ellas se encontraba en el interior de los países, que es donde his tóricamente siempre se vio más cine nacional. Actualmente, existen en la re gión entre 5 y 20 salas de cine –40 en el caso de México— por cada millón de habitantes –tres veces menos que hace tres décadas (GONZÁLEZ, 2012: 119). De esta manera, el incremento del 65% promedio que el parque exhibidor regional mostró en la década del 2000 —empujado por México, Brasil y Co lombia (en el resto de los países, la cantidad de salas casi no ha variado)— hay que ubicarlo en el contexto de una alta concentración geográfica y clasista y del alto costo de las entradas, que representan –considerando una única sali da familiar al cine— alrededor del 10% del ingreso mensual medio latinoame ricano (GONZÁLEZ, 2012: 120). ricano (GONZÁLEZ, 2012: 120). En este escenario aparece el cine digital a través de su nave insignia: el 3D. Hacia finales de 2008, y especialmente desde 2009, comenzaron a abrirse de cenas de salas con proyección digital de alta gama (2K de resolución mínima5). Todas estas salas digitales están equipadas para exhibir películas tridimen sionales, la gran esperanza de las multinacionales y las grandes compañías de la industria cinematográfica, para incrementar la asistencia a las salas y, sobre todo, para elevar la recaudación: debido a que las entradas para ver películas en 3D cuestan un 30% más, desde 2009 y 2010 se apreció como tendencia firme que aproximadamente el 10% de los estrenos —filmes hollywoodenses en tercera dimensión dirigidos al público infanto-juvenil— significan alrededor En este escenario aparece el cine digital a través de su nave insignia: el 3D. Hacia finales de 2008, y especialmente desde 2009, comenzaron a abrirse de cenas de salas con proyección digital de alta gama (2K de resolución mínima5). Comercialización Si los latinoamericanos –que en promedio concurren 0,8 veces al cine al año— tuvieran una circulación cinematográfica regional verdaderamente efi ciente, tendrían —en teoría— entre 400 y 550 filmes regionales anuales en tre los cuales escoger –dependiendo el país-. Sin embargo, ello no ocurre: anualmente se estrenan entre 3 y 20 filmes latinoamericanos no nacionales –dependiendo del país—, cuyo público suele ser menor al 1% del total de los espectadores de cine (GONZÁLEZ, 2012: 107). A N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 En 2013 se vendieron en América Latina 581 millones de entradas —que en promedio cuestan 5,5 dólares cada una— para asistir a alguna de las 11.300 213 Revista Brasileira de Estudos de Cinema e Audiovisual salas existentes en el subcontinente para elegir entre los 210 estrenos que, en promedio, se estrenan anualmente.3 A su vez, en el mismo año América Latina sumó casi 2.700 millones de dólares de recaudación por entradas de cine — 2,7 billones de dólares, según la nomenclatura anglosajona que en América Latina utiliza Brasil.4 Esta cifra se debe, principalmente, al constante aumento de las entradas—que duplicó su precio durante el último decenio (en los últimos dos años empujado por los precios premium de las salas 3D)— y a la alta inflación registrada en los últimos años en Argentina y Venezuela –estos países se encuentran entre los países con mayores tasas de inflación del mundo. Como ejemplo de lo anterior: la revista Variety, en un artículo del 6 de abril de 2013, llegó a publicar con bombos y platillos que el box office argentino tuvo en 2012 un crecimiento interanual del 35%; pero esta apreciación olvidó de tomar en cuenta la mencionada inflación argentina. Analizando los resultados de 2012 para Argentina y Venezuela, se apre cia —en efecto— un aumento interanual del 35% tanto para la recaudación argentina —tomando en cuenta todas las películas exhibidas, no sólo los estrenos- como para la venezolana. Sin embargo, si se observa el aumento de espectadores para los años 2011-2012, en Argentina el crecimiento fue de tan sólo el 6% y en Venezuela no hubo ningún crecimiento: se vendió la misma cantidad de entradas (alrededor de 30 millones). En 2013 las dis paridades de aumento entre la cantidad de espectadores y la recaudación fueron similares. 214 Concentración en la exhibición Todas estas salas digitales están equipadas para exhibir películas tridimen sionales, la gran esperanza de las multinacionales y las grandes compañías de la industria cinematográfica, para incrementar la asistencia a las salas y, sobre todo, para elevar la recaudación: debido a que las entradas para ver películas en 3D cuestan un 30% más, desde 2009 y 2010 se apreció como tendencia firme que aproximadamente el 10% de los estrenos —filmes hollywoodenses en tercera dimensión dirigidos al público infanto-juvenil— significan alrededor A N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 215 del 40% de la taquilla —situación que se da tanto en América Latina como en el mundo (GONZÁLEZ, 2011: 142). Sin embargo, las promesas que circundan alrededor del cine digital sobre democratización, abaratamiento de costos y mayor diversidad, entre otras, están quedando en la teoría: si bien el cine digital reduce considerablemente los costos por copias –cada copia en 35 milímetros cuesta entre 1500 y 2000 dólares—, la instalación de estas salas digitales—que engloban proyector, pe riféricos, sistema operativo, equipamiento para exhibir en tercera dimensión y en algunos casos, hasta una pantalla distinta— ronda en América Latina casi el doble que en el “Primer Mundo”. En la práctica, la digitalización la están pudiendo cubrir las empresas exhibidoras más grandes, mientras que las “in dependientes”—empresas pequeñas, locales, suburbanas, que generalmente programan cine nacional, iberoamericano, mundial de calidad, corren serio riesgo de tener que cerrar, o ser cooptadas por las más grandes. La presencia del cine nacional en América Latina La difícil situación por la que atraviesan gran parte de los cines naciona les en América Latina –sobre todo en el sentido de su muy escasa llegada al público y de su poca sustentabilidad (salvo contadísimas excepciones)— no se basa en la cantidad de producciones realizadas sino en la distribución y en la exhibición. Justamente, estos eslabones de la cadena de valor audio visual están profundamente descuidados por los Estados latinoamericanos —como también ocurre en otras regiones del mundo, como la Unión Euro pea (a pesar de sus abultados fondos de ayuda, destinados mayormente a la producción cinematográfica). —como también ocurre en otras regiones del mundo, como la Unión Euro pea (a pesar de sus abultados fondos de ayuda, destinados mayormente a la producción cinematográfica). Las diez películas más vistas —casi en su totalidad blockbusters de Ho llywood (9 de 10 en casi toda América Latina)— suelen representar en Lati noamérica entre el 35% y el 50% del total, tanto de espectadores como de recaudación en los distintos mercados. A su vez, los filmes de Hollywood con centran desde el 80% de los espectadores y taquilla en los distintos mercados latinoamericanos –en algunos mercados este porcentaje excede el 90%. 216 Revista Brasileira de Estudos de Cinema e Audiovisual Por su parte, la presencia de los filmes nacionales en las pantallas latinoa mericanas varía de acuerdo a la capacidad, la tradición cinematográfica y las políticas de fomento de cada país. Aunque las políticas públicas de cine hayan incrementado la producción del mismo durante la década, las audiencias de los distintos filmes nacionales son muy bajas, las películas nacionales alcanzan un market share de entre el 1% y el 15% de los espectadores según la capacidad productiva de cada país y la coyuntura —algún éxito esporádico. En el caso de los países de mayor desarrollo cinematográfico, como Argentina, Brasil y México fluctúa entre el 5% y el 15% -salvo años excepcionales, en que ese market share puede acer carse al 16%-18%. En el resto de los países, ese porcentaje ronda entre el 1% y el 5% (GONZÁLEZ, 2012: 124), salvo excepciones. Esta nula presencia del cine latinoamericano en las pantallas de los mismos países es una constante en los últimos 25 años en toda la región. La presencia del cine nacional en América Latina Durante la última década la oferta de cine nacional se ha incrementado notablemente en los casos de Argentina, México y Brasil, duplicando en el primer caso, y triplicando en los dos restantes el número de películas nacio nales estrenadas. Chile, Colombia y Perú también han visto incrementar su producción cinematográfica notablemente (de entre 5 y 10 en el año 2000 a entre 35 y 50 en 2013) –en el caso de los dos primeros, fueron muy importan tes las legislaciones nacionales de fomento al cine aprobadas en la década de 2000; en el caso peruano, se destaca una importante producción en el interior del país, por fuera de los circuitos oficiales. El resto de los países ha mostrado una tendencia a incrementar levemente el número de estrenos locales (GON ZÁLEZ, 2012: 125). Las majors son las distribuidoras con mayor presencia entre los “Top 10” –y, por lo tanto, son las empresas que se llevan la mayor parte de la torta en las recaudaciones de toda América Latina. A pesar de que se puede suponer que las distribuidoras nacionales comer cializan preferentemente filmes nacionales (UNESCO, 2007: 6), ello no ocurre en la cinematografía latinoamericana con mayor potencial de convocatoria. “Stefan vs Kramer”, que en 2012 resultó el segundo filme más visto en su país, fue comercializado por Fox, mientras que “Nosotros los nobles”, que por 217 Revista Brasileira de Estudos de Cinema e Audiovisual algunos meses de 2013 fue el filme mexicano más visto de toda la historia, se distribuyó por la Warner. “Metegol”, la animación de Juan José Campanella (producida en gran parte por el canal de televisión Telefé, que a su vez, per tenece a Telefónica), fue la película argentina más vista en ese país durante 2013: se distribuyó por UIP. Al mismo tiempo, los filmes argentinos más taqui lleros ese año se distribuyeron por Fox y Disney —ésta última major está te niendo una participación destacada en la distribución de filmes argentinos con potencialidad de ser “taquilleros” desde hace algunos años (especialmente, a partir de las erráticas políticas del gobierno argentino: intentando ayudar al cine nacional, el kirchnerismo exigió a las majors que comercializaran cine argentino; Hollywood terminó distribuyendo contados estrenos nacionales, los más taquilleros, hiriendo gravemente el reducido puñado de distribuidoras argentinas dedicadas al cine local). La presencia del cine nacional en América Latina “Tesis sobre un homicidio” también se ubicó en otro “Top 10”: en el uru guayo, se distribuyó también por Disney, mientras que “Metegol” y “Séptimo” (una coproducción argentino-española protagonizada por Ricardo Darín) fue ron estrenadas en Chile por la Warner. Retomando una ironía de Octavio Geti no, la integración regional pareciera que está siendo abordada por empresas extranjeras... (GETINO, 2000). Aunque no hubo variación en el número total de estrenos comercia les la hubo en incrementos significativos en la cantidad de películas naciona les estrenadas comercialmente en Perú, Colombia, Uruguay y Perú, aunque —como lamentablemente sucede desde hace más de veinte años a nivel re gional— el público no sigue las producciones nacionales, salvo en contadas excepciones –unas dos o tres películas nacionales explican alrededor del 90% de los resultados de mercado de todos los estrenos locales: esta situación se produce en toda América Latina. Los porcentajes del market share durante 2012 se ubicaron en los núme ros habituales que se vienen dando desde hace años: entre el 2% en Perú y Uruguay y el 13% en Chile —cifra inusual para este país, alcanzada gracias al éxito de “Stefan vs Kramer” (similar a lo que sucede en los países latinoame ricanos, muy esporádicamente, cuando surge algún filme nacional que resulta éxito de taquilla, tal como sucedió, por ejemplo, con “Tropa de elite 2” en 218 Revista Brasileira de Estudos de Cinema e Audiovisual Brasil, en el 2010; con “El viaje” y “El viaje 2” en Colombia, en 2010 y 2012, respectivamente, o con el “El secreto de sus ojos” en Argentina, en 2009, que representó por sí sola el 47% de los espectadores del centenar de estrenos nacionales de ese año). Se toma el año 2012 porque 2013 fue un año poco común, con estrenos nacionales que rompieron récords históricos, especialmente en México, Perú y Venezuela –en menor medida, en la Argentina (con tres filmes na cionales que superaron el millón de espectadores) y Brasil (que alcanzó un market share de 18,6%)-, dando porcentajes que no son representati vos de los resultados de mercado para los filmes latinoamericanos en los últimos 25 años. Fuente: Elaboración propia basada en datos del Incaa, Imcine, Ancine, CNCA, CNAC, Conacine, Dirección de Cinematografía (Colombia), Nielsen, Filme-B, Deisica. La presencia del cine nacional en América Latina Un ejemplo de estas altas y bajas se puede apreciar en México: si en 2013 se estrenaron dos películas que rompieron los récords históricos de asistencia para un filme nacional (“No se admiten devoluciones” y “Nosotros los nobles”), en 2012 el mercado azteca se había presenciado uno de los market share más bajos para el cine nacional desde el año 2000 (4,8%) aunque no tan lejos de los porcentajes de otros años, con un rango que varía entre el 4,3% y el 7,6% —salvo los años 2000, 2001 y 2002, con los éxitos de “Amores perros”, “Y tu mamá también” y “El crimen del Padre Amaro”, donde el cine nacional llegó a alcanzar el 8,9%, 8,5% y el 11,1%, respectivamente. Por otro lado, una buena manera de apreciar la participación de las pelícu las nacionales en sus respectivos mercados es dividir la cantidad de especta dores que fueron a ver cine local por la cantidad de estrenos nacionales. 19 A N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 219 América Latina (2012) - Cantidad de espectadores por estreno nacional # País Cantidad de espectadores por filme nacional estrenado comercialmente 1 Brasil 187.683 2 México 162.687 3 Colombia 161.905 4 Venezuela 113.077 5 Chile 95.385 6 Perú 77.500 7 Argentina 26.818 8 Uruguay 4.118 América Latina (2012) - Cantidad de espectadores por estreno nacional Fuente: Elaboración propia basada en datos del Incaa, Imcine, Ancine, CNCA, CNAC, Conacine, Dirección de Cinematografía (Colombia), Rentrak, Filme-B. América Latina (2007) - Cantidad de espectadores por estreno nacional # País Cantidad de espectadores por filme nacional estrenado comercialmente 1 México 311.628 2 Colombia 206.000 3 Venezuela 166.667 4 Brasil 132.179 5 Chile 78.333 6 Uruguay 74.000 7 Perú 62.500 8 Argentina 33.763 América Latina (2007) - Cantidad de espectadores por estreno nacional 220 De esta manera, se puede apreciar que cinematografías que son consi deradas exitosas en su país —como suele verse a Argentina más allá de sus fronteras—, en realidad se ubican en los últimos lugares de este “ranking”, mientras que cinematografías en recuperación —como la mexicana o bra sileña—, o que ha ido consolidándose lentamente durante la última década —como la colombiana o venezolana—, se ubican en los primeros lugares. La presencia del cine nacional en América Latina Para tratar de comprender mejor el caso argentino, muchas veces sobre valorado, vayamos al análisis de mercado del año 20126: de los 132 estrenos comerciales locales, ninguna película alcanzó un millón de espectadores — cifra que varios blockbusters de Hollywood superan cada año en este país; la última vez que un filme nacional superó el millón de espectadores fue en 2009, con “El secreto de sus ojos”. A su vez, dos filmes argentinos convo caron el 50% de todos los espectadores que fueron a ver cine nacional en este país en 2012, mientras que sólo ocho filmes nacionales superaron los 100.000 espectadores, y 75 no llegaron a convocar 2000 espectadores cada uno (33 películas ni siquiera fueron vistas por 500 espectadores y una doce na de estrenos argentinos no pudieron convencer ni a 100 personas de que las vean…) Estos porcentajes se vienen repitiendo en Argentina desde hace muchos años.7 7 Elaboración propia basada en datos del Incaa, de las consultoras Rentrak, Ultracine, de Deisica y de medios especializados. 6 Insistimos con que se toman los números de 2012, puesto que este año es más representativo –con respecto a los resultados de mercado de la última década- que 2013 (un año atípico). Filmes latinoamericanos no nacionales En el caso de películas iberoamericanas no nacionales, el panorama es peor: la oferta y el consumo de éstas se ubican entre el 0,02% y el 2% —salvo la presencia de algún éxito excepcional, generalmente distribuído por algu na major norteamericana (GONZÁLEZ, 2012: 126). La circulación de películas iberoamericanas al interior de la región es muy limitada, de tal modo que se estrenan en número reducido en los distintos 221 países, variando entre 3 y 20 estrenos anuales, dependiendo del mercado (GONZÁLEZ, 2012: 127). Uruguay tiene una “alta” cantidad de filmes iberoamericanos exhibidos en sus salas comerciales, debido a la tradición cinéfila de su público —si se con taran las exhibiciones en los variados espacios alternativos existentes en el país, sobre todo en Montevideo (destacándose una entidad señera como la Cinemateca Uruguaya), el número de estrenos iberoamericanos registrados en la presente investigación se duplicaría. En Brasil también existe una destacada red de salas de “cine arte”, en es pecial en Rio de Janeiro, São Paulo y Porto Alegre, que es donde se exhiben mayormente los filmes iberoamericanos. Tomando el promedio de los últimos años, el 40% de los filmes iberoameri canos estrenados en los países analizados fueron argentinos. Esta importante presencia de los filmes argentinos en toda la región puede explicarse porque el país sudamericano es el que más largometrajes realiza y también el que más coproduce —lo hace prácticamente con todos los países del subcontinente—, amén de su tradición fílmica de décadas, presente en el imaginario del público latinoamericano (GONZÁLEZ, 2012: 99). Por su parte, un cuarto de los estrenos iberoamericanos correspondió en la década pasada a películas españolas; estos filmes son los que recaudan más, en comparación con los filmes latinoamericanos, debido a que se suelen estrenar los “tanques” de esa filmografía (El laberinto del fauno, Rec, Planet 51, entre otros). Las películas mexicanas representaron el 16% de los filmes iberoameri canos estrenados durante la última década (GONZÁLEZ, 2012: 127). Esto muestra cómo el esfuerzo del país azteca en incrementar su producción va rindiendo sus frutos –amén de que, como en el caso argentino, la cinemato grafía mexicana posee una importante tradición en toda América Latina (las películas clásicas mexicanas todavía se ven en la televisión de varios países de la región, algo que no sucede con cinematografías de otros países). 222 Políticas públicas cinematográficas en América Latina La conformación y el desarrollo de la actividad cinematográfica y audiovi sual sólo puede llevarse a cabo con el firme apoyo del Estado, debido a las altas y riesgosas inversiones necesarias que este sector requiere, en un marco de concentración oligopólica de la distribución y la exhibición —básicamente, en manos de las majors (Buena Vista, UIP, Warner, Fox, Sony), o de grandes empresas asociadas a éstas—, amén de los —relativamente— reducidos mer cados. Esto es cierto en todo el mundo, a excepción de los Estados Unidos, China y la India, países que cuentan con un enorme mercado interno que hace po sible la amortización de sus productos audiovisuales —e inclusive en el caso de los Estados Unidos, el sector cinematográfico se beneficia de distintos apoyos estatales (al contrario de lo que el sentido común indica), tales como subsidios directos (desgravaciones y exenciones fiscales, pagos diferidos, amortizaciones aceleradas) e indirectos (incontables estrategias y recursos políticas y económicos que presionan a nivel mundial en favor de sus propias empresas) (WASKO, 2003; GUBACK, 1980). En China el Estado también se encuentra presente a la hora de fomentar su cine nacional. En América Latina, el neoliberalismo ortodoxo imperante (desregulación estatal, liberalización, privatización) desde las décadas de 1970 y 1980 (de pendiendo del país) dio paso, a lo largo del decenio 2000-2010, a un renovado impulso de políticas públicas heterodoxas destinadas al estímulo del mercado interno, tanto a nivel general, como el ámbito del cine, en particular. Un nuevo “neofomentismo” tuvo lugar al interior de los distintos Estados latinoameri canos a lo largo de esta década, en contra de lo sucedido durante la década de 1990, cuando los cines latinoamericanos (en especial, Argentina, Brasil y México) vieron desmantelar la ayuda estatal al cine. Durante la primera década del siglo XXI en toda América Latina fueron san cionándose legislaciones nacionales dedicadas al cine, como en Chile, Co lombia, Venezuela, Ecuador, Panamá, Uruguay y Nicaragua. Con excepción 223 Revista Brasileira de Estudos de Cinema e Audiovisual de Paraguay y las Guayanas, todos los países de América del Sur y México8 poseen legislación nacional de fomento a la cinematografía9 y órgano rector de la actividad. A nivel general, estas políticas públicas de fomento al cine en América Latina se basan en ayudas directas –sobre todo a la producción, aunque en algunos casos, también en la distribución, exhibición y promoción—, prin cipalmente, a través de subsidios o créditos blandos. 9 En algunos países, como Perú, Bolivia y Venezuela, existen debates al interior del sector para reformular sus respectivas leyes de cine. 8 En el caso de los países centroamericanos y caribeños, el fomento al cine es más endeble (exceptuando el caso cubano), basado principalmente en exenciones fiscales o en incentivos para la utilización de los escenarios naturales locales como escenarios para rodajes de producciones extranjeras. Políticas públicas cinematográficas en América Latina En algunos países los incentivos se extienden a otras actividades audiovisuales, como la producción y difusión de contenidos televisivos y audiovisuales –tal como sucede en Chile o en Colombia (en los últimos años, Brasil aprobó una ley de televisión paga que obliga a emitir contenido brasileño, buscando fomentar la producción na cional, mientras que en la Argentina se lanzaron programas específicos para producir contenidos destinados a la futura televisión digital). Sin embargo, en la práctica, las políticas “neofomentistas” en América La tina se concentran mayoritariamente en la producción cinematográfica con el fin de potenciar el buen uso de la infraestructura disponible en cada país dedicada a esta actividad. Por otro lado, buscan aplicar incentivos fiscales para atraer la inversión, tanto del sector privado –como sucede, por ejemplo, en Brasil, Colombia y México— como de los propios contribuyentes del país a través de impuestos a las taquillas o de partidas provenientes del presu puesto nacional –como ocurre, de distintas maneras, en Argentina, Uruguay, Venezuela, Perú y Bolivia. En la letra de las legislaciones latinoamericanas de fomento al cine se inclu yen todos los eslabones de la cadena productiva, desde el desarrollo hasta la difusión y exhibición. A nivel general, las políticas públicas de fomento al cine en América Latina se basan en ayudas directas –sobre todo a la producción, aunque en algunos casos, también a la distribución, exhibición y promoción—, 224 Revista Brasileira de Estudos de Cinema e Audiovisual principalmente, a través de subsidios o créditos blandos. En algunos países los incentivos se extienden a otras actividades audiovisuales, como la producción y difusión de contenidos televisivos y audiovisuales –tal como sucede en Chile o en Colombia (recientemente, en Argentina se lanzaron programas específicos para producir contenidos destinados a la futura televisión digital). Una de las medidas más antiguas de protección –e inclusive, de promo ción— de la producción cinematográfica a nivel mundial es la institución de la “cuota de pantalla” en las salas de cine –es decir, la reserva de un tiempo mínimo de programación anual o periódica en las salas de cine de un país, destinado a la exhibición obligatoria de filmes nacionales. A pesar de que las legislaciones de distintos países latinoamericanos contemplan la cuota de pantalla –como México, Chile, Perú—, sólo en Argentina, Brasil y Venezuela el Estado tiene una posición más activa con esta prerrogativa. Políticas públicas cinematográficas en América Latina En algunos casos, como en Chile, la televisión tiene la obligación efectiva de programar en sus pantallas cierta cantidad de películas nacionales. Los principales mecanismos de fomento al cine en América Latina Las distintas medidas de apoyo a la actividad cinematográfica fueron, y son muy importantes para mantener la producción cinematográfica en América Lati na y los distintos países que realizan cine, en un contexto de creciente e intensa competencia global –con las majors hollywoodenses como protagonistas. Las medidas más frecuentes de fomento al sector cinematográfico se dan en la producción, aunque también existen —pero en mucha menor me dida— apoyos a la pre producción, a la post producción, la distribución y la exhibición, ayudas que no son homogéneas y varían entre la regulación, el mecenazgo y la promoción (HARVEY, 2005: 428). Estas ayudas se basan en variados sistemas, principalmente constituídos por operaciones no reintegrables, como subsidios, adelantos sobre ingresos de ta quilla y subvenciones de distintos tipos. En América Latina la mayor parte de las ayudas son selectivas, aunque también existen varias de carácter automático. 225 Por otro lado, también fueron y son muy utilizadas las políticas fiscales de exención impositiva para estimular la inversión de capitales privados, princi palmente, en la producción cinematográfica —aunque también se han aplica do líneas en este sentido para la exhibición y otros eslabones de la cadena de valor. Se destacan los casos de Gran Bretaña, Canadá y, en América Latina, Puerto Rico, Brasil —con la sanción de las leyes “Rouanet” (1991) y del Audio visual (1993)— y, desde 2003, Colombia —cuando se dio la aprobación de su ley de cine, que pone mucho énfasis en este tipo de ayudas. Otras medidas no menos importantes son los sistemas basados en el otor gamiento de créditos a la actividad cinematográfica, un sector en donde el ca pital y la banca privada son poco propensos a atender —debido al alto riesgo inherente a esta actividad. Este tipo de ayudas se ofrecen en Francia, España, México, Argentina y Brasil, entre otros países. En este breve compendio sobre ayudas al sector cinematográfico no pue de faltar la “banca cinematográfica”, es decir, experiencias en donde el Estado asume el rol de banquero de la actividad cinematográfica a través de líneas reintegrables de créditos, o la cobertura de riesgos a través de avales o ga rantías. Se destaca la experiencia francesa, la mexicana (entre 1947 y 1979), la brasileña (a través de Embrafilme, entre 1969 y 1990) y, en alguna medida, los sistemas español, portorriqueño y colombiano. Los principales mecanismos de fomento al cine en América Latina Como se mencionó, en Améri ca Latina se destacó la actividad del Banco Cinematográfico de México; y en Italia descolla la Banca Nazionale del Lavoro, con su sección cinematográfica (HARVEY, 2005: 19). 26 A N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 Intentos de políticas públicas regionales en Iberoamérica Desde hace más de dos décadas existen intentos para articular legislacio nes, políticas y acciones gubernamentales de fomento al cine y al audiovisual a nivel regional. Los antecedentes más importantes son la Conferencia Ibe roamericana de Autoridades Cinematográficas (Caci) y la Reunión Especiali zada de Autoridades Cinematográficas y Audiovisuales del Mercosur (Recam). 226 10 Reunión Informal de Ministros y de Responsables de las Políticas Culturales en Iberoamérica. Madrid, 25 y 26 de junio de 1997. La Conferencia de Autoridades Cinematográficas de Iberoamérica (Caci) En noviembre de 1989 los representantes de distintos gobiernos iberoame ricanos firmaron en Caracas tres importantes documentos dirigidos a promover el cine iberoamericano: el Convenio de Integración Iberoamericana, el Acuerdo Latinoamericano de Coproducción Cinematográfica y el Acuerdo para la Crea ción del Mercado Cinematográfico Latinoamericano. A partir de estas firmas nació la Conferencia de Autoridades Cinematográficas de Iberoamérica (Caci) –luego se le agregó el término “audiovisual” al “cinematográfico”, cambiando la sigla a Caaci. Sin embargo, desde hace un par de años, el nombre de este orga nismo volvió a remitir solamente a lo cinematográfico (volviendo a la sigla Caci). En octubre de 1995, la V Cumbre Iberoamericana de Jefes de Estado y de Gobierno, celebrada en Bariloche, Argentina, aprobó de un programa de fomento de la industria audiovisual iberoamericana que sería el único que al canzaría cierta trascendencia en el sector: Ibermedia. Este programa se lanzó buscando dar impulso “a la distribución, promoción, formación, a coproduc ciones y al desarrollo de proyectos”.10 Ibermedia, cuya primera etapa abarcó desde enero de 1998 hasta diciem bre de 2002, se constituyó inicialmente con la adhesión institucional y el apor te monetario de nueve países: Argentina, Brasil, Colombia, Cuba, España, México, Portugal, Uruguay y Venezuela. Luego, a lo largo de los años, se irían incorporando Chile, Perú, Ecuador, Puerto Rico, Costa Rica, República Domi nicana, Panamá y Paraguay. También es de mencionar el programa DocTV Iberoamérica —que se en cuentra enmarcado en la Caci—, integrado por las agencias nacionales de cine y las televisoras públicas-estatales de casi todos los países de la región, cuyo objetivo es fomentar la creación y difusión de documentales latinoamericanos. De manera paradójica el nacimiento de la Caci, y posteriormente, de Iber media, se dio en años en los que la política neoliberal de desguace del Es 227 Revista Brasileira de Estudos de Cinema e Audiovisual tado y de baja en las medidas de protección y fomento a distintos sectores productivos ocurría en la mayor parte de América Latina, con las consecuen cias de desmantelar las industrias locales y facilitar el ingreso irrestricto de bienes y servicios extranjeros —especialmente, de compañías transnacio nales. Todas las cinematografías que tenían algún respaldo estatal tuvieron severos recortes y hasta disolución de las medidas de fomento estatales, dejándolas al borde de la desaparición —especialmente, durante el primer lustro de la década de 1990. “crear la Reunión Especializada de Autoridades Cinematográficas y Audiovisuales (Recam), con la finalidad de analizar, desarrollar e implementar mecanismos destinados a promover la complementación e integración de dichas industrias en la región, la armonización de políticas públicas del sector, la promoción de la libre circulación de bienes y servicios cinematográficos en la región y la armonización de los aspectos legislativos” (GMC, 2003). La Reunión Especializada de Autoridades Cinematográficas y Audiovisuales del Mercosur (Recam) En marzo de 1995 se realizó en Buenos Aires la Primera Reunión Especia lizada de Cultura del Mercosur. Allí se conformaron siete Comisiones Técnicas para tratar distintos aspectos de la cultura subregional; una de ellas se dedi có al sector de las industrias culturales. Desde ese momento comenzaron a realizarse distintas reuniones periódicas, dentro de las cuales tuvo un lugar destacado el sector cinematográfico y audiovisual. Sin embargo, hubo que esperar hasta diciembre de 2003 para que el Grupo Mercado Común del Mercosur —uno de los más altos órganos decisorios de esta instancia supranacional— creara la Reunión Especializada de Autori dades Cinematográficas y Audiovisuales (Recam), invocando el Tratado de Asunción y el Protocolo de Ouro Preto—ambos, documentos fundantes del Mercosur—, además del Protocolo de Integración Cultural del Mercosur, el Protocolo de Montevideo sobre Comercio de Servicios y las Decisiones del Consejo del Mercado Común. 228 A N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 Así, el GMC acordó: Así, el GMC acordó: “crear la Reunión Especializada de Autoridades Cinematográficas y Audiovisuales (Recam), con la finalidad de analizar, desarrollar e implementar mecanismos destinados a promover la complementación e integración de dichas industrias en la región, la armonización de políticas públicas del sector, la promoción de la libre circulación de bienes y servicios cinematográficos en la región y la armonización de los aspectos legislativos” (GMC, 2003). 228 Pocos meses después, en marzo de 2004, la Recam realizó su primera reunión ordinaria. Desde entonces forman parte de la Recam, como países miembros, Argentina (Incaa), Brasil (Secretaría del Audiovisual y Ancine), Pa raguay (Dirección Nacional del Audiovisual) y Uruguay —Instituto Nacional del Audiovisual (INA), que desde 2008 se llama Instituto del Cine y el Audiovisual del Uruguay (Icau). Y como países asociados se encuentran Bolivia —Con sejo Nacional del Cine (Conacine)—, Chile —Consejo Nacional del Arte y la Industria Audiovisual— y Venezuela —Centro Nacional Autónomo de Cinema tografía (CNAC)—; desde 2012 Venezuela es país miembro —en el contexto de similar incorporación de Venezuela al Mercosur, luego de la suspensión de Paraguay a partir del “golpe institucional” contra el presidente Fernando Lugo. En 2011 Ecuador se sumó a la Recam como país asociado, a través del Consejo Nacional de Cinematografía (CNCine). Aunque el nombre de este organismo refiere tanto a cine como a “audiovi sual”, en lo concreto, sólo el ámbito cinematográfico intentó tener cabida en la Recam: nunca hubo un acercamiento serio ni participación sostenida de autoridades relacionadas con el ámbito televisivo u otro relacionado con el quehacer audiovisual no cinematográfico. Esbozo de balance de las iniciativas de integración cinematográfica La cooperación audiovisual depende de la cooperación política, económi ca y cultural de los gobiernos y espacios regionales en donde se inserta. Así, mientras de éstas administraciones suelen centrarse en temáticas económi cas, comerciales, financieras y políticas, la cultura suele quedar relegada. En el ámbito cultural se observa que los organismos regionales existentes suelen actuar sin coordinación, solapadamente, superponiéndose. A su vez, los acuerdos que se toman suelen ser meras declaraciones de buenas inten ciones que no son vinculantes —la Carta Cultural Iberoamericana es un buen ejemplo de ello: las decisiones tomadas en los organismos regionales de cul tura escasamente se trasladan a las normativas nacionales. En el ámbito au 229 Revista Brasileira de Estudos de Cinema e Audiovisual diovisual, esta deficiencia es aún mayor –la nula coordinación entre la Caci y la Recam, es un ejemplo de ello (son varios los países que pertenecen a ambas instancias, con repetidos objetivos y declaraciones de intenciones similares, pero que no dialogan ni se potencian entre sí). Por otra parte, se continúa imponiendo una visión nacional que busca im poner sus intereses por sobre el resto: reuniones que debieran ser de coope ración terminan siendo feroces competencias, en donde la fortaleza de los países más grandes termina imponiéndose. Esto es lo que sucede en las distintas reuniones de Ibermedia —el progra ma regional más exitoso en lo que hace al audiovisual—, según relatan varias autoridades presentes en esas reuniones11: allí, el éxito es medido por sus parti cipantes por la cantidad de proyectos nacionales seleccionados y premiados, y sobre todo, por la suma de apoyos monetarios recibidos en relación a la cuota aportada. A su vez, esto es así porque —merced a las limitaciones presupues tarias para el sector— las asociaciones nacionales de productores y realiza dores interpelan fuertemente a los representantes nacionales de sus agencias nacionales de cine: Ibermedia termina perdiendo la perspectiva, y finalmente se reduce a reuniones con duras negociaciones para tratar de obtener los mayores recursos posibles, más que un lugar de reflexión y acción sobre la construcción y fortalecimiento de un espacio audiovisual regional, con su valor agregado a partir de sinergias y potencialidades que vayan más allá de un mero agregado de realidades nacionales y tengan en cuenta una mayor integración, circulación y espacio común de producción, conocimiento y consumo. La mayoría de los recursos de Ibermedia se destinan a la coproducción. 11 Entrevistas personales realizadas con carácter confidencial a algunos funcionarios y autoridades nacionales de cine presentes en algunas de esas reuniones (entrevistas realizadas en Córdoba, Argentina, 2006; México, 2008; Bogotá, 2011; Río de Janeiro, 2011; Buenos Aires, 2012; y Madrid, 2013). Esbozo de balance de las iniciativas de integración cinematográfica La diferencia con el resto de áreas del programa —distribución, el desarrollo de proyectos y la formación— es bastante significativa. A su vez, Argentina, Brasil, México, Chile y España son los países con más proyectos apoyados —es decir, los países con mayores recursos—, mientras que un porcentaje im portante de los recursos se destina a proyectos que no se concretan y menos 30 A N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 230 Revista Brasileira de Estudos de Cinema e Audiovisual del 30% de los filmes resultantes de este programa se estrenan allende las fronteras de su(s) país(es) productor(es) (GONZÁLEZ, 2012: 103). Otro aspecto importante a tener en cuenta es la profesionalización de los funcionarios de estos organismos públicos. La Caci tiene una Secretaría Ejecutiva en Caracas, ejercida por el presiden te de la agencia venezolana de cine —el Consejo Nacional de Cinematografía (CNAC)—, y a su vez, su principal programa Ibermedia tiene en su oficina de Madrid a profesionales con post grados o gestores culturales con experiencia. El caso de la Reunión Especializada de Autoridades Cinematográficas y Au diovisuales es bien diferente. Aunque la Recam posee una secretaría técnica, esta institución —que fi gura oficialmente en el organigrama del Mercosur— ha estado comandada en la práctica por la Argentina, bajo la influencia de su Agencia Nacional de Cine —el Instituto Nacional de Cine y Artes Audiovisuales (Incaa). En casi diez años de funcionamiento la Recam no ha conseguido ningún logro sustentable e importante: el que pudo haber sido su mayor logro, el Observatorio del Mercosur Audiovisual —constituído por el reconocido inves tigador hispano-argentino Octavio Getino—, quedó congelado en 2008 ante la salida de la Recam por parte de Getino debido a discrepancias políticas. En 2003, en la etapa de conversaciones para crear la Recam entre las dis tintas Agencias Nacionales de Cine del Mercosur, Argentina y Brasil firmaron un acuerdo de codistribución —uno de los primeros en el mundo— para fomentar el estreno de filmes argentinos en Brasil y brasileños en la Argentina —seis por año en cada país— con apoyo de las respectivas Agencias Nacionales de Cine. 12 Argentina es un país que no tiene examen de ingreso para ingresar a la función pública, por lo que la enorme mayoría de los funcionarios –inclusive secretarios de Estado y hasta ministros- apenas tienen educación secundaria. Casi la totalidad de los funcionarios públicos ingresan por contactos políticos o por tener un familiar o conocido con poder trabajando en el Estado. A su vez, los funcionarios comienzan a trabajar sin tener conocimiento alguno para el cargo que fueron asignados. La única Esbozo de balance de las iniciativas de integración cinematográfica Sin embargo, y aunque se incrementó momentáneamente la oferta de estos filmes en ambos mercados, la calidad de los lanzamientos y la respuesta del público fue pobre en Argentina, en donde la ejecución de este convenio dejó mucho que desear por parte de los funcionarios del Incaa12: se hizo un pobre 31 A N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 231 Revista Brasileira de Estudos de Cinema e Audiovisual seguimiento a la comercialización, al marketing y a la difusión de las películas brasileñas —en el lado brasileño se manejaron más profesionalmente. Por ello, tras dos años de intentos, Brasil decidió dejar de aplicar el acuerdo. En 2004, la Recam había intentado avanzar en un Sello Cultural del Mercosur dedicado al audiovisual, pero ante falta de personal capacitado que trabaja ra mancomunadamente con Cancillerías, Ministerios de Economía, Hacienda y Aduanas, entre otras instituciones, el proyecto quedó en la nada. Tampoco fructificaron ideas sobre una “cuota de pantalla regional” y sobre un “certificado de nacionalidad” regional para las películas de países miembros del Mercosur. A su vez, en 2009 la Recam firmó un convenio con la Unión Europea por un monto de 1,86 millón de euros para reforzar el espacio cinematográfico mercosureño —1,5 millón de euros aportados por la Unión Europea, y 360 mil euros aportados por la Recam (a través de los aportes de los países inte grantes). El programa para llevar a cabo este convenio se denominó Programa Mercosur Audiovisual (PMA) e iba a durar tres años (GMC, 2009). Se estable cieron objetivos que, a abril de 2014, no se cumplieron. Uno de los principales objetivos del Programa Mercosur Audiovisual era reforzar el observatorio; cuando se comenzó a implementar, fue lo primero que se dio de baja. Otro de los objetivos importantes era crear una red de unas 30 salas digita les en todo el Mercosur para proyectar exclusivamente películas de los países que conforman la Recam; desde 2008 se viene anunciando que “en breve” se comenzaría a implementar (Mercosur Radio, 2008; UPI, 2012). A abril de 2014 todavía no se construyó ni una sala de esa red. excepción dentro del Estado argentino es el ámbito de Cancillería: allí sí existen duros exámenes para acceder a los cargos diplomáticos –aunque durante el gobierno kirchnerista, también se está dejando a un lado a los funcionarios de carrera para reemplazarlos por funcionarios sin experiencia puestos por afinidad política. Esbozo de balance de las iniciativas de integración cinematográfica 32 A N O 4 • E D 7 \| J A N E I R O J U N H O 2 0 1 5 Otro de los objetivos del PMA era la realización de estudios e investigacio nes. No se hicieron públicas las convocatorias, a pesar de que la Unión Euro pea así lo mandaba. El organismo europeo también pedía que los consultores tuvieran —mínimamente— título universitario y experiencia en el tema sobre el que iban a investigar: las investigaciones terminaron recayendo en produc 232 Revista Brasileira de Estudos de Cinema e Audiovisual tores afines a la jefatura del Incaa y hasta en empleados de ese organismo ar gentino que no tenían título universitario ni experiencia en la temática—según la Unión Europea, ningún empleado de cualquier Agencia Nacional de Cine del Mercosur podía haberse presentado; por este tema, existe una denuncia ante la Oficina Anti-fraude de la Unión Europea.13 En 2009, año de la firma del convenio entre la Recam y la Unión Europea, se anunció que el Programa Mercosur Audiovisual iba a comenzar a implementar se el 1 de julio de ese año (GMC, 2009). Sin embargo, el inicio de la implementa ción se dio recién en febrero de 2011 (PMA, 2011) – momento en el que tendría que haber estado transcurriendo el penúltimo año de implementación. Hasta abril de 2014 lo único concreto que se realizó fueron talleres y en cuentros dispersos –especialmente a partir de 2012, cuando la Unión Europea comenzó a pedir a la Recam que concretara el Programa Mercosur Audiovi sual, que debería haber concluído a mediados de 2012. 13 Denuncia realizada ante la Oficina Europea de Lucha contra el Fraude (OLAF) el 16 de julio de 2012. 14 Un claro ejemplo en este sentido es el caso del área francófona, con un foro institucionalizado de televisoras de distintos países de habla francesa, existente desde hace años. A modo de conclusión Las distintas Agencias Nacionales de Cine latinoamericanas casi no han variado sus políticas de fomento, volcadas casi exclusivamente a la produc ción, con criterios que no varían mucho de las de hace medio siglo. La presencia de los filmes nacionales y latinoamericanos en las pantallas es ínfima: 5% promedio para las películas nacionales y 0,5% promedio para los filmes latinoamericanos —en este último caso, vistos casi exclusivamente por pocos miles de espectadores de buen nivel económico y educativo (es decir, una reducida minoría dentro de la población de la región). Sin embargo, en la televisión de América Latina los contenidos locales son mayoría, en un medio en que el habitante latinoamericano mira entre tres y cuatro horas diarias, a diferencia de la hora y media que, en promedio, dedica cada dos años para ver una película en el cine, o de los 16 años que —según marca el promedio— transcurren para que elija ver una película nacional en 233 las salas... No obstante, la presencia del cine latinoamericano en la televisión es prácticamente nula. Desde hace muchos años se vienen realizando periódicamente llamadas a plantear unas políticas integrales del audiovisual, que engloben al cine, a la televisión y a las nuevas tecnologías audiovisuales. Existen casos aislados, muy voluntariosos, loables y esforzados, impulsados en ocasiones por pro fesionales probos y bien intencionados, como el mencionado Programa Iber media, DocTV Iberoamérica, TAL TV o de la fallida Reunión Especializada de Autoridades Cinematográficas y Audiovisuales del Mercosur (Recam), amén de distintos foros y encuentros que se crean y se esparcen por toda América Latina convocados por productores, realizadores, documentalistas, exhibido res, funcionarios y distintos agentes del sector. Sin embargo, nada concreto ha salido de ello —excepto algunas medidas en pro de la cooperación cinematográfica14 (siendo Ibermedia la que mayores logros ha conseguido) y de la televisión educativa y documental (sin mayor repercusión en la industria ni en las audiencias). A pesar de variados esfuerzos, declaraciones y romantizaciones, el cine latinoamericano continúa en su laberinto, situación incentivada por el des conocimiento mutuo existente entre nuestras variadas cinematografías y sus realidades y que mina la posibilidad de acometer una necesaria integra ción del séptimo arte latinoamericano, para potenciar sinergias, capacida des y públicos. Fuentes estadísticas Agencia Nacional de Cinema (Brasil), Consejo de la Cultura y las Artes (Chile), Consejo Nacional Autónomo de Cinematografía (Venezuela), Consejo Nacional de Cinematografía (Perú), Dirección de Cinematografía (Colombia), Instituto Nacional de Cine (México), Instituto Nacional de Cine y Artes Audiovi 234 suales (Argentina), Deisica, Filme-B, Media Salles, Nielsen-Rentrak, Ultracine, empresas exhibidoras. suales (Argentina), Deisica, Filme-B, Media Salles, Nielsen-Rentrak, Ultracine, empresas exhibidoras. www.programaibermedia.com www.recam.org 235 Bibliografía BALIO, Tino. Hollywood as a Modern Business Enterprise, 1930-1939. Nueva York: Scribners, 1993. CASTAÑEDA LÓPEZ, Liliana. “Agentes del sector cinematográfico y la diversidad cultural en Colombia” en: Comunicación y sociedad. Número 15, enero-junio de 2011, pgs. 143 a 168. GETINO, Octavio. Cine iberoamericano: los desafíos del nuevo siglo. San José de Costa Rica: Editorial Veritas, 2005. GETINO, Octavio. Cine iberoamericano: los desafíos del nuevo siglo. San José de Costa Rica: Editorial Veritas, 2005. __________ “Sin imágenes dónde mirarse” en Le Monde Diplomatique, edición Cono Sur, número 16, octubre de 2000. GONZÁLEZ, Roque. “Cine latinoamericano: entre la pantalla de plata y las pantallas digitales (2000-2009)”. In: Octavio GETINO (coordinador), América Latina: producción y mercados en la primera década del siglo XX. Buenos Aires: Ciccus, 2012, pgs. 61 a 182. __________ Cine latinoamericano y nuevas tecnologías audiovisuales. La Habana: Fundación del Nuevo Cine Latinoamericano, 2011. 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https://openalex.org/W4389834014	https://www.ressat.org/index.php/ressat/article/download/717/188	English	null	Factors Influencing Intention and Behaviour of Agricultural Extension Staff Concerting Post-Covid-19 Digital Technical Guidance: A Study in Yogyakarta, Indonesia	Research in social sciences and technology	2,023	cc-by	10,473	Research in Social Sciences and Technology Factors Influencing Intention and Behaviour of Agricultural Extension Staff Concerting Post-Covid-19 Digital Technical Guidance: A Study in Yogyakarta, Indonesia Rahima Kalikya, Hafni Amalia Juniartia, & Septi Kusumawatia Corresponding author Email: rahima.kaliky@ugm.ac.id * Corresponding author Email: rahima.kaliky@ugm.ac.id ABSTRACT Agriculture extension staff are encouraged to attempt more efficient methods to enhance their competency through digital webinars or technical guidance, in Bahasa Bimbingan Teknis (Bimtek) programs. The research aims to explore factors influencing the intention and behavior of agricultural extension staff towards the Technical guidance online Ministry of Agriculture in Special Region Yogyakarta, Indonesia. This research included the Behavior Intention (BI) and Use Behavior (UB) models with significant construct. The research used the quantitative method and analyzed PLS-SEM. One hundred eighty extension staffs participated, chiefly 34-42 years old. All of the indicators passed the outer model assessment. The analysis results conclude that significant effects are Attitude to BI, Control Behavior to BI, Perceived Usefulness to BI, and Behavior Intention to UB. The perception of extension staffs about Technical guidance online can enhance their performance to continue using it in obtaining agricultural technology information, extension materials, information on agricultural policies, and the latest agricultural development programs. The insignificant constructs are Subjective Norm, Motivation, and Perceived Ease to Use. The validated research model explains 61% of the variance (R2 = 0.61) in Attitude, Perceived Usefulness, and Control Behaviour towards Behavior Intention (BI). Moreover, BI explains 8.9% of the variance (R2 = 0.089) in UB of Technical guidance online. This research found that BI and UB have Q2 values of 0.538 and 0.077. The practical implication could be applied as preliminary development planning studies to enhance the efficiency of Technical guidance online as Bank Data. a. Extension and Communication Development, Graduate School of Gadjah Mada University, Yogyakarta, Indonesia. 10.46303/ ressat.2023.35 10.46303/ ressat.2023.35 Article Info Received: March 14, 2023 Accepted: October 23, 2023 Published: November 1, 2023 How to cite Kaliky, R., Juniarti, H. A., & Kusumawati, S. (2023). Factors influencing intention and behaviour of agricultural extension staff concerting Post-Covid-19 digital technical guidance: A study In Yogyakarta, Indonesia. Research in Social Sciences and Technology, 8(4), 147-171. Kaliky, R., Juniarti, H. A., & Kusumawati, S. (2023). Factors influencing intention and behaviour of agricultural extension staff concerting Post-Covid-19 digital technical guidance: A study In Yogyakarta, Indonesia. Research in Social Sciences and Technology, 8(4), 147-171. https://doi.org/10.46303/ressat.2023.35 Copyright license This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0). KEYWORDS Digital guidance; behavior; dissemination; extension agriculture staff. 148 Kaliky et al. INTRODUCTION Agricultural extension staff are communicators of agricultural development who have the task of helping farmers to be able to farm well and develop their farming businesses to be productive through increasing knowledge, changing farmers' attitudes towards a more positive direction, and improving farmers' skills in farm management. Therefore, agricultural extension staffs must have adequate hard skills (technical knowledge) and soft skills such as communication skills, critical thinking, integrity, creativity, and emotional intelligence. The ability to interact as well to farmers was suggested by (Huda, 2015) is the social competency of extensionst. These competencies are essential for an extension worker, considering that agricultural extension is an active and dynamic learning process between communicators (extension staff) and communicants (farmer and agricultural business actor) that aims to educate farmers and business actors so that they can consciously change their mindset, attitude, and behavior in farming and farming to obtain the benefits as expected (Kaliky & Hariadi, 2013). Some occupations become outdated as revolutions change, while others become crucial. Over the preceding few decades, new technological advances have replaced machinery with telecommunications and electricity (WEF & ADB, 2017). These technologies and trends distort physical, digital, and biological obstacles in many disciplines, businesses, and economies. These advanced technologies will transform future employment (Horáková et al., 2017). Accomplishing the duties and functions as a disseminator, an extension worker is required to have competence in disseminating innovative information. Meanwhile, the results of several studies on the qualification extension staffs in various regions categorized as low to moderate. The conclusion of Muliady's (2009) research in West Java is that the competence of agricultural extension staffs in Karawang, Subang, and Sukabumi districts in developing rice paddy farming is low. The research results (Yusneli & Tanjung, 2021) in Pasaman Regency, West Sumatra, show that the competence of agricultural extension staffs in innovation communication, learning management, and entrepreneurship management is only moderate. Based on (Huda, 2015), factors that significantly affect the ability to communicate agricultural extension innovations are learning facilities, training, and coverage of extension materials. On the other hand, training activities for extension staff are minimal due to restrictions on activities and budget reductions for extension activities, including training for agricultural extension staff in the Covid-19 pandemic. KEYWORDS To overcome these problems, extension staff are expected to follow new efficient ways to get extension material while improving hard skills and soft skills to improve their competence through digital webinars or technical guidance, in bahasa Bimbingan Teknis (Bimtek) programs. Based on Arifin et al. (2021), the agricultural sector can grow positively by 1.75% in 2020 or during the COVID-19 pandemic due to increased agricultural production, fair commodity prices, and aspects of resilience agriculture. According to the standard theory of development economics, the more developed the country, the share of income from the agricultural sector will decrease, while the share of the manufacturing and service sectors will increase. In a RESSAT 2023, 8(4): 147-171 149 recession or economic crisis, the process of structural transformation of the economy is generally disrupted, mainly because quite several people make the agricultural sector the last resort to survive. The agricultural workforce increased significantly from 36.71 million (27.53% of the total workforce of 133.36 million people) in August 2019 to 41.13 million people (29.76% of the total workforce of 138.22 million people) in August 2020 (Arifin et al., 2021). The increased agricultural labor burdens the agricultural sector because agricultural labor productivity is relatively low. Therefore, the agricultural sector must take advantage of the technological changes in production, harvesting, and post-harvesting in the future. The Covid-19 pandemic positively impacted the communication of development information in various fields, including agricultural extension. Based on a survey (Prisma, 2020), 37% of farmers actively use the internet to get agricultural information, primarily through Google and YouTube, and 58% of farmers plan to learn and use the internet. Research (Indraningsih et al., 2020) shows that during the Covid-19 pandemic, agricultural extension staffs had more accessible access to various topics of information ranging from cultivation to marketing because, during the pandemic, there were various webinars and digital technical guidance (Bimtek Online) that were easily accessible without paying. The phenomenon shows that digital webinars or technical guidance is an alternative solution to the problem mentioned. The Ministry of Agriculture has made various breakthroughs in disseminating information, innovations, and agricultural technology during the Covid-19 pandemic intensively by utilizing digital media to accelerate advanced, independent, and modern Indonesian agriculture. Dissemination in the pandemic era is carried out using the digital guidance method. Kaliky et al. The participation of extension staff in the digital guidance is a personal choice. Whether or not to participate in the digital guidance depends on personal intentions and will be manifested in behaviour. This research identifies the intentions and behaviour of agricultural extension staff to utilize Technical guidance online as a medium for obtaining information and knowledge about agricultural technology innovations to improve hard skills and soft skills as an endeavour to improve competence to support extension performance. It is expected that post-Covid-19, the extension staff will utilize various information, innovations, and agricultural technology that have been and will be presented in the Ministry of Agriculture digital guidance. These efforts are inseparable from strong behavioural intentions. Thus, what factors influence the behavioural intentions of agricultural extension staff in the Special Region of Yogyakarta (SRY). Thus, it is necessary to conduct a scientific study to obtain a model of the intention and behaviour of agricultural extension staffs towards digital guidance and the effectiveness of guidance on improving the competence and performance of agricultural extension staffs for supporting the success of post-Covid-19 agricultural development in the SRY, Indonesia. KEYWORDS Some of the echelons that conduct intensive digital guidance are the Directorate General of Food Crops (in Bahasa: Direktorat Jenderal Tanaman Pangan or Ditjen TP), the Agricultural Extension and Human Resources Agency (in Bahasa: Badan Penyuluhan dan Sumber Daya Manusia Pertanian or BPSDMP), and Assessment Institute for Agricultural Technology of Yogyakarta (in Bahasa: Balai Pengkajian Teknologi Pertanian or BPTP DIY). Ditjen TP organizes Technical Guidance and Socialization or Bimbingan Teknis dan Sosialisasi "BTS Propaktani", BPSDMP organizes digital guidance with the concept of Ngobrol Asyik "Ngobras", and BPTP DIY organizes digital guidance "Jumpa Teknologi Virtual (JTV)". The Ministry of Agriculture's digital guidance presents science-based information, technological innovations, and success stories to target audiences of agricultural extension staffs, farmers, researchers, lecturers, agricultural service providers, and other audiences to improve knowledge, skills, and attitudes. Hopefully, agricultural extension staff's competence can improve by participating in digital guidance. Competence is the workability of each individual that includes aspects of knowledge, skills, and attitudes that follow the expected standardization (Undang-Undang (UU) Tentang Sistem Pendidikan Nasional [Law on the National Education System], 2003) and a person's competence in their field of work is inseparable from the personal characteristics that underlie someone behaviour related to a person's work effectiveness (McClelland, 1973). RESSAT 2023, 8(4): 147-171 150 Kaliky et al. Research Question Agricultural extension staffs as disseminators of information and technological innovations are required to have adequate hard and soft skills to support the competence and performance of extension services. It is alleged that the competence of extension staff as disseminators of technological innovation is not optimal in supporting their performance as disseminators. Based on the problem the follo ing research q estions can be form lated Based on the problem, the following research questions can be formulated. Based on the problem, the following research questions can be formulated. • What factors influence agricultural extension staff's intention and behaviour towards the Ministry of Agriculture's digital technical guidance?; • What factors influence agricultural extension staff's intention and behaviour towards the Ministry of Agriculture's digital technical guidance?; • What is the intention and behaviour Technical guidance online model validated with significant construct? • What is the intention and behaviour Technical guidance online model validated with significant construct? The hypothetical model for the research construct is in Figure 1. The following specific hypotheses are put out by this research, which is aligned with the introduction in the preceding section: H1: Attitude has a positive and significant effect on behaviour intention H2: Subjective Norm has a positive and significant effect on behaviour intention H3: Control Behavior has a positive and significant effect on behaviour intention H4: Motivation has a positive and significant effect on behaviour intention H5: Perceived Usefulness has a positive and significant effect on behaviour intention H6: Perceived Ease to Use has a positive and significant effect on Behavior Intention H7: Behavior Intention has a positive and significant effect on Use Behavior RESSAT 2023, 8(4): 147-171 151 Figure 1. Figure 1. Hypothetical Model Intention and Behaviour Digital Guidance Hypothetical Model Intention and Behaviour Digital Guidance Hypothetical Model Intention and Behaviour Digital Guidance Table 1. Table 1. Total population of agricultural extension staff in the district and sample size of the research Working Area Population Sample Bantul District 95 44 Gunungkidul District 129 48 Kulon Progo District 82 42 Sleman District 86 46 Total 392 180 Source: SRY Agriculture and Food Security Office (2022) and primary data. ion of agricultural extension staff in the district and sample size of the research Total population of agricultural extension staff in the district and sampl Agriculture and Food Security Office (2022) and primary data. Source: SRY Agriculture and Food Security Office (2022) and primary data. Location and Sample Selection Technique The research location consists of all districts in Yogyakarta. The sub-district sample unit is an extension institution, namely the Agricultural Extension Centre/ Agricultural Extension Centre for Food and Fisheries (in Bahasa: Balai Penyuluhan Pertanian/ Balai Penyuluhan Pertanian Pangan dan Perikanan or BPP/BP4). The sampling technique was accidental sampling (Sugiyono, 2019). The location of BPP/ BP4 in each district or stratum is not stratified, and population members are considered homogeneous. The population (N) are 392 extension workers, and the total sample is 180 extension staff suitable for the PLS-SEM analysis criteria. The sample distribution is listed in Table 1. Data Analysis y The research data will be analyzed using a PLS-SEM structural model analysis to test the hypotheses. The advantages of PLS-SEM are that it can be used when the sample size is small, the data is not normally distributed, and the model used is complex with many indicators (Hair et al., 2019). When the underlying theory has not been adequately explored, PLS-SEM applies in beneficial (Gotthardt & Mezhuyev, 2022; Rigdon, 2012). The PLS evaluation model is based on prediction measurements. When substantial effects emerge, PLS-SEM has an enhanced probability to identify them (Sarstedt et al., 2017). Based on Chin (2010), when theoretical information is limited and the focus is on revealing significant effects, more accurate statistical power enables utilisation in studies that are exploratory (Chin, 2010; Sarstedt et al., 2017). Ghozali (2014) stated that the evaluation of the PLS model is carried out by evaluating the outer model and inner model. This research aims to identify the Intention and Behaviour Model extension staffs usage Technical guidance online Ministry of Agriculture. This study's outer model evaluation of reflective measurements consists of Cronbach's alpha, Dillon-Goldstein's rho, Composite reliability, and AVE. The inner or structural model consists of R2 value, Q2 or predictive relevance, and path coefficient are measured. The data collection technique uses an The research data will be analyzed using a PLS-SEM structural model analysis to test the hypotheses. The advantages of PLS-SEM are that it can be used when the sample size is small, the data is not normally distributed, and the model used is complex with many indicators (Hair et al., 2019). When the underlying theory has not been adequately explored, PLS-SEM applies in beneficial (Gotthardt & Mezhuyev, 2022; Rigdon, 2012). The PLS evaluation model is based on prediction measurements. When substantial effects emerge, PLS-SEM has an enhanced probability to identify them (Sarstedt et al., 2017). Based on Chin (2010), when theoretical information is limited and the focus is on revealing significant effects, more accurate statistical power enables utilisation in studies that are exploratory (Chin, 2010; Sarstedt et al., 2017). Ghozali (2014) stated that the evaluation of the PLS model is carried out by evaluating the outer model and inner model. This research aims to identify the Intention and Behaviour Model extension staffs usage Technical guidance online Ministry of Agriculture. This study's outer model evaluation of reflective measurements consists of Cronbach's alpha, Dillon-Goldstein's rho, Composite reliability, and AVE. RESEARCH METHOD Quantitative research is conducted with a survey descriptive design approach (adjustable measurement of phenomena) using structured questionnaires that are tested for validity and reliability, and the unit of analysis is the individual (Effendi & Singarimbun, 2012; Sugiyono, 2017). The procedure for gathering data began with focus groups conducted in each regency to acquire information in group circumstances, either through planned interview questions that the moderator asks of each participant in turn or through a script to encourage group conversations in understanding contemporary issues. Focus groups also enable researchers to observe participants' reactions to the comments and opinions expressed by other participants, allowing them to identify parallels and variances in viewpoints. The social phenomena are utilized as the fundamental theoretical and questioning framework. In addition, the questionnaire link is shared with the leader of every sub-district sample unit via WhatsApp. RESSAT 2023, 8(4): 147-171 152 Kaliky et al. Agricultural extension staffs who fulfilled the respondent criterion as SRY extension staff and had experience following the Bimtek Online Program filled out the questionnaire voluntarily. L ti d S l S l ti T h i Agricultural extension staffs who fulfilled the respondent criterion as SRY extension staff and had experience following the Bimtek Online Program filled out the questionnaire voluntarily. Sample Description The total of 180 extension staffs participated, consisting of men and women. The respondents at the research location are mostly 34-42 years old, totalling 65 people, with a percentage of 36%. Respondents aged 25-33 years were 18 people with a percentage of 10%. Extension workers who are quite old, aged 43-51 years, are 54 extension workers or 30%. As many as 24% or 43 agricultural extension workers in Yogyakarta who are categorized as old (52-60 years old) will enter retirement within the next 1-8 years. The complete description of the respondent's age is obtainable in Table 3. Data Analysis The inner or structural model consists of R2 value, Q2 or predictive relevance, and path coefficient are measured. The data collection technique uses an RESSAT 2023, 8(4): 147-171 153 online questionnaire, Google Form, which the construct and indicators questions shown in Appendix 4. Total The age of SRY agricultural extension workers varies, affecting performance. Based on Widakdo et al. (2021), age and performance are interconnected with decreasing performance. A person's expertise, speed, and energy will shrink over time, which adds to the saturation of work, thus affecting the decline in productivity. The tenure is the duration of the respondent's work as an agricultural extension staff member. The situation generally shows that the tenure of agricultural extension staff is relatively moderate. The complete description of the tenure agriculture staffs concluded at Table 4. Table 3. The age categories of respondent Ages Categories Frequency Percentages (%) Very young (25-33 years old) 18 10 Young (34-42 years old) 65 36 Quite old (43-51 years old) 54 30 Old (52-60 years old) 43 24 Total 180 100 Source: Primer Data, 2022 The age categories of respondent Kaliky et al. Most extension workers have a working period of 11-20 years with a frequency of 100 people or 56% or moderate. There were 58 extension workers, or 32 per cent, with a new tenure of fewer than ten years. There are 9 or 5% of extension workers with a working period of 21-30 years or a fairly old category. Extension workers, classified as an old category, have a working period of 31-40 years, as many as 13 people or 7%. According to Siagian (2008), the working period shows how long a person has worked in each job or position. According to Kreitner and Kinicki (2004), a length of service will make an employee feel more completely at ease in an organization. Due to having adapted to the environment, a worker will feel comfortable with his job. This statement is supported by research conducted by Karima et al. (2018) that the tenure factor positively and significantly affects employee performance. The user behaviour of the Ministry of Agriculture's Online Technical Guidance represents the accessibility of information disseminated to agricultural extension staff. Based on the descriptive analysis of the research results, 158 extension staffs or 88% of respondents participated in the BTS Propaktani. There were 62% of respondents or 112 extension staffs following Ngobras. Seventy-three extension staffs, or 40.6% of respondents, followed JTV. The graph of user behaviour is shown in Figure 2. Fi 2 User Behaviour Technical guidance online Ministry of Agriculture Indonesia Source: primer data analysis, 2022 158 112 73 ProPakTani Ngobras Jumpa Teknologi Virtual (JTV) BPTP Yogyakarta User Behavior Technical Guidance Online User Behavior Technical Guidance Online User Behavior Technical Guidance Online Jumpa Teknologi Virtual (JTV) BPTP Yogyakarta Source: primer data analysis, 2022 Based on qualitative data using Focus Group Discussions in four districts, BTS ProPakTani has more positive attention to accessing agriculture innovation and information from the Ministry of Agriculture Indonesia than Ngobras and JTV. BTS Propaktani held the Technical guidance online twice daily and five days per week. The webinar media BTS Propaktani are ZoomMeeting and Live Streaming features on YouTube. They used social media Instagram, YouTube, TikTok, Telegram, and WhatsApp to disseminate the weekly topic list. Most users or extension staffs pick out the topic related to the advisory materials in their fostered area. Moreover, Ngobras held the Technical guidance online once weekly, every Tuesday. The webinar media Ngobras are ZoomMeeting and Live Streaming features on YouTube. Table 4. Table 4. The tenure of agriculture staffs in SRY Tenure Categories Frequency Percentage (%) New (1-10 years) 58 32 Moderate (11-20 years) 100 56 Fairly old (21-30 years) 9 5 Old (31-40 years) 13 7 Total 180 100 Source: Primer Data 2022 RESSAT 2023, 8(4): 147-171 Table 4. The tenure of agriculture staffs in SRY Tenure Categories Frequency Percentage (%) New (1-10 years) 58 32 Moderate (11-20 years) 100 56 Fairly old (21-30 years) 9 5 Old (31-40 years) 13 7 Total 180 100 Source: Primer Data, 2022 The tenure of agriculture staffs in SRY RESSAT 2023, 8(4): 147-171 154 Kaliky et al. Kaliky et al. They used social media, Facebook, Instagram and WhatsApp to inform the event. Ngobras was the RESSAT 2023, 8(4): 147-171 155 communication media among extension staffs and the Head of Indonesia Extension Agriculture Agency. Additionally, Technical guidance online JTV was held and lastly accessible in August 2022. The webinar media JTV are ZoomMeeting and Live Streaming features on YouTube. They used social media, Facebook, Instagram and WhatsApp to inform the event. JTV is used as the innovation dissemination media specific location on Special Region of Yogyakarta, the innovation and information adjust the potential resources on Special Region of Yogyakarta. Scholars have explored how social media supports social mobilization over the last few decades. Indeed, social media allows both individuals and organizations to communicate with their followers and the general public (Forde, 2017; Stein, 2009) Table 5. Outer Model Evaluation Constructs Cronbach's Alpha rho_A Composite Reliability Average Variance Extracted (AVE) Attitude 0.917 0.923 0.934 0.670 Subjective Norm 0.880 0.881 0.918 0.736 Control Behavior 0.689 0.718 0.829 0.619 Motivation 0.892 0.905 0.933 0.822 Perceived Usefulness 0.824 0.831 0.896 0.741 Perceived Ease to Use 0.890 0.893 0.920 0.696 Behavior Intention 0.945 0.946 0.965 0.902 Use Behavior 1.000 1.000 1.000 1.000 Source: primer data analysis, 2022 Source: primer data analysis, 2022 Convergent validity occurs when the scores obtained from two different instruments measuring the same construct have a high correlation. The rule of thumb for convergent validity is outer loading > 0.708 and average variance extracted (AVE) is > 0.5. The findings of SmartPLS 3.0 processing reveal that all indicators have a loading factor value more than 0.708 and an AVE value greater than 0.5. Appendix 1 is the valid model of loading factors. Twelve reflective indicators which drop out from each construct are ATT7, CB1, CB2, CB3, MOV2, MOV3, PEOU4, PEOU5, PU1, PU2, UB1, and UB2. Ensure that every latent variable's conceptualization varies from other latent variables that employed discriminant validity. Discriminant validity is evaluated by assessing the cross- loadings among constructs, the Fornel-Larcker criterion, and the Heterotrait-Monotrait Ratio of Correlation (HTMT). The model has adequate discriminant validity if the AVE squared value of each exogenous construct (the value on the diagonal) is larger than the correlation between the construct and other constructs (the value below the diagonal) (Ghozali, 2014) (Fornell & Larcker, 1981; Ghozali, 2014). Based on the Fornell-Larcker Criterion Value derived as indicated in Appendix 2, the calculation of discriminant validity test utilizing the AVE quadratic value. According to the discriminant validity test, the AVE square root values of all constructs are larger than their connection with other latent details (as assessed by the Fornell-Larcker criteria). Analysis of the structural model Evaluation Outer Model Outer model validity measurement consists of Internal consistency reliability, Convergent validity, and Discriminant Validity. The latent variable is modelled in reflective models as a single predictor of the values of each dependent indicator variable. Multicollinearity is not a problem in an outer reflective measuring model (Garson, 2016). Internal consistency reliability is assessment based on Cronbach's alpha, Dillon- Goldstein's rho, and Composite reliability. Hair et al. (2014) describe Cronbach's alpha as a reliability indicator for internal consistency that counts on equal indicator loadings. Cronbach's alpha assumes equal population covariances among indicators in a single block, which is rare in actual study. However, it can be utilized as a lower constraint on reliability (Guttman, 1945; Sijtsma, 2009). The recommended value of Cronbach's alpha is 0.70-0.90, and the minimum is 0.70 or 0.60 in exploratory research (Hair et al., 2019). Cronbach's alpha is obtained using the indicator variance-covariance matrix, whereas Dillon-alpha Goldstein's alpha is obtained using factor loadings. Subsequently, consistent factor loading estimations should be utilized to calculate Dillon-Goldstein's. Another metric used to assess the unidimensionality of a reflective block is Dillon-Goldstein's rho, which is based on the variance of the sum of variables in the component of relevance. A block is generally considered unidimensional when the Dillon-rho Goldstein's is greater than 0.7. It considers how much the latent variable explains its block of indicators. This index is a stronger indication than Cronbach's alpha (Sanchez, 2018). Composite reliability is considered a more appropriate reliability criterion in the context of PLS-SEM. Higher values imply higher reliability (Gotthardt & Mezhuyev, 2022). The recommended value of composite reliability is the same as Cronbach's alpha. Cronbach's alpha is the lower constraint for internal consistency reliability, whereas composite reliability is the higher bound (Hair et al., 2019). Cronbach's alpha values are unsupported and less accurate, whereas composite reliability is more dependable since indicators are weighted based on the construct's specific indicator loadings (Gotthardt & Mezhuyev, 2022). Cronbach's alpha is likely too conservative, whereas composite reliability is likely too permissive. The ultimate reliability may lie between the two measures (Al-Emran et al., 2019; Hair et al., 2019). RESSAT 2023, 8(4): 147-171 156 Kaliky et al. The Internal consistency reliability test results in Table 5 that all constructs have met the required reliability. The Internal consistency reliability test results in Table 5 that all constructs have met the required reliability. Table 5 Analysis of the structural model The structural model or inner model aims to predict the relationship between latent variables. The coefficient of determination (R2), the blindfolding-based cross-validated redundancy, the statistical significance, and the path coefficients are an example of assessment criteria that should be measured for inner model evaluation. At the structural level, multicollinearity is possible in either a reflective or a formative model. In other words, the latent variables represented as causes of an endogenous latent variable might be multicollinear. Structural RESSAT 2023, 8(4): 147-171 157 multicollinearity is an issue in both reflective and formative models and a problem in OLS regression models (Garson, 2016). The structural Variance Inflation Factor (VIF) coefficients in ideally show VIF < 3, possible collinearity issues when VIF ≥ 3-5, and probable collinearity issues when VIF ≥ 5 (Hair et al., 2019). Based on the inner VIF value, all constructs have VIF values below 5. The VIF value is attainable in Appendix 3. The structural model or inner model is evaluated by looking at the significant structural path coefficient, which eliminated the unsupported hypotheses depicted in Figure 3 and described in Table 7. It resulted in the validated hypotheses that are illustrated in Figure 4 and reported in Table 8. Figure 3. Structural Model Intention and Behaviour Technical guidance online Structural Model Intention and Behaviour Technical guidance online Source: primer data analysis, 2022 Hypotheses Result The stage for assessing the inner model all postulated for the strength and direction of path coefficient values, and produced t-values for relationship significance. Based on PLS-SEM analysis in Table 7 exogenous variables significantly affect endogenous variables: Attitude to Source: primer data analysis, 2022 Table 7. Research hypotheses tested Model Intention and Behaviour Technical guidance online Path Original Sample or β Sample Mean Standar d Deviatio n T Statistics P Values Decision H1 ATT→ BI 0.156 0.156 0.091 1.719 0.043 Supported H2 SN → BI 0.093 0.095 0.105 0.890 0.187 Unsupport ed H3 CB → BI 0.216 0.223 0.100 2.146 0.016 Supported H4 MOV → BI 0.077 0.082 0.081 0.958 0.169 Unsupport ed H5 PU → BI 0.223 0.218 0.088 2.531 0.006 Supported H6 PEOU → BI 0.134 0.127 0.119 1.122 0.131 Unsupport ed H7 BI → UB 0.299 0.303 0.068 4.367 0.000 Supported Source: primer data analysis, 2022 ch hypotheses tested Model Intention and Behaviour Technical guidance online Research hypotheses tested Model Intention and Behaviour Technical guidance Researchers used a bootstrapping technique to assess the statistical significance of the path coefficient values while calculating t-values. Hair et al. (2017) state actual t-values must be higher than critical t-values. This research used 5% significance level, so the critical t-value > 1.96 or the p-value < 0.05. T-values were subsequently utilized to investigate the proposed hypotheses. Based on this research, there are the hypotheses results: 1. Attitude has positive and significant effect on behaviour intention. 2. Subjective Norm has positive and insignificant effect on behaviour intention 3. Control Behavior has positive and significant effect on behaviour intention 4. Motivation has positive and insignificant effect on behaviour intention 5. Perceived Usefulness has positive and significant effect on behaviour intention 6. Perceived Ease to Use has positive and insignificant effect on Behavior Intention Behavior Intention has positive and significant effect on Use Behavior 7. Behavior Intention has positive and significant effect on Use Behavior Validated Model of Intention and Behaviour Technical guidance online Hypotheses Result The stage for assessing the inner model all postulated for the strength and direction of path coefficient values, and produced t-values for relationship significance. Based on PLS-SEM analysis in Table 7, exogenous variables significantly affect endogenous variables: Attitude to Behavior Intention, Control Behavior to Behavior Intention, Perceived Usefulness to Behavior Intention, and Behavior Intention to Usage Behavior. These coefficients represent the proposed correlations between the constructs. The numbers are typically between +1 and 1, with positive signifying a favourable connection and negative signifying an unfavourable connection. The greater the link, the closer the values are to one. As a result, the closer the values are to 0, the weaker the correlation. Bootstrapping was RESSAT 2023, 8(4): 147-171 158 Kaliky et al. used to calculate values and confidence intervals for statistical significance (Hair et al., 2017, 2019). Bootstrapping to 5000, Bias Correct and Accelerated (BCa) Bootstrap, and the one-tailed test type were applied. p g Validated Model of Intention and Behaviour Technical guidance online Validated Model of Intention and Behaviour Technical guidance online The structural relationship of the examined constructs showed that not all path models are significant. Alternatively, we compiled the Validated Research Model of Intention and Behaviour Technical guidance online (with a significant path only). The path model is seen in Figure 4. RESSAT 2023, 8(4): 147-171 159 Figure 4. Figure 4. Validated Model Intention and Behaviour Technical guidance online (with significant path only) Validated Model Intention and Behaviour Technical guidance online (with significant path only) Source: primer data analysis, 2022 Source: primer data analysis, 2022 The inner model assessment described the Validated Path Model of Intention and Behaviour Technical guidance online. The quantity of R2 value and predictive relevance (Q2) were calculated (obtained in Table 8). The validated research model explains 61% of the variance (R2 = 0.61) in Attitude, Perceived Usefulness, and Control Behaviour towards Behavior Intention (BI). Moreover, BI explain 8.9% of the variance (R2 = 0.089) in (Usage Behavior) UB of Technical guidance online, which means it has moderate and very weak explanatory power. Table 8. f Validated Research Model Intention and Behaviour Technical guidance online Coefficient of Validated Research Model Intention and Behaviour Technical guidance online (with significant path only) Construct R Square R Square Adjusted Behavior Intention 0.61 0.603 Use Behavior 0.089 0.084 Source: primer data analysis, 2022 Coefficient of Validated Research Model Intention and Behaviour Technical guid The Q2 value larger than zero means that the preliminary theoretical model is sufficiently predictive, whereas a Q2 score less than zero implies that the theoretical model is insufficiently predictive (Sarstedt et al., 2017). These Q2 values are obtained in Table 9. Blindfolding is calculated from Q2. Construct Cross-validated redundancy. Determining the structural model's prediction accuracy for a certain endogenous component, Q2 values for that construct should RESSAT 2023, 8(4): 147-171 160 Kaliky et al. be greater than zero. Q2 values greater than 0, 0.25, and 0.50 respectively the PLS-path model's small, medium, and large predictive relevance (Hair et al., 2019). T bl 9 be greater than zero. Q2 values greater than 0, 0.25, and 0.50 respectively the PLS-path model's small, medium, and large predictive relevance (Hair et al., 2019). Figure 4. Q2 Value of Validated Model Intention and Behaviour Technical guidance online (with significant path only) Constructs SSO SSE Q² Attitude 1260.000 1260.000 Control Behavior 540.000 540.000 Perceived Usefulness 540.000 540.000 Behavior Intention 540.000 249.631 0.538 Use Behavior 180.000 166.184 0.077 Source: primer data analysis, 2022 alidated Model Intention and Behaviour Technical guidance online (with significant alidated Model Intention and Behaviour Technical guidance online (with significant Q2 Value of Validated Model Intention and Behaviour Technical guidance online (wi path only) Source: primer data analysis, 2022 The value of Q2 has a range of 0 < Q2 < 1, where the closer to 1 means the better the model. This research found that consequently BI and UB have Q2 values of 0.538 and 0.077, calculated in Table 6. That means the predictive relevance category is large and small. Moreover, these revealed Q2 value and R2 value imply that the theoretical model has strong predictive relevance and significant explanatory power. The amount of Q2 is equivalent to the coefficient of total determination in path analysis. The importance of the path's strength is used to analyze t-values to know the value of the relation. The techniques are the same as the t-statistic test before. Table 10 calculates the t-value Validated Model Intention and Behaviour Technical guidance online. Table 10. T-value Validated Model Intention and Behaviour Technical guidance online Construct Original Sample or β Sample Mean Standard Deviation T Statistics P Values Decision ATT-> BI 0.228 0.223 0.072 3.178 0.001 Supported CB -> BI 0.310 0.314 0.079 3.917 0.000 Supported PU -> BI 0.346 0.348 0.071 4.875 0.000 Supported BI -> UB 0.299 0.300 0.066 4.540 0.000 Supported Source: primer data analysis, 2022 T-value Validated Model Intention and Behaviour Technical guidance online DISCUSSION All research hypotheses were tested using PLS-SEM by assessing the outer and inner models. There are four hypotheses that are significant and three hypotheses that are insignificant (see Table 7). Only validated hypotheses are discussed in this section, consisting of Attitude towards Behavior Intention (H1), Control Behavior towards Behavior Intention (H3), Perceived Usefulness towards Behavior Intention (H5), and Behavior Intention towards Use Behavior (H7). RESSAT 2023, 8(4): 147-171 161 Attitude towards Behavior Intention (H1) Attitude is an aspect of cognition (thinking or knowledge), affection (feeling), and predisposition to action (conation) of extension staff towards Technical guidance online Ministry of Agriculture. The results show a p-value < α (0.001 < 0.05), which means that attitude positively affects behavioural intention. The β value is 0.228, which means were attitude rose by one-unit, behavioural intention can increase by 22.8%. This effect is positive. In structural modelling terminology, attitude is a latent variable, where manifest variables (indicators) include cognitive, affective, and conative, as well as verbal and nonverbal reactions. The three-component hierarchical attitude model is shown schematically in the following matrix (Table 11). Table 11. Responses categories of attitudes Response Categories Mode Response Cognition Affection Conation Verbal Expressions of beliefs Expressions of feelings Expressions of intentions Nonverbal Perceptual reactions Physiological reactions Motoric responses Source: Adapted from the theory of attitudes and attitude-behaviour relationships (Ajzen Icek, 1993). Responses categories of attitudes Responses categories of attitudes Table 11 depicts the many sorts of answer categories used in attitude analysis. Perceptions of attitude and object information are contained in cognitive categories. Attitude cognitive indicators include vocal displays of beliefs as well as nonverbal perceptual reactions. Affective responses include spoken displays of feelings toward the attitude object, as well as physical responses, facial expressions, and nonverbal markers of positive or negative emotions. Conative reactions include behavioral inclinations, plans, intentions, commitments, and different overt motor activities that include the attitude object. This research explored the extension staff's cognitive, including the purpose of Technical guidance online and the conviction that Technical guidance online will increase the effectiveness and efficiency of job performance. Technical guidance online provides applicable innovations under field conditions. Moreover, the affective indicator inquired in this research is the feeling of happiness to join Technical guidance online rather than offline/on-site technical guidance because it is more time and cost-efficient. Even though it requires an internet fee (data plan) to access Technical guidance online, the extension workers feel happy because the speakers are RESSAT 2023, 8(4): 147-171 162 Kaliky et al. competent in their innovation fields. The significant indicator that measures the conative is the action plan to prioritize their time to join Technical guidance online. The Role of Control Behavior on Behavior Intention (H3) Control Behavior in this study is a control belief that includes the perception of extension workers regarding the skills needed to successfully carry out activities (participating in Technical guidance online) as measured by two indicators: control beliefs and perceived power. The results show a p-value < α (0.000 < 0.05), which means that Control Behavior positively affects the Behavioural Intention of Technical guidance online. The β value is 0.310, if Control Behavior increased one-unit, Behavioural Intention can increase by 31%. This effect is positive. According to Jogiyanto (2007) perceived behavioral control is the perceived ease or challenge of completing an action. The perceived ease reflects prior experience, expected problems, and difficulties that a someone has surmount obstacles to engaging in a certain behaviour, such as financial, time, and skill restrictions (Ajzen, 1991; Deng et al., 2016). In accordance with the TRA concept developed by Ajzen (1993) into the Theory of Planned Behavior (TPB) that determines intention, not only attitudes towards behavior and subjective norms, but also aspects of perceived behavioral control. Agricultural extension staffs have a cognitive representation in the form of readiness to participate in Technical guidance online. They are influenced by the belief that extension staff can have the strength to strive for facilities and infrastructure that can support the accessibility of digital guidance. Efforts to provide laptops, android phones, laptops, and internet networks are attempted by extension staff to maximize activities to participate. The power felt by extension workers forms the intention of extension workers to continue using digital guidance in obtaining agricultural technology information, obtaining extension materials, and obtaining information on agricultural policies and the latest agricultural development programs. Based on another agriculture research, Jin et al. (2022), self-efficacy and resource convenience have the greatest effect on farmers perceived behavioral control. The degree that people feel their mastery of new technology is expressed in their self-confidence and personal convenience of somatosensory capability of new technologies (Bandura, 1982). The more materials farmers have and the fewer hurdles they anticipate, the greater their perceptual and behavioral control regarding technology adoption and more potent their behavioral intention. (H. Wang et al., 2019). Perceived Usefulness Significantly Effects on Behavior Intention (H5) g y Perceived usefulness is a person's view that using certain technology or innovation (following online technical guidance) will help improve their job performance. Perceived usefulness was discovered as a perception that was directly impacted by perceived ease of use, suggesting that if users thought the system was easy to use, they would believe ICT was beneficial and would be willing to employ the technology (Purnomo & Kusnandar, 2019). The results of this study show a p-value < α (0.000 < 0.05), which means that perceived usefulness has a positive RESSAT 2023, 8(4): 147-171 163 significant effect on the behavior intention of Technical guidance online. The β value is 0.346, were Perceived usefulness increased in one-unit, Behavioural Intention can increase by 34.6%. Extension staffs believe that online technical guidance can increase the knowledge of agricultural extension staffs and can be used to prepare advisory materials. Technical guidance online materials can be accessed again via Google Drive and YouTube. This makes the performance of extension workers easy in carrying out their extension activities. The perception of extension staffs about Technical guidance online that can enhance their performance as extension workers affects the behavioral intention of extension workers to continue using Technical guidance online in obtaining agricultural technology information, extension materials, and information on agricultural policies and the latest agricultural development programs, especially after the Covid-19 Pandemic. The intention of agricultural extension staffs towards digital guidance also needs to be reviewed from the perspective of the Technology Acceptance Model (TAM), a development of the TRA and TPB theories. Technical guidance online can be seen from the perspective of Perceived Usefulness and Perceived Ease of Use. TAM can analyze why Technical guidance online or technology systems are implemented because it includes psychological factors in behavior. The TAM model has been tested and compared with TRA and TPB, concluding that there is consistency that TAM is quite good (Jogiyanto, 2007). The construct of perceived usefulness with indicators works more quickly (work becomes faster), benefits (useful), increases productivity, enhances effectiveness, and improves job performance. Moreover, Perceived Ease of Use with indicators of easy to learn, controllable, clear and understandable, flexible, easy to become skillful, and easy to use (Davis, 1989). Personal variables such as experience and computer anxiety influenced users' beliefs about the perceived usefulness of ICT (Hadi Purnomo & Lee ii, 2010). Perceived Usefulness Significantly Effects on Behavior Intention (H5) TAM pointed out the relevance of PU above PEOU as the main factor of intentional usage in the acceptance model's process, PU was projected to be impacted by PEOU due to the simpler a technology was to utilise, the more advantageous it could be if every other factor was similar (Venkatesh, 2000). Purnomo and Kusnandar's (2019) research shows that perceived usefulness influences the intention to utilize ICT. According to prior TAM research, perceived usefulness is a major factor of technology adoption. (Lee et al., 2014; Ong & Lai, 2006; Park et al., 2009; Venkatesh & Davis, 2000). Perceived usefulness is "the degree of which a person believes that using a particular system would enhance his or her job performance" and Perceived Ease of Use is "the degree of which a person believes that using a particular system would be free of effort" (Davis, 1987). The attitudes of a user toward use a system are determined by PU, which in turn determines behavioural intents and leads to actual system use (Folorunso & Ogunseye, 2008). Behavior Intention and Use Behavior (H7) The intention is a cognitive representation of a person's readiness to perform a certain behavior or action, and this intention can be used as a strong predictor of behavior. The Theory of Planned Behavior (TPB) suggests that behavioral intentions will influence a person's behavior. The TPB RESSAT 2023, 8(4): 147-171 164 Kaliky et al. contains a number of characteristics that help to understand people's behavioral motives and intentions, and it has been frequently employed in studies in social science (H. Wang et al., 2019). The results of this research show a p-value < α (0.00 < 0.05), which means that behavior intention significantly affects the use behavior of Technical guidance online. The β value is 0.299, that means were Behaviour Intention increased by one unit, Use Behavior can increase by 29.9%. Behavioural Intention will determine whether agricultural extension workers will take action to participate Technical guidance online as measured by the intensity. The intention of agricultural extension staff in the form of their readiness to continue using Technical guidance online to obtain information on agricultural technology, extension materials, policy information, and the latest agricultural development programs affects the behavior of agricultural extensionist in the form of length and focus in following Technical guidance online. Interesting Technical guidance online material will affect how long and focused agricultural extension workers participated. Perceived Usefulness Significantly Effects on Behavior Intention (H5) The Theory of Planned Behavior (TPB) is a socio-psychological model that explains human behavioral intentions and analyzes how certain intentions transform into specific behaviors (Tama et al., 2021). TPB's founder, Ajzen (Ajzen, 1991, 2015), defines intention as the immediate act or determinant of a someone's behavior. TPB is a popular socio-psychological model for analyzing factors that influence farmers' intentions toward their actions, according to various studies (Deng et al., 2016; Despotović et al., 2019; Judge et al., 2019; Liao et al., 2018; Y. Wang et al., 2018). In line with use behaviour in agriculture, Jin et al. (2022) described that Tanzanian farmers' willingness to adopt new agricultural technology reflects their technological awareness, social group influence, and control capacities. Practical Implications The participation of extension staff in Technical guidance online is a personal choice. This personal choice depends on intention and behavior. The data and information are important to know as input in managing agricultural extension human resources in SRY related to strategies to enhance the capability of agricultural extension post-Covid-19 pandemic in supporting the competence and performance of extension staff in supporting sustainable agricultural development in SRY. Based on the results of the research, some points to consider compiling practical implications are: • Increased positive Attitudes to use Technical guidance online can be encouraged by raising awareness of cognitive, affective, and conative aspects. There are varied strategies to obtain a positive attitude, such as human resources attitude and capacity building to adapt the digital transformation of the extension system. • Aspects of perceived behavioral control can be improved through cognitive representations in the form of facilities and infrastructure that can support the accessibility of participating in Technical guidance online. Supporting facilities for internet access and adequate equipment must be provided in areas far from the Regency center and with stable and fast internet. The availability of facilities can increase positive perceptions of accessing Technical guidance online. • Perceived Usefulness beneficial of Technical guidance online is a behavior intention that supports the continuation of participating in Technical guidance online. Extension staff feel the benefits of increasing knowledge, using Technical guidance online materials to prepare advisory materials, and improving their job performance. SRY extension workers represent the behavior of using online repositories through Google Drive and YouTube in re-accessing extension materials. • Increased behavioral intention will accomplish the usage behavior Technical guidance online. Optimization of the relevant behavior intention element according to significant constructs consisting of attitude, control behavior, perceived usefulness, and related constructs consisting of Subjective Norm, Motivation, and Perceived Ease to Use are the keys to continuously increasing usage behavior. Synthesizing based on research, data analysis, and the literature review related to Technical guidance online and post-Covid-19 agricultural development in SRY. It have three practical implications in effectiveness of online Ministry of Agriculture Technical guidance in Indonesia. The practical implication of the research can be refer to the policy resources in the Agriculture Extension Digitalization Agenda as initial research, notably the Web 2.0 generation and Web 3.0 in particular. CONCLUSION This research has explored the related constructs that significantly affect Behavior Intention. Based on path analysis PLS-SEM, these constructs significantly affect Behavior Intention are Attitudes, Control Behavior, and Perceived Usefulness. The extension development must be considered another aspect of personality extension staffs and system information infrastructure. The Behavior Intention significantly affects the Usage Behavior. Thus, the Technical guidance online organizer could encourage the behavior intention for the successful online guidance impact. The findings of this study explored the significant validated model of Behavior Intention and Use Behavior of Technical guidance online accessed by agriculture extension staff in the Special Region of Yogyakarta. The significant constructs that affect Behaviour Intention are Attitude, Control Behavior, and Perceived Usefulness. The insignificant constructs are Subjective Norm, Motivation, and Perceived Ease to Use. In addition, Behavior Intention significantly affected the Use Behavior. The practical implication could be preliminary research in the Digitalisation Extension System development agenda in the Ministry of Agriculture, especially in the Post-Covid-19 era. RESSAT 2023, 8(4): 147-171 165 Practical Implications The limited internet access facilities can be mitigated by personalizing the topics and materials of the Technical guidance online, so extension staff can efficiently search for information. In the field of business communication, based on Parravano et al. research (2019), RESSAT 2023, 8(4): 147-171 166 Kaliky et al. the significance of providing personalized content is determined by the importance of producing a highly relevant approach to specific target audience members. The benefits of digital technology and its inclusion into the "Internet of Things" system are utilized. The "Internet of Things" technology, which enables customer communication, is a contemporary digitalization approach with considerable potential to support the successful development of enterprises and benefits. Personalization of Technical guidance online users needs to be implemented to support the Internet of Behaviours (IoB). The gathering and interpreting of data based on user habits and interests is known as IoB. IoB connects the digital environment to human behaviour, traits objectives, and communications, and it enables an adequate adjustment or exchange between the quality of experience (QoE) and the quality of service (QoS). IoB expansion operations may have ramifications for developing knowledge as a service software (De Kerckhove & Saracco, 2021) in agricultural advisory. The behavior of using the online repository can be optimized by providing Bank Data using a knowledge management system (KMS). The Bank Data is a collection of recordings and materials (presentation materials) of Technical guidance online that agricultural extension workers can access at any time. The organization of the Bank Data must be categorized based on agricultural commodities, agricultural topics (cultivation, post-harvest, sales, management, and branding), and the location of agricultural innovations. Knowledge Management can efficiently use Technical guidance online organizers to share knowledge about agricultural process problems at each extension staff location and share experiences about things outside of work that benefit the knowledge development of Technical guidance online users. Based on its definition, Knowledge Management is a process that helps organizations identify, select, organize, disseminate, and transfer important information and experiences that are part of the organization (Mathew, 2010). Limitations of the Study Despite everything, this study has some shortcomings that might be addressed in subsequent studies. First, gender differences were not obtained in this data and analysis. This research is not concerned with gender perception in behavior social science analysis. 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https://openalex.org/W1989835660	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0013629&type=printable	English	null	New Cobalt-Mediated Radical Polymerization (CMRP) of Methyl Methacrylate Initiated by Two Single-Component Dinuclear β-Diketone Cobalt (II) Catalysts	PloS one	2,010	cc-by	6,221	Abstract Two dinuclear cobalt complexes based on bis-diketonate ligands (ligand 1: 3,39-(1,3-phenylene)bis(1-phenylpropane-1,3- dione); ligand 2: 3,39-(1,4-phenylene)bis(1-phenylpropane-1,3-dione)) were successfully synthesized. The two neutral catalysts all showed satisfactory activities in the cobalt-mediated radical polymerization (CMRP) of methyl methacrylate (MMA) with the common initiator of azodiisobutyronitrile (AIBN). The resulting polymerizations have all of the characteristics of a living polymerization and displayed linear semilogarithmic kinetic plots, a linear correlation between the number-average molecular weight and the monomer conversion, and low polydispersities. Mono- or dicomponent low polydispersity polymers could be obtained by using the two dinuclear catalysts under proper reaction conditions. All these improvements facilitate the implementation of the acrylate CMRP and open the door to the scale-up of the syntheses and applications of the multicomponent low polydispersity polymers. Citation: Bao F, Feng L, Gao J, Tan Z, Xing B, et al. (2010) New Cobalt-Mediated Radical Polymerization (CMRP) of Methyl Methacrylate Initiated by Two Single- Component Dinuclear b-Diketone Cobalt (II) Catalysts. PLoS ONE 5(10): e13629. doi:10.1371/journal.pone.0013629 Editor: Matthew H. Todd, University of Sydney, Australia Received February 18, 2010; Accepted July 13, 2010; Published October 26, 2010 Copyright: 2010 Bao 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: 2010 Bao et al. This is an open-access article distributed under the terms of the Creative Commons Attrib use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Fundamental Research Funds for the Central Universities (CUGL100407) and the National Natural Science Foundation of China (50903077, 20872044), the Natural Science Foundation of Hubei Province (2009CDB163, 2008CDZ065). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: polymerbaofeng@yahoo.com.cn (FB); marui.cug@gmail.com (RM) The first example of cobalt-mediated radical polymerization (CMRP), reported by Wayland et al., was the radical polymeri- zation of acrylates in the presence of cobalt porphyrin complexes such as (tetramesitylporphyrinato)cobalt complex 1 [Co(TMP)] [5]. The cobalt complex reversibly end-caps the growing polymeric radicals under heating, which accounts for the equilibrium between dormant (P-CoIIIL) and active species (P?) (Scheme S2). Abstract Later, more efficient halogenated cobalt porphyrin complexes were designed, synthesized, and used to improve the polymerization kinetics. Moreover, the process was extended to alkylcobaloximes 2, which are suitable photoinitiators for CMRP of acrylates [6]. One of the newest promising cobalt-mediated radical polymerization (CMRP) processes for conducting a successful controlled polymerization of VOAc is the recently reported [Co(acac)2]-mediated polymerization. This was initiated with 2,29-azobis(4-methoxy-2,4-dimethyl-valeronitrile) (V-70) in the presence of bis(acetylacetonate)-cobalt(II) ([Co(acac)2]), result- ing in the formation of poly-(vinyl acetate) (PVOAc) with predetermined Mn and low polydispersity. This system has been proposed to follow the organometallic radical polymerization mechanism. There is, however, an important feature of this efficient CMRP system that needs clarification [7–13]. The cobalt complexes used for ATRP had been reported by a few groups. One example was reported by Wang et al. using cobaltocene(II) as a catalyst, which, well known as a 19-electron compound, reportedly reduced the initiator and was oxidized from Co(II) to Co (III) to initiate the radical polymerization of MMA [14–16]. New Cobalt-Mediated Radical Polymerization (CMRP) of Methyl Methacrylate Initiated by Two Single-Component Dinuclear b-Diketone Cobalt (II) Catalysts Feng Bao1, Lingling Feng1, Jie Gao1, Zhifang Tan1, Bin Xing1, Rui Ma2, Chunjie Yan2 1 College of Chemistry, Central China Normal University, Wuhan, China, 2 Department of Materials Science and Chemical Engineering, China University of Geosciences, Wuhan China Feng Bao1, Lingling Feng1, Jie Gao1, Zhifang Tan1, Bin Xing1, Rui Ma2, Chunjie Yan2 1 College of Chemistry, Central China Normal University, Wuhan, China, 2 Department of Materials Science and Chemical Engineering, China University of Geosciences, Wuhan, China Cobalt-mediated radical polymerization of the dinuclear catalytic systems Recently, R. Je´roˆme and his coworkers reported a system based on cobalt acetylacetonate [Co(acac)2] that imparts control to the radical polymerization of vinyl acetate initiated by 2,29-azobis(4- methoxy-2,4-dimethyl valeronitrile), V-70, in the bulk at 30uC. The molar mass of poly(vinyl acetate) indeed changes linearly with monomer conversion, in good agreement with the predicted values. Moreover, the polydispersity is as low as 1.2 [7–13]. These observations are consistent with a mechanism based on reversible addition of the growing radicals to the cobalt complex and establishment of an equilibrium between alkylcobalt(III) and cobalt(II) complexes, that is, the dormant and the active species, respectively. A similar mechanism was previously proposed for acrylate polymerization mediated by cobalt porphyrin and cobaloxime complexes. However, the CMRP of vinyl acetate was initiated at higher temperature (60uC) by AIBN instead of V- 70 in the presence of [Co(acac)2]. Under these conditions, the molar mass again increases with monomer conversion, the polymerization rate is higher, but the experimental molar masses are higher than predicted (Mn,theor/Mn,SEC <0.5), which is evidence for higher probability of irreversible chain termination. Consistently, the molar mass distribution is much broader (Mw/ Mn = 2.0–3.5). Seemingly, the AIBN is not a preferable initiator for CMRP of MMA up to now. In this report, we make an attempt to use a common initiator instead of V-70 in the presence of CMRP. Under the using of the new bis(b-ketone) dinuclear cobalt complexes, the AIBN is an effective initiator in CMRP. The most attractive approach to reduce the catalyst residue in CRPs products is to substantially increase the catalyst activity so that only a very small amount of catalyst is needed to catalyze the polymerization. Thus, no post-purification or catalyst recovery is necessary and the catalyst can be safely and economically left in the polymer products, as in polyolefins [17]. One approach to increase the catalytic activities is to introduce suitable ligands with multiple binding sites. Herein we report the synthesis, reactivity, and polymerization behavior of two dinuclear cobalt catalyst (Scheme S1) capable of controlled radical polymerization of methyl methacrylate. In a comparison of bis(b-diketone) [ligand 1: 3,39-(1,3-phenylene)bis(1- phenylpropane-1,3-dione), ligand 2: 3,39-(1,4-phenylene)bis(1- phenylpropane-1,3-dione)] with b-diketone (acetylacetonate) li- gands, the former have two binding sites and the latter have only one. Interestingly, the structures of the two bis(b-diketone) ligands could be regarded as a joint model of two acetylacetone ligands. Results and Discussion Up to now, the catalyst activity and efficiency of cobalt catalyst are still not so high for CRPs (CMRP or ATRP). These facts mean that these catalysts coprecipitate with the polymer products after polymerization, coloring and contaminating the products. Thus, post-polymerization purification methods and in situ catalyst separations by liquid-liquid and solid-liquid (i.e., solid-supported catalysts) biphasic polymerization have been explored for CRPs to reduce the catalyst residue concentration in the polymers. However, these techniques lead to high costs and scale-up difficulties. Introduction In recent years the development of the controlled/living techniques of radical polymerization (CRPs) was developed as an answer to the steadily increasing demand for new materials with controlled properties [1–4]. This concept is indeed a valuable strategy to provide a large range of polymers with well-defined molecular characteristics (length, composition and architecture), under non-very demanding conditions. The past few years have witnessed the rapid growth in the development and understanding of new CRP methods. All of these methods are based on establishing a rapid dynamic equilibration between a minute amount of growing free radicals and a large majority of the dormant species. Nowadays, nitroxide-mediated polymerization (NMP), atom transfer radical polymerization (ATRP), and radical addition fragmentation chain transfer (RAFT) are among the more popular CRP techniques [4]. NMP and ATRP rely on the same concept, which consists of decreasing the radical concentra- tion in the medium and thus the probability of irreversible termination. This is achieved by reversible conversion of the growing macroradicals P? to dormant species PX. The persistent- radical effect (PRE) is the origin of the high propensity of the radicals to undergo reversible deactivation rather than self- coupling reactions. The lability of the cobalt–carbon bond under thermal and photolytic treatment, and thus the reversibility of its cleavage, makes cobalt complexes suitable candidates for PRE and regulation of CRP. October 2010 \| Volume 5 \| Issue 10 \| e13629 1 PLoS ONE \| www.plosone.org Living Radical Polymerization trinuclear compound [Co3Py6L3]*4.11(CHCl3). Another recent paper reports the same compound as a water solvate and argues that it must be trinuclear, like other characterized members having the same stoichiometry [20–24]. Cobalt-mediated radical polymerization of the dinuclear catalytic systems Therefore, this communication could be considered as a continuous research to the former paper (Scheme S2). The ligands and Co(II) complexes were synthesized by an improved procedure of the literature [18], as shown in Scheme S1. The synthetic way is easily performed to the previous report [19]. The single crystal of ligand 2 was grown in appropriate mixed solvents, and they were fit for X-ray crystallography description. In addition, we also expected that the polymerization could be controlled. The results well coincided with our expection of obtaining higher activity in CMRP. Beyond our expectations, the molecular weight distributions of the obtained polymers showed an uncommon result. The polymerization results were reported in the following text. The structure of ligand 2 has already been reported by Soldatov et al [19]. Two different crystalline forms of the H2L2 were isolated in that paper. As the forms were distinct (as attested by solid state experimental characterization), they were thus con- firmed as polymorphs. The crystal structure of H2L2 (Figure 1) shows a planar structure with the two diketonate units in trans- arrangement to maximized conjugation. The two crystal cell parameters of the ligand 2 obtained by Soldatov group are distinct to the ligand 2 reported by our group. Similarly, the ligand 2 of this paper could be seen as the third polymorphs. We carried out the polymerization of MMA catalyzed by 4–5 (Scheme S2) under conditions similar to those used with the cobalt catalyst reported by Matyjaszewski and Je´roˆme et al and the results were summarized in Table 1. When the polymerization of methyl methacrylate (MMA) is conducted in the presence of complex 5 under the same experimental conditions, clear polymer is formed for only one hour at 80uC (Table 1, sample 6). More interestingly, the polymerization of MMA starts after this short period of time, Not long before, a cobalt complex with ligand 2 has already been reported and crystallographically characterized [19] as a Figure 1. X-ray structure of ligand 5. Hydrogen atoms are omitted for clarity. doi:10.1371/journal.pone.0013629.g001 Figure 1. X-ray structure of ligand 5. Hydrogen atoms are omitted for clarity. doi:10.1371/journal.pone.0013629.g001 PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13629 PLoS ONE \| www.plosone.org 2 Living Radical Polymerization Table 1. Bulk cobalt mediated radical polymerization of methyl methacrylate in the presence of dinuclear complex 4 and complex 5. Cobalt-mediated radical polymerization of the dinuclear catalytic systems Complex Sample number [M]/[Co] [Co]/ [AIBN] t [h] Z[b] [%] Peak #1 Peak #2 Mn,sec [c] [gmol21] Mn,theor [d]/ Mn,sec [c] MW/Mn Content [%] Mn,sec [c] [gmol21] Mw/Mn Content [%] 4 1 320 3 4 66 16400 1.28 1.441 97.95 5950 1.008 2.05 4 2 320 3 8 92 25100 1.17 1.632 95.37 9420 1.032 4.63 4 8 320 2 6 65 17900 1.16 1.382 100 / / 0 4 3 320 2 8 76 24400 1.00 1.566 100 / / 0 4 9 320 2 2 39 12100 1.03 1.421 100 / / 0 4 10 320 1 4 77 24300 1.02 1.273 100 / / 0 5 4 600 3 6 78 34500 1.35 1.076 89.17 30700 1.001 10.83 5 6 600 2 1 29 20500 0.85 1.285 96.54 4800 1.021 3.46 5 5 600 2 2 54 23200 1.40 1.310 87.58 7820 1.015 12.42 5 7 600 2 4 66 29200 1.36 1.594 76.90 9470 1.007 23.10 5 11 600 2 5 73 33100 1.32 1.602 72.58 11890 1.010 27.42 5 12 600 1 4 55 30800 1.08 1.083 98.62 3100 1.025 1.38 [a] Bulk polymerization of MMA at 80uC. [b] The monomer conversion Z is determined gravimetrically after removal of the unconverted monomer in vacuo. [c] Determined by size-exclusion chromatography (SEC). [d]Mn,theor = ([M]0/[Co II]0)6Mmono6Z. doi:10.1371/journal.pone.0013629.t001 Table 1. Bulk cobalt mediated radical polymerization of methyl methacrylate in the presence of dinuclear complex 4 and complex 5. Table 1. Bulk cobalt mediated radical polymerization of methyl methacrylate in the presence of dinuclear complex 4 and complex 5. [a] Bulk polymerization of MMA at 80uC. [b] The monomer conversion Z is determined gravimetrically after removal of the unconverted monomer in vacuo. [c] Determined by size-exclusion chromatography (SEC). [d]Mn,theor = ([M]0/[Co II]0)6Mmono6Z. doi:10.1371/journal.pone.0013629.t001 As far as our knowledge goes, many dinuclear catalysts had been reported for using in ethylene or norbornene polymerization through coordination mechanism [25–27]. We are interested in comparing our results with those applications of dinuclear late transition metal complexes. In ethylene or norbornene polymer- ization, the results showed that the catalytic activity evidently increased and molecular weight distributions of the obtained polymers broadened. The observed effects can be explained on the basis of and steric and electronic effects and cooperative and two independent controlled processes is observed by judging from the GPC curve of the polymer as portrayed in Figure 2. Cobalt-mediated radical polymerization of the dinuclear catalytic systems The polymer shows two separate narrow distribution peaks in the GPC curve. The Mn of the polymer of peak #1 is 20500 g/mol and the Mw/Mn is 1.285. The content of the peak #1 in the total polymer is estimated to 96.54% by using Wyatt Technology (Astra 473) software. The Mn of the polymer of peak #2 is 8400 g/mol and the Mw/Mn is 1.021. Similar results are also found in many other polymerization products. and two independent controlled processes is observed by judging from the GPC curve of the polymer as portrayed in Figure 2. The polymer shows two separate narrow distribution peaks in the GPC curve. The Mn of the polymer of peak #1 is 20500 g/mol and the Mw/Mn is 1.285. The content of the peak #1 in the total polymer is estimated to 96.54% by using Wyatt Technology (Astra 473) software. The Mn of the polymer of peak #2 is 8400 g/mol and the Mw/Mn is 1.021. Similar results are also found in many other polymerization products. Figure 2. Size-exclusion chromatogram for Sample 6. doi:10.1371/journal.pone.0013629.g002 PLoS ONE \| www.plosone.org 3 October 2010 \| Volume 5 \| Issue 10 \| e13629 Figure 2. Size-exclusion chromatogram for Sample 6. doi:10.1371/journal.pone.0013629.g002 October 2010 \| Volume 5 \| Issue 10 \| e13629 Living Radical Polymerization interactions of the two adjacent metal centers in dinuclear complexes. These two metal centers are electronically coupled through the ligand bridge. When one of the centers was activated at a given time, it had an electronwithdrawing effect on another center. The interaction between two metals made it possible to create more than one kind of active species during the polymerization. The two separate late metal complex units act as the mutual bulky steric effects for each other, which leads to the excellent catalytic performance. dinuclear catalysts, it has the same possibilities to create two kinds of active species during the CMRP for the electronwithdrawing effect on another center. As described in Scheme S3, a cobalt complex corresponds to polymerization equilibrium in CMRP of acrylates. In an individual polymerization process, the two kinds of active species catalyzed two independent cobalt-mediated radical polymerization processes. That is why we got the peculiar polymer of two distinct molecular weights as the GPC curve shown in Figure 2. The very narrow polydispersities of the two polymer components indicate the two independent cobalt-mediated radical polymerization processes are controlled and living. Cobalt-mediated radical polymerization of the dinuclear catalytic systems Not long ago, a significant research about dinuclear catalyst system arouses our attention. Matyjaszewski group developed a highly active dinuclear copper-based catalyst of copper-based complex CuBr/N,N,N’,N’-tetrakis(2-pyridylmethyl)- ethylenedi- amine (TPEN) for atom transfer radical polymerization [17]. The activator CuIBr/TPEN existed in solution as dinuclear and mononuclear complexes in equilibrium but as a dinuclear complex in its single crystals. Therefore, both mononuclear and dinuclear complexes coexisted in the solution of CuBr/TPEN at a 1/1 molar ratio. There was fast exchange equilibrium between the dinuclear and mononuclear complexes. The facts of the real active species of CuBr/TPEN catalyst in solution might be the mononuclear species were found by the large differences in catalytic activity between CuBr/TPEN and CuBr/BPMPrA. Therefore, only single narrow molecular weight distribution peak was found in GPC curve. Other bulk CMRP results are summarized in Table 1 and the other GPC curves are shown in Figure 3 and Figure 4. The results of two peaks in an individual GPC curves are found in most bulk CMRPs. Nonetheless, there still some polymers had only single narrow molecular weight distribution peak in GPC curve. It could be observed in Figure 3 (Sample 3) and Table 1 (Sample 8, 9, 10). From the above-mentioned GPC results, some unimodal distri- bution means only one active species worked in the CMRP with the catalyzation of a dinuclear cobalt complex under certain reaction conditions. Take all the GPC results shown in Table 1 into account, we could find the monocomponent polymer obtained by CMRP are all catalyzed by complex 4 (shown in Scheme S2) system. Similar results were not found in complex 5 systems. When will we get monocomponent narrow distribution polymers as well as when will we get two-component narrow distribution polymers, that are very significant questions to us. It is natural for us to explain these questions from the molecular structures of the two dinuclear complexes (complex 4 and complex 5). From Scheme S2, in complex 4, one benzoylacetone substituent group is in the para position to the other benzoyla- cetone substituent. Relatively, in complex 5, the benzoylacetone substituent group is in the ortho position to the other Originally, we expected to get higher activity systems in CMRP by using the dinuclear cobalt complexes. It’s a pleasant surprise to us, higher catalytic activities in CMRP is successfully obtained as expected. Cobalt-mediated radical polymerization of the dinuclear catalytic systems On the contrary to the ethylene polymerization, the molecular weight distributions of the obtained CMRP polymers did not broaden. Moreover, the polydispersity (PDI) of the two different constituents in Sample 6 (Table 1) are all very narrow (1.285 and 1.021). Similar to ethylene polymerization catalyzed by Figure 3. Size-exclusion chromatograms for Sample 1–3. doi:10.1371/journal.pone.0013629.g003 PLoS ONE \| www.plosone.org 4 October 2010 \| Volume 5 \| Issue 10 \| e13629 Figure 3. Size-exclusion chromatograms for Sample 1–3. doi:10.1371/journal.pone.0013629.g003 October 2010 \| Volume 5 \| Issue 10 \| e13629 October 2010 \| Volume 5 \| Issue 10 \| e13629 Living Radical Polymerization Figure 4. Size-exclusion chromatograms for Sample 4–7. doi:10.1371/journal.pone.0013629.g004 Living Radical Polymerization Figure 4. Size-exclusion chromatograms for Sample 4–7. doi:10.1371/journal.pone.0013629.g004 Figure 4. Size-exclusion chromatograms for Sample 4–7. doi:10.1371/journal.pone.0013629.g004 benzoylacetone substituent. These facts mean the two cobalt metal center in complex 5 is closer than in complex 4. Therefore, for the higher electrophilic and steric effect, the complex 5 is more liable to generate two active species in CMRP. With a comparison to the bi-component polymers catalyzed by complex 4, the mono- component polymers catalyzed by the same complex are all high molecular weight constituent. polymerization of MMA starts after this period of time, and a controlled process is observed, as assessed by the linear increase of the molar mass with monomer conversion (Figure 5 and Figure 6). As a rule the polydispersity is very low (1.076#Mw/Mn#1.632) throughout the polymerization. The slight but significant increase in polydispersity with monomer conversion is observed in any controlled radical processes as result of irreversible termination reactions and, in this case, possible transfer reactions to monomer and/or polymer. Further evidence for the pronounced efficacy of benzoylacetone substituent. These facts mean the two cobalt metal center in complex 5 is closer than in complex 4. Therefore, for the higher electrophilic and steric effect, the complex 5 is more liable to generate two active species in CMRP. With a comparison to the bi-component polymers catalyzed by complex 4, the mono- component polymers catalyzed by the same complex are all high molecular weight constituent. As a rule the polydispersity is very low (1.076#Mw/Mn#1.632) throughout the polymerization. The slight but significant increase in polydispersity with monomer conversion is observed in any controlled radical processes as result of irreversible termination reactions and, in this case, possible transfer reactions to monomer and/or polymer. Conclusion In conclusion, we have developed the first dinuclear catalyst for rapid, living MMA polymerization as demonstrated by kinetics studies and GPC data. The two unique, symmetrical, dinuclear cobalt complexes can be easily synthesized in one step from the bis- diketonate ligands. The neutral catalysts show satisfactory activities in MMA living polymerization. Interestingly, one or two low polydispersity polymers could be obtained by using the two dinuclear catalysts under proper reaction conditions. As we know, in precision gel permeation chromatographic (GPC) work, the calibration curve is obtained by using standard polystyrenes or other low polydispersity polymers. A general idea is to make ‘‘cocktails’’ – mixtures that contain several standards. That way, we don’t have to make so many injections. But don’t make the polymers too close. Therefore, the syntheses of multicomponent low polydispersity polymers have significant values in polymer chemistry. In this study, our successful producing two-component low polydispersity PMMA confirm the feasibility of multicomponent low polydispersity polymers syntheses. The further study of the multinuclear catalysts in CRP is currently under investigation and will be published shortly. The kinetics of this CMRP of MMA are first-order in monomer, as assessed by the linear dependence of ln([M]0/[M]) versus time (Figure 5 and 6), which confirms that the macroradical concentration remains essentially constant and that irreversible chain termination reactions are quite restricted. In contrast to vinyl acetate polymerization reported by Je´roˆme et al, an induction period is not evidently observed in the radical polymerization of MMA, which would be almost simultaneous of the radicals generated in situ to convert CoII into CoIII_P with the polymerization beginning. During this beginning period of CMRP, a drastic change in the color of the medium, from purple to dark brown-green, is observed, consistent with the suggested change in oxidation state of the cobalt complex. MMA oligomers (Mn = 4300 gmol21, Mw/Mn = 1.21), pre- formed in bulk at 50uC with AIBN in the presence of complex 4, were collected and purified by repeated precipitation in heptane, and finally used as macroinitiators for MMA polymerization. As shown in Figure 7, resumption of the MMA polymerization was successful, as assessed by the shift of the SEC chromatograms with time towards lower elution volumes. A new MMA homopolymer (Mn = 11650 g/ mol) was obtained with its maintained narrow polydispersity (PDI= 1.17), and a very small amount of unconverted macro- initiator. Conclusion This observation paves the way for the synthesis of block Cobalt-mediated radical polymerization of the dinuclear catalytic systems Further evidence for the pronounced efficacy of As expected, the molar mass of primary constituent of PMMA, is controlled by the [MMA]/[Co2+] ratio. Moreover, the Figure 5. Dependence of PMMA molar mass Mn on monomer conversion Z for bulk polymerization of MMA at 80uC (for the primary constituent of the obtained PMMA). [MMA]/[Co(II)] = 600 (Table 1). (% complex 4,N complex 5) doi:10.1371/journal.pone.0013629.g005 Figure 5. Dependence of PMMA molar mass Mn on monomer conversion Z for bulk polymerization of MMA at 80uC (for the primary constituent of the obtained PMMA). [MMA]/[Co(II)] = 600 (Table 1). (% complex 4,N complex 5) doi:10.1371/journal.pone.0013629.g005 October 2010 \| Volume 5 \| Issue 10 \| e13629 October 2010 \| Volume 5 \| Issue 10 \| e13629 PLoS ONE \| www.plosone.org 5 Living Radical Polymerization Living Radical Polymerization Figure 6. Time dependence of ln([M]0/[M]) for the bulk polymerization of MMA at 80uC (for the primary constituent of the obtained PMMA). [MMA]/[Co(II)] = 600 (Table 1). [M]0 and [M] are the MMA concentration at times 0 and t, respectively. (% complex 5; &complex 4) doi:10.1371/journal.pone.0013629.g006 Living Radical Polymerization Figure 6. Time dependence of ln([M]0/[M]) for the bulk polymerization of MMA at 80uC (for the primary constituent of the obtained PMMA). [MMA]/[Co(II)] = 600 (Table 1). [M]0 and [M] are the MMA concentration at times 0 and t, respectively. (% complex 5; &complex 4) doi:10.1371/journal.pone.0013629.g006 copolymers by sequential polymerization of MMA and any comonomer whose polymerization is controlled by the same initiator/Co complex system. In that case, exchange of Co complexes is required between the consecutive polymerization steps. this cobalt complex in mediating the radical polymerization of MMA can be found in the good agreement between experimental and theoretical molar masses (Mn,theor/Mn,SEC = 160.40, Table 1, for the peak #1). The theoretical values were calculated under the assumption that one polymer chain is growing per cobalt complex unit, according to the mechanism proposed by Wayland and co- workers for the CMRP of acrylates (Scheme S3). 1H NMR and FT-IR analyses showed that the obtained polymers were more or less syndiotactic, similar to samples prepared with ordinary CRPs. There were almost no effects of AIBN on their tacticity. Lowering the temperature increased the racemic (rr) content, which would be expected for a free-radical polymerization. These also support the radical propagation mechanism with the cobalt catalysts. PLoS ONE \| www.plosone.org Supporting Information Scheme S1 Syntheses of dinuclear cobalt(II) complexes Found at: doi:10.1371/journal.pone.0013629.s001 (0.42 MB TIF) Scheme S1 Syntheses of dinuclear cobalt(II) complexes Found at: doi:10.1371/journal.pone.0013629.s001 (0.42 MB TIF) Scheme S1 Syntheses of dinuclear cobalt(II) complexes Found at: doi:10.1371/journal.pone.0013629.s001 (0.42 MB TIF) Scheme S2 Structure of cobalt(II) complexes (1–5) Found at: doi:10.1371/journal.pone.0013629.s002 (0.39 MB TIF) Scheme S2 Structure of cobalt(II) complexes (1–5) Found at: doi:10.1371/journal.pone.0013629.s002 (0.39 MB TIF) Scheme S2 Structure of cobalt(II) complexes (1–5) Found at: doi:10.1371/journal.pone.0013629.s002 (0.39 MB TIF) , , , , , , , , , Complex 4: The ligand 2 of 3,39-(1,4-phenylene)bis(1-phenyl- propane-1,3-dione) was synthesized improving to the synthesis of ligand 1. Elemental analysis calculated for C24H18O4: C 77.82, H 4.90; found: C 77.90, H 5.01. The single-crystals (yellow crystals) of Ligand 2 were grown in ethanol, and the ORTEP plot of structure is shown in Fig. 1. (CCDC reference number is 678685.) The complex 4 (as brown crystal) was obtained analogously to the preparation of complex 1. Elemental analysis calculated for C48H32Co2O8: C 67.46, H 3.77; found: C 67.72, H 3.85. However, attempts to get mono-crystal structure of complex 4 were Complex 4: The ligand 2 of 3,39-(1,4-phenylene)bis(1-phenyl- propane-1,3-dione) was synthesized improving to the synthesis of ligand 1. Elemental analysis calculated for C24H18O4: C 77.82, H 4.90; found: C 77.90, H 5.01. The single-crystals (yellow crystals) of Ligand 2 were grown in ethanol, and the ORTEP plot of structure is shown in Fig. 1. (CCDC reference number is 678685.) Th l 4 ( b t l) bt i d l l t th Complex 4: The ligand 2 of 3,39-(1,4-phenylene)bis(1-phenyl- propane-1,3-dione) was synthesized improving to the synthesis of ligand 1. Elemental analysis calculated for C24H18O4: C 77.82, H 4.90; found: C 77.90, H 5.01. The single-crystals (yellow crystals) of Ligand 2 were grown in ethanol, and the ORTEP plot of structure is shown in Fig. 1. (CCDC reference number is 678685.) Scheme S3 Equilibrium between dormant and active species in CMRP of acrylates Scheme S3 Equilibrium between dormant and active species in CMRP of acrylates y Found at: doi:10.1371/journal.pone.0013629.s003 (0.06 MB TIF) y Found at: doi:10.1371/journal.pone.0013629.s003 (0.06 MB TIF) Materials and Methods All manipulations involving air- and moisture-sensitive com- pounds were carried out under an atmosphere of dried and purified nitrogen using standard Schlenk and vacuoline techniques. Solvents were purified using standard procedures. Reagents of the 2,29- azobisisobutyronitrile (AR) were obtained from Aldrich and used without further purification. Methyl methacrylate were dried over CaH2, and then freshly distilled under vacuo prior to use. Molecular weights and molecular weight distributions were determined using PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13629 6 Living Radical Polymerization Living Radical Polymerization Figure 7. Size-exclusion chromatogram for the macroinitiator and block copolymer. doi:10.1371/journal.pone.0013629.g007 Figure 7. Size-exclusion chromatogram for the macroinitiator and block copolymer. doi:10.1371/journal.pone.0013629.g007 unsuccessful. 1H NMR of Complex 4 (400M Hz, CDCl3): 8.70 (6H, m), 7.57–7.64(12H, m), 6.99(6H, s). FT-IR(KBr): 2986, 1709, 1611, 1497, 1439, 1313, 1229, 1146, 1089, 1016, 750, 713 cm21. gel permeation chromatography (GPC) coupled with multiangle laser-light scattering (MALLS). The system included a StyragelH HMW 6E GPC column (7.86300 mm), a Wyatt OPTILAB RI detector, and a Wyatt multiangle laser-light scattering detector (DAWN E). The MALLS operated at 18 angles, from 26u to 149u and was equipped with a He-Ne laser (690 nm). The column and the RI detector were set at 40uC. The mobile phase was THF and the polymer solutions of injected sample were filtered through 0.2 mm syringe filters before injection. The molecular weights and polydispersity indexes were measured for the main peaks and were calculated using Wyatt Technology (Astra 473) software. The MALLS detector allowed the calculation of absolute molecular weights. Infrared spectra were recorded on polymer-KBr pellets with a Bruker EQUINOX55 FT-IR spectrophotometer in the region of 4000,400 cm21. NMR spectra were obtained using a Varian 400 MHz at room temperature in CDCl3 solution using tetramethylsilane as internal standard. General polymerization procedure AIBN (98.4 mg, 0.60 mmol) and cobalt complex 4 (85.4 mg, 0.1 mmol) and solvent (xylene, 10 ml) were placed in a 50 mL flask and degassed by three vacuum/nitrogen cycles. Dry, degassed methyl methacrylate (11 mL, 120 mmol) was then added by syringe under nitrogen. The purple mixture was stirred after heated at 80uC. After a few hours, the color changed from purple to dark brown-green. Polymerization occurred for no less than 4 h, after which the viscosity of the solution increased substan- tially. After a given time, the mixture was diluted with THF and poured into 10-fold methanol. The resulting precipitated polymers were treated by filtering, washing with methanol several times, and drying under vacuum at 40uC to a constant weight. Some samples were preceded at different reaction times, and monomer conversion was determined by weighing the collected polymer after removal of the unconverted monomer in vacuo. Synthesis of two cobalt complexes Complex 5: The ligand 1 of 3,39-(1,3-phenylene)bis(1-phenyl- propane-1,3-dione) was synthesized according to Pikramenou and his coworkers’ previous work. PMMA oligomers of only one constituent (460 mg, Mn,SEC = 4300 gmol21, Mw/Mn = 1.21) were prepared at 50uC with AIBN in the presence of complex 4 and collected at low monomer conversion by repeated precipitation in heptane. They were dissolved in degassed MMA (2.5 mL, 26.5 mmol) and heated at 50uC to resume the controlled radical polymerization. his coworkers previous work. To a DMF (5 ml) solution of 1,3-bis(3-phenyl-3-oxopropanoyl)- benzene (40 mg, 0.11 mmol) were added triethylamine (0.31 ml, 0.22 mmol) and cobalt(II) chloride (16 mg, 0.12 mmol) dissolved in DMF (5 ml). The brown precipitate was collected for further characterized in about 80% yield. Elemental analysis calculated for C48H32Co2O8: C 67.46, H 3.77; found: C 67.52, H 3.68. However, attempts to get mono-crystal structure of complex 5 were unsuccessful. 1H NMR of Complex 5 (400M Hz, CDCl3): 8.59 (H, m), 7.50–7.59(12H, m), 6.94(6H, s). FT-IR(KBr): 2924, 1661, 1599, 1508, 1454, 1424, 1292, 1208, 1106, 1057, 744, 700 cm21. Complex 4: The ligand 2 of 3,39-(1,4-phenylene)bis(1-phenyl- propane-1,3-dione) was synthesized improving to the synthesis of ligand 1. Elemental analysis calculated for C24H18O4: C 77.82, H 4.90; found: C 77.90, H 5.01. The single-crystals (yellow crystals) of Ligand 2 were grown in ethanol, and the ORTEP plot of structure is shown in Fig. 1. (CCDC reference number is 678685.) The complex 4 (as brown crystal) was obtained analogously to the preparation of complex 1. Elemental analysis calculated for C48H32Co2O8: C 67.46, H 3.77; found: C 67.72, H 3.85. However, attempts to get mono-crystal structure of complex 4 were p To a DMF (5 ml) solution of 1,3-bis(3-phenyl-3-oxopropanoyl)- benzene (40 mg, 0.11 mmol) were added triethylamine (0.31 ml, 0.22 mmol) and cobalt(II) chloride (16 mg, 0.12 mmol) dissolved in DMF (5 ml). The brown precipitate was collected for further characterized in about 80% yield. Elemental analysis calculated for C48H32Co2O8: C 67.46, H 3.77; found: C 67.52, H 3.68. However, attempts to get mono-crystal structure of complex 5 were unsuccessful. 1H NMR of Complex 5 (400M Hz, CDCl3): 8.59 (H, m), 7.50–7.59(12H, m), 6.94(6H, s). FT-IR(KBr): 2924, 1661, 1599, 1508, 1454, 1424, 1292, 1208, 1106, 1057, 744, 700 cm21. PLoS ONE \| www.plosone.org References 1. Matyjaszewski K, Xia J (2001) Atom transfer radical polymerization. Chem Rev 101: 2921–2990. and fluorous media—A versatile route to catalyst recycling and oligomer formation. J Polym Sci Part A: Polym Chem 43: 3804–3813. 2. Patten TE, Xia J, Abernathy T, Matyjaszewski K (1996) Polymers with very low polydispersities from atom transfer radical polymerization. Science 272: 866–868. 17. Tang H, Arulsamy N, Radosz M, Shen Y, Tsarevsky NV, et al. (2006) Highly active copper-based catalyst for atom transfer radical polymerization. J Am Chem Soc 128: 16277–16285. . Percec V, Barboiu B (1995) ‘‘Living’’ radical polymerization of styre 3. Percec V, Barboiu B (1995) ‘‘Living’’ radical polymerization of styrene initiated by arenesulfonyl chlorides and CuI(bpy)nCl. Macromolecules 28: 7970–7972. 18. Bassett AP, Magennis SW, Glover PB, Lewis DJ, Spencer N, et al. (2004) Highly luminescent, triple- and quadruple-stranded, dinuclear Eu, Nd, and Sm(III) lanthanide complexes based on bis-diketonate ligands. J Am Chem Soc 126: 9413–9424. 4. Goto A, Fukuda T (2004) Kinetics of living radical polymerization. Prog Polym Sci 29: 329–385. 5. Wayland BB, Poszmik G, Mukerjee SL, Fryd M (1994) Living radical polymerization of acrylates by organocobalt porphyrin complexes. J Am Chem Soc 116: 7943–7944. 19. Soldatov DV, Zanina AS, Enright GD, Ratcliffe CI, Ripmeester JA (2003) A new bridging chelating ligand for crystal engineering: synthesis, polymorphism, and two modes of assembly of 1,4-bis(3-phenyl-1,3-propanedion)benzene with metal cations resulting in either discrete or polymeric complexes. Crystal Growth & Design 3: 1005–1013. 6. Arvanitopoulos LD, Greuel MP, Harwood HJ (1994) ‘‘Living’’ free radical polymerization using alkyl cobaloximes as photoinitiators. Polym Prepr 35: 549–550. 20. Clegg JK, Bray DJ, Gloe K, Gloe K, Jolliffe KA, et al. (2008) Synthetic, structural, electrochemical and solvent extraction studies of neutral trinuclear cobalt(II), nickel(II), copper(II) and zinc(II) metallocycles and tetrahedral tetranuclear iron(III) species incorporating 1,4-aryl-linked bis-b-diketonato ligands. Dalton Transactions. pp 1331–1340. 7. Debuigne A, Caille J, Je´roˆme R (2005) Highly efficient cobalt-mediated radical polymerization of vinyl acetate. Angew Chem Int Ed 44: 1101–1104. 8. Debuigne A, Caille J, Detrembleur C, Je´roˆme R (2005) Effective cobalt mediation of the radical polymerization of vinyl acetate in suspension. Angew Chem Int Ed 44: 3439–3442. 21. Cotton FA, Soderberg RH (1964) A spectroscopic study of the polymeric nature of bis(acetylacetonato)cobalt(II). Inorg Chem 3: 1. 9. Wayland BB, Basickes L, Mukerjee S, Wei M, Fryd M (1997) Living radical polymerization of acrylates initiated and controlled by organocobalt porphyrin complexes. Macromolecules 30: 8109–8112. 22. Author Contributions Conceived and designed the experiments: JG BX RM. Performed the experiments: lF ZT BX RM CY. Analyzed the data: lF ZT CY. Wrote the paper: FB. PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13629 7 Living Radical Polymerization References Burgess J, Fawcett J, Ressel DR, Gilani SR (2000) Monomeric bis(2,4- pentanedionato)cobalt(II). Acta Crystallogr Sect C 56: 649–650. p 10. Bryaskova R, Detrembleur C, Debuigne A, Je´roˆme R (2006) Cobalt-mediated radical polymerization (cmrp) of vinyl acetate initiated by redox systems: toward the scale-up of CMRP. Macromolecules 39: 8263–8268. 23. Wang LC, Jang HY, Roh Y, Iynch V, Schultz AJ, et al. (2002) Diastereoselective cycloreductions and cycloadditions catalyzed by co(dpm)2-silane (dpm = 2,2,6,6-tetramethylheptane-3,5-dionate): mechanism and partitioning of hydro- metallative versus anion radical pathways. J Am Chem Soc 124: 9448–9453. p 11. Lu Z, Fryd M, Wayland BB (2004) New life for living radical polymerization mediated by cobalt(II) metalloradicals. Macromolecules 37: 2686–2687. metallative versus anion radical pathways. J Am Chem Soc 124: ediated by cobalt(II) metalloradicals. Macromolecules 37: 2686–26 24. Maria S, Kaneyoshi H, Matyjaszewski K, Poli R (2007) Effect of electron donors on the radical polymerization of vinyl acetate mediated by [Co(acac)2]: degenerative transfer versus reversible homolytic cleavage of an organocobal- t(III) complex. Chem Eur J 13: 2480–2492. 12. Peng CH, Scricco J, Li S, Fryd M, Wayland BB (2008) Organo-cobalt mediated living radical polymerization of vinyl acetate. Macromolecules 41: 2368–2373. 13. Kaneyoshi H, Matyjaszewski K (2005) Effect of ligand and n-butyl acrylate on cobalt-mediated radical polymerization of vinyl acetate. Macromolecules 38: 8163–8169. 25. Hu T, Tang LM, Li XF, Li YS, Hu NH (2005) Synthesis and ethylene polymerization activity of a novel, highly active single-component binuclear neutral nickel(II) catalyst. Organometallics 24: 2628–2632. 14. Wang B, Zhuang Y, Luo X, Xu S, Zhou X (2003) Controlled/‘‘living’’ radical polymerization of MMA catalyzed by cobaltocene. Macromolecules 36: 9684–9686. 26. Zhang D, Jin GX (2003) Novel highly active bimuclear 2, 5-disubstituted amino- p-benzoquinone nickel (II) ethylene polymerization catalysts. Organometallics 22: 2851–2854. 15. Matsubara K, Matsumoto M (2006) Cobalt(I)-mediated living radical polymer- ization of methyl methacrylate. J Polym Sci Part A: Polym Chem 44: 4222–4228. 27. Huang YB, Tang GR, Jin GY, Jin GX (2008) Binuclear nickel and copper complexes with bridging 2,5-diamino-1,4-benzoquinonediimines: synthesis, structures, and catalytic olefin polymerization. Organometallics 27: 259–269. 16. Weiser MS, Mu¨lhaupt R (2005) Cobalt(II) octanoate and cobalt(II) perfluor- ooctanoate catalyzed atom transfer radical polymerization of styrene in toluene PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13629 8
https://openalex.org/W3024639852	https://europepmc.org/articles/pmc7281454?pdf=render	English	null	Modulatory Effect of Nicotinic Acid on the Metabolism of Caco-2 Cells Exposed to IL-1β and LPS	Metabolites	2,020	cc-by	7,498	Received: 15 April 2020; Accepted: 14 May 2020; Published: 16 May 2020 Received: 15 April 2020; Accepted: 14 May 2020; Published: 16 May 2020 Abstract: Inﬂammatory bowel diseases (IBD) are the most common gastrointestinal inﬂammatory pathologies. Previous work evidenced a lower content of nicotinic acid (NA) in feces of IBD patients compared to healthy subjects. In the present study, we aimed to understand the eﬀects of NA on intestinal inﬂammation, as several studies reported its possible beneﬁcial eﬀect, and investigate its inﬂuence on inﬂammation-driven metabolism. NA was tested on a Caco-2 in-vitro model in which inﬂammation was induced with interleukin-1β (IL-1β) and lipopolysaccharide (LPS), two mayor proinﬂammatory compounds produced in IBD, that stimulate the production of cytokines, such as interleukin 8. A metabolomics approach, with gas chromatography–mass spectrometry (GC-MS) and nuclear proton magnetic resonance (1H-NMR), was applied to study the metabolic changes. The results showed that NA signiﬁcantly reduced the level of IL-8 produced in both LPS and IL-1β stimulated cells, conﬁrming the anti-inﬂammatory eﬀect of NA also on intestinal inﬂammation. Moreover, it was demonstrated that NA treatment had a restoring eﬀect on several metabolites whose levels were modiﬁed by treatments with IL-1β or LPS. This study points out a possible use of NA as anti-inﬂammatory compound and might be considered as a promising starting point in understanding the beneﬁcial eﬀect of NA in IBD. Keywords: inﬂammation; metabolomics; IBD; nicotinic acid metabolites H OH OH metabolites H OH OH metabolites Modulatory Eﬀect of Nicotinic Acid on the Metabolism of Caco-2 Cells Exposed to IL-1β and Department of Biomedical Sciences, University of Cagliari, Metropolitan City of Cagliari, 09042 Monserrato, Italy; cristina.piras@unica.it (C.P.); federica.murgia@unica.it (F.M.); martina.spada@unica.it (M.S.); lauratronci90@gmail.com (L.T.); vera.leoni@tiscali.it (V.P.L.); gabrieleserreli@hotmail.it (G.S.); mdeiana@unica.it (M.D.); latzori@unica.it (L.A.) * Correspondence: marialaurasantoru@gmail.com; Tel.: +39-3498615876 www.mdpi.com/journal/metabolites 1. Introduction Inﬂammatory bowel disease (IBD) is a chronic inﬂammatory disease responsible for profound changes in the gastrointestinal tract. These changes are due, at least in part, to the recruitment of inﬂammatory cells, particularly myeloid cells such as neutrophils and monocytes, and include exhaustion of nutrients, increased oxygen consumption, and generation of large quantities of reactive nitrogen and oxygen intermediate [1]. The dysregulation and the inappropriate response of the immune system against the microﬂora of the gut are typical features of the IBD, too. Another important feature of gut inﬂammation is the disruption of the homeostasis between microbes and host, the so-called dysbiosis [2]. Upon activation of the immune system, cytokines and chemokines, are produced [3], triggering a cascade of downstream reactions [4]. Most of the changes happening during these processes are well known, but some still need to be fully elucidated, especially those responsible for the metabolic alteration. Diﬀerent cytokines produced during intestinal inﬂammation seem to have a direct eﬀect on metabolism. One of the most important cytokines secreted is interleukin-1β (IL-1β). This cytokine is responsible for the activation of lipolysis, inhibition of gluconeogenesis, and increase of vascular permeability to ﬂuids and solutes [3]. Considering the preponderance of Metabolites 2020, 10, 204; doi:10.3390/metabo10050204 www.mdpi.com/journal/metabolites 2 of 11 ring the Metabolites 2020, 10, 204 gluconeogenesis, a Gram negative bacteria in the gastrointestinal tract, lipopolysaccharide (LPS) plays a crucial role in the activation of immune response and consequently in the progression of the disease [5]. Moreover, LPS can stimulate the release of interleukin 8 (IL-8) and other inﬂammatory cytokines in diﬀerent cell types, leading to an acute inﬂammatory response [6]. The inﬂuence on metabolism carried on by the inﬂammatory process is particularly important when talking about the gastrointestinal tract [7]. Indeed, the intestinal epithelium is composed of a very dynamic barrier that is regulated in a complex way to both accommodate the transport of nutrients and ﬂuids, and then select and exclude any antigens from the luminal interface [8]. In our previous work, we applied a multi-omics approach, both metabolomic and metagenomic, to study feces samples coming from IBD patients [9]. This study evidenced a low content of NA in feces of patients aﬀected by IBD, suggesting an anti-inﬂammatory role of NA. 1. Introduction Indeed, as NA seems to exert anti-inﬂammatory eﬀects in diﬀerent tissues [10,11], in the present study we aimed to investigate its eﬀect on intestinal inﬂammation and metabolism using an inﬂammation in-vitro model based on Caco-2 cells stimulated with IL1-β and LPS [12]. a crucial role in the activation of immune response and consequently in the progression of the disease [5]. Moreover, LPS can stimulate the release of interleukin 8 (IL-8) and other inflammatory cytokines in different cell types, leading to an acute inflammatory response [6]. The influence on metabolism carried on by the inflammatory process is particularly important when talking about the gastrointestinal tract [7]. Indeed, the intestinal epithelium is composed of a very dynamic barrier that is regulated in a complex way to both accommodate the transport of nutrients and fluids, and then select and exclude any antigens from the luminal interface [8]. In our previous work, we applied a multi-omics approach, both metabolomic and metagenomic, to study feces samples coming from IBD patients [9]. This study evidenced a low content of NA in feces of patients affected by IBD, suggesting an anti-inflammatory role of NA. Indeed, as NA seems to exert anti-inflammatory effects in different tissues [10,11], in the present study we aimed to investigate its effect on intestinal inflammation and metabolism using an inflammation in-vitro model based on Caco-2 cells stimulated with IL1-β and LPS [12]. 2.1. Evaluation of Cell Viability 2.1. Evaluation of Cell Viability To investigate the eﬀect of LPS, IL-1β, and NA on diﬀerentiated Caco-2 cell monolayers, experiments were initially carried out to assess cell viability after treatment and to choose a non-toxic concentration for each compound. As reported in Figure 1, cell viability remained unchanged in the presence of the tested compounds. LPS was tested in the concentration range 10–100 µg/mL, IL-1β in a concentration of 10 and 25 ng/mL and NA in the concentration range 100–300 µg/mL. The cell viability was tested after 48 h of treatment for each compound. To investigate the effect of LPS, IL-1β, and NA on differentiated Caco-2 cell monolayers, experiments were initially carried out to assess cell viability after treatment and to choose a non-toxic concentration for each compound. As reported in Figure 1, cell viability remained unchanged in the presence of the tested compounds. LPS was tested in the concentration range 10–100 μg/mL, IL-1β in a concentration of 10 and 25 ng/mL and NA in the concentration range 100–300 μg/mL. The cell viability was tested after 48 h of treatment for each compound. Figure 1. Effects of LPS (a), IL-1β (b), and NA (c) on cell viability. Cell viability was evaluated after 48 h of incubation of each compound. Data, expressed as % of control, are presented as means ± standard deviation. Statistical analysis was performed using an unpaired Student’s t-test. Figure 1. Eﬀects of LPS (a), IL-1β (b), and NA (c) on cell viability. Cell viability was evaluated after 48 h of incubation of each compound. Data, expressed as % of control, are presented as means ± standard deviation. Statistical analysis was performed using an unpaired Student’s t-test. Figure 1. Effects of LPS (a), IL-1β (b), and NA (c) on cell viability. Cell viability was evaluated after 48 h of incubation of each compound. Data, expressed as % of control, are presented as means ± standard deviation. Statistical analysis was performed using an unpaired Student’s t-test. Figure 1. Eﬀects of LPS (a), IL-1β (b), and NA (c) on cell viability. Cell viability was evaluated after 48 h of incubation of each compound. Data, expressed as % of control, are presented as means ± standard deviation. Statistical analysis was performed using an unpaired Student’s t-test. 2.2. Evaluation of IL-8 Production 2.2. Evaluation of IL-8 Production 2.3. Metabolomics Analysis 2.3. Metabolomics Analysis To investigate the metabolic changes induced by NA, Caco-2 cells were exposed to LPS (50 μg/mL), IL-1β (25 ng/mL), and NA (200 μg/mL) alone or in combination for 48 h. Cells and cell culture media metabolites extracts were analyzed with both GC-MS and 1H-NMR, two of the most common techniques used in metabolomics. A total of 47 compounds were identified and quantified with the NMR approach in cells and 21 compounds in media, while 44 and 34 compounds were identified respectively in cells and media with the GC-MS (supporting Figures 1–8 and supporting Table S1). Metabolomics results show that IL-1β had a remarkable effect on energetic metabolism, compared to the LPS effect on the same metabolic pathways (Figure 3, Table 1): intracellular levels of acetic acid, glutamic acid, citric acid, creatine, creatine phosphate, and malic acid were significantly increased after the 48 h treatment with IL-1β, while 2-hydroxybutyric acid, 3-hydroxybutyric acid, alanine, fructose, fumaric acid, glutamine, threonine, isoleucine, lactic acid, ornithine, serine, and glycerophosphocholine were decreased. On the other hand, the LPS treatment induced an increase in malic acid levels and a decrease in 2-hydroxybutyric acid, fumaric acid, serine, glycerophosphocholine, and pyruvic acid. Extracellular metabolites were measured in the cell culture To investigate the metabolic changes induced by NA, Caco-2 cells were exposed to LPS (50 µg/mL), IL-1β (25 ng/mL), and NA (200 µg/mL) alone or in combination for 48 h. Cells and cell culture media metabolites extracts were analyzed with both GC-MS and 1H-NMR, two of the most common techniques used in metabolomics. A total of 47 compounds were identiﬁed and quantiﬁed with the NMR approach in cells and 21 compounds in media, while 44 and 34 compounds were identiﬁed respectively in cells and media with the GC-MS (supporting Figures S1–S8 and supporting Table S1). Metabolomics results show that IL-1β had a remarkable eﬀect on energetic metabolism, compared to the LPS eﬀect on the same metabolic pathways (Figure 3, Table 1): intracellular levels of acetic acid, glutamic acid, citric acid, creatine, creatine phosphate, and malic acid were signiﬁcantly increased after the 48 h treatment with IL-1β, while 2-hydroxybutyric acid, 3-hydroxybutyric acid, alanine, fructose, fumaric acid, glutamine, threonine, isoleucine, lactic acid, ornithine, serine, and glycerophosphocholine were decreased. On the other hand, the LPS treatment induced an increase in malic acid levels and a decrease in 2-hydroxybutyric acid, fumaric acid, serine, glycerophosphocholine, and pyruvic acid. 2.2. Evaluation of IL-8 Production 2.2. Evaluation of IL-8 Production f To estimate the effect of LPS, IL-1β, and NA on inflammation, an ELISA assay was carried out to measure the levels of IL-8 released after treatments with each compound. In the first experiment, cells were treated with LPS (50 μg/mL) and LPS in combination with NA (100 μg/mL) and with IL- 1β (25 ng/mL) and IL-1β plus NA (100 μg/mL; Figure 2a). As shown in the figure, the levels of IL-8 were significantly increased after treatment with both LPS and IL-1β, while NA at the concentration of 100 μg/mL did not reduce the production of IL-8 when compared with LPS and IL-1β alone. Therefore, a second experiment was performed with a higher concentration of NA (200 μg/mL). As shown in Figure 2b, LPS and IL-1β increased IL-8 production, and NA reduced the IL-8 levels. To estimate the eﬀect of LPS, IL-1β, and NA on inﬂammation, an ELISA assay was carried out to measure the levels of IL-8 released after treatments with each compound. In the ﬁrst experiment, cells were treated with LPS (50 µg/mL) and LPS in combination with NA (100 µg/mL) and with IL-1β (25 ng/mL) and IL-1β plus NA (100 µg/mL; Figure 2a). As shown in the ﬁgure, the levels of IL-8 were signiﬁcantly increased after treatment with both LPS and IL-1β, while NA at the concentration of 100 µg/mL did not reduce the production of IL-8 when compared with LPS and IL-1β alone. Therefore, a second experiment was performed with a higher concentration of NA (200 µg/mL). As shown in Figure 2b, LPS and IL-1β increased IL-8 production, and NA reduced the IL-8 levels. 3 of 11 Metabolites 2020, 10, 204 Figure 2. IL-8 measurement. Detection of IL-8 levels in Caco-2 cell culture medium after treatment with LPS (50 μg/mL) and LPS (50 μg/mL) +NA (100 μg/mL) and after treatment with IL-1β (25 ng/mL) and IL-1β (25 ng/mL)+NA (100 μg/mL) (a) and detection of IL-8 levels in Caco-2 cell culture medium after treatment with LPS (50 μg/mL) and LPS (50 μg/mL) +NA (200 μg/mL) and with IL-1β (25 ng/mL) and IL-1β (25 ng/mL) +NA (200 μg/mL) (b). Statistical analysis was performed by a Student’s t-test. Results were considered significant when p < 0.05 and p < 0.01. NS = Non-significant. Figure 2. IL-8 measurement. 2.2. Evaluation of IL-8 Production 2.2. Evaluation of IL-8 Production Detection of IL-8 levels in Caco-2 cell culture medium after treatment with LPS (50 µg/mL) and LPS (50 µg/mL) +NA (100 µg/mL) and after treatment with IL-1β (25 ng/mL) and IL-1β (25 ng/mL)+NA (100 µg/mL) (a) and detection of IL-8 levels in Caco-2 cell culture medium after treatment with LPS (50 µg/mL) and LPS (50 µg/mL) +NA (200 µg/mL) and with IL-1β (25 ng/mL) and IL-1β (25 ng/mL) +NA (200 µg/mL) (b). Statistical analysis was performed by a Student’s t-test. Results were considered signiﬁcant when p < 0.05 and ** p < 0.01. NS = Non-signiﬁcant. Figure 2. IL-8 measurement. Detection of IL-8 levels in Caco-2 cell culture medium after treatment Figure 2. IL-8 measurement. Detection of IL-8 levels in Caco-2 cell culture medium after treatment Figure 2. IL-8 measurement. Detection of IL-8 levels in Caco-2 cell culture medium after treatment with LPS (50 μg/mL) and LPS (50 μg/mL) +NA (100 μg/mL) and after treatment with IL-1β (25 ng/mL) and IL-1β (25 ng/mL)+NA (100 μg/mL) (a) and detection of IL-8 levels in Caco-2 cell culture medium after treatment with LPS (50 μg/mL) and LPS (50 μg/mL) +NA (200 μg/mL) and with IL-1β (25 ng/mL) and IL-1β (25 ng/mL) +NA (200 μg/mL) (b). Statistical analysis was performed by a Student’s t-test. Results were considered significant when p < 0.05 and p < 0.01. NS = Non-significant. Figure 2. IL-8 measurement. Detection of IL-8 levels in Caco-2 cell culture medium after treatment with LPS (50 µg/mL) and LPS (50 µg/mL) +NA (100 µg/mL) and after treatment with IL-1β (25 ng/mL) and IL-1β (25 ng/mL)+NA (100 µg/mL) (a) and detection of IL-8 levels in Caco-2 cell culture medium after treatment with LPS (50 µg/mL) and LPS (50 µg/mL) +NA (200 µg/mL) and with IL-1β (25 ng/mL) and IL-1β (25 ng/mL) +NA (200 µg/mL) (b). Statistical analysis was performed by a Student’s t-test. Results were considered signiﬁcant when p < 0.05 and ** p < 0.01. NS = Non-signiﬁcant. 2.3. Metabolomics Analysis 2.3. Metabolomics Analysis Extracellular metabolites were measured in the cell culture medium. glycerophosphocholine, and pyruvic acid. Extracellular metabolites were measured in the cell culture medium. Results show that alanine, glutamine, lactic, and uric acid were increased in the cell culture medium after IL-1β treatment, showing an opposite trend compared to the intracellular levels. Conversely, glycine, ornithine, and glucose levels were decreased. As IL-1β, LPS increased serine, glutamine, and cholesterol levels in the cell culture medium while glucose levels were decreased. (Figure 4, Table 1). g g The eﬀect of NA treatment alone on Caco-2 model was also investigated. NA treatment alone caused the increase of ATP, glycerolphosphate, lactic acid, and glycerophosphocholine and a decrease of fumaric acid, glycine, pyruvic acid, and serine at the intracellular level (Figure 3, Table 1), while at the extracellular level, there were increased levels of serine and tyrosine and lower levels of alanine (Figure 4, Table 1). Regarding the eﬀects of NA on rescuing metabolism after inﬂammation, levels of glutamine, isoleucine, ornithine, and glycerophosphocholine were increased and glutamic acid levels were reduced after IL-1β+NA treatment compared to the IL-1β treatment alone (Figure 3, Table 1). 4 of 11 12 Metabolites 2020, 10, 204 Figure 3. Significantly different intracellular metabolites measured in Caco-2 cells by 1H-NMR and GC-MS. Metabolites are indicated as peak areas normalized for total protein content and total area (n = 3). Statistical analysis was performed using an unpaired Student’s t-test. p < 0.5, p < 0.01. Figure 3. Signiﬁcantly diﬀerent intracellular metabolites measured in Caco-2 cells by 1H-NMR and GC-MS. Metabolites are indicated as peak areas normalized for total protein content and total area (n = 3). Statistical analysis was performed using an unpaired Student’s t-test. p < 0.5, ** p < 0.01. Figure 3. Significantly different intracellular metabolites measured in Caco-2 cells by 1H-NMR and GC-MS. Metabolites are indicated as peak areas normalized for total protein content and total area (n = 3). Statistical analysis was performed using an unpaired Student’s t-test. p < 0.5, p < 0.01. Figure 3. Signiﬁcantly diﬀerent intracellular metabolites measured in Caco-2 cells by 1H-NMR and GC-MS. Metabolites are indicated as peak areas normalized for total protein content and total area (n = 3). Statistical analysis was performed using an unpaired Student’s t-test. p < 0.5, ** p < 0.01. 2.3. Metabolomics Analysis 2.3. Metabolomics Analysis Extracellular Metabolites Proinﬂammatory Stimuli Proinﬂammatory Stimuli + NA Alanine ↑ Alanine ↓ Cholesterol ↑ - Glycine ↓ Glycine ↓ Glucose ↓ - Glutamine ↑ Glutamine ↓ Lactic acid ↑ - Ornithine ↓ Ornithine ↑ Serine ↑ Serine ↓ - Tyrosine ↑ Uric acid ↑ - tabolites 2020, 10, x FOR PEER REVIEW 6 of Figure 4. Significantly different extracellular metabolites measured in the medium of Caco-2 cell by 1H-NMR and GC-MS. Metabolites are presented as peak areas normalized for total protein content and total area (n = 3). Statistical analysis was performed using an unpaired Student’s t-test. * p < 0.05, ** p < 0.01. Figure 4. Signiﬁcantly diﬀerent extracellular metabolites measured in the medium of Caco-2 cell by 1H-NMR and GC-MS. Metabolites are presented as peak areas normalized for total protein content and total area (n = 3). Statistical analysis was performed using an unpaired Student’s t-test. * p < 0.05, ** p < 0.01. Extracellular Metabolites Figure 4. Significantly different extracellular metabolites measured in the medium of Caco-2 cell by 1H-NMR and GC-MS. Metabolites are presented as peak areas normalized for total protein content and total area (n = 3). Statistical analysis was performed using an unpaired Student’s t-test. * p < 0.05, ** p < 0 01 Figure 4. Signiﬁcantly diﬀerent extracellular metabolites measured in the medium of Caco-2 cell by 1H-NMR and GC-MS. Metabolites are presented as peak areas normalized for total protein content and total area (n = 3). Statistical analysis was performed using an unpaired Student’s t-test. * p < 0.05, p < 0.01. Moreover, at the intracellular level, the IL-1β+NA treatment was able to reduce levels of alanine, 3-hydroxybutyric acid and to increase ATP and proline levels when compared to the IL-1β treatment alone. The treatment with LPS+NA caused an increase in the levels of glycerophosphocholine, ATP, glycerolphosphate, glycine, and proline and a decrease of 3-hydroxybutyric acid and alanine levels, compared to LPS treatment alone at the intracellular level (Figure 3, Table 1). At the extracellular level, results showed a decrease of alanine and glutamine, contrarily to ornithine and tyrosine, which were increased in IL-1β+NA treated samples compared to IL-1β treatment alone. At the extracellular level, LPS+NA treatment also induced a decrease of serine, alanine, and glycine and an increased level of ornithine when compared to LPS alone (Figure 4, Table 1). 2.3. Metabolomics Analysis 2.3. Metabolomics Analysis Moreover, at the intracellular level, the IL-1β+NA treatment was able to reduce levels of alanine, 3-hydroxybutyric acid and to increase ATP and proline levels when compared to the IL-1β treatment alone. The treatment with LPS+NA caused an increase in the levels of glycerophosphocholine, ATP, l l h h t l i d li d d f 3 h d b t i id d l i l l Table 1. Intracellular and extracellular metabolites signiﬁcantly altered by treatment with proinﬂammatory stimuli (LPS and IL-1β) and by treatment with proinﬂammatory stimuli in combination with NA in Caco-2 cells. lphosphate, glycine, and proline and a decrease of 3-hydroxybutyric acid and alanine ed to LPS treatment alone at the intracellular level (Figure 3, Table 1). At the extra esults showed a decrease of alanine and glutamine, contrarily to ornithine and tyrosine creased in IL-1β+NA treated samples compared to IL-1β treatment alone. At the extra PS+NA treatment also induced a decrease of serine, alanine, and glycine and an inc f ornithine when compared to LPS alone (Figure 4, Table 1). Overall, metabolites ally perturbed in stimulated Caco-2 cells were mostly associated with energetic pa s glycolysis, tricarboxylic acid (TCA) cycle, urea cycle, ketone bodies metabolism, an lism) at the intracellular level (Figure 5a). After IL-1β and LPS stimuli plus NA th ys appeared to be modified, but in the opposite direction (Figure 5b). Intracellular Metabolites Proinﬂammatory Stimuli Proinﬂammatory Stimuli + NA 2-Hydroxybutyrate ↓ - 3-Hydroxybutyrate ↓ 3-Hydroxybutyrate ↓ Acetate ↑ - Alanine ↓ Alanine ↓ - ATP ↑ Citrate ↑ - Creatine ↑ - Creatine Phosphate ↑ - Fructose ↓ - Fumarate ↓ - Glutamic acid ↑ Glutamic acid ↓ Glutamine ↓ Glutamine ↑ - Glycerolphosphate ↑ Glycerophosphocholine ↓ Glycerophosphocholine ↑ - Glycine ↑ Isoleucine ↓ Isoleucine ↑ Lactic acid ↓ - Malate ↑ - Ornithine ↓ Ornithine ↑ - Proline ↑ Pyruvate ↓ - Serine ↓ - Threonine ↓ - lphosphate, glycine, and proline and a decrease of 3-hydroxybutyric acid and alanin ed to LPS treatment alone at the intracellular level (Figure 3, Table 1). At the extra sults showed a decrease of alanine and glutamine, contrarily to ornithine and tyrosin Intracellular Metabolites Proinﬂammatory Stimuli Proinﬂammatory Stimuli + NA 5 of 11 Metabolites 2020, 10, 204 Table 1. Cont. 2.3. Metabolomics Analysis 2.3. Metabolomics Analysis Overall, metabolites found statistically perturbed in stimulated Caco-2 cells were mostly associated with energetic pathways (such as glycolysis, tricarboxylic acid (TCA) cycle, urea cycle, ketone bodies metabolism, and lipid metabolism) at the intracellular level (Figure 5a). After IL-1β and LPS stimuli plus NA the same pathways appeared to be modiﬁed, but in the opposite direction (Figure 5b). 6 of 11 05, Metabolites 2020, 10, 204 and total area (n = < 0 01 Figure 5. Relevant metabolic pathways that were found significantly altered after treatment with proinflammatory stimuli (LPS and IL-1β) (a) and after treatment with proinflammatory stimuli in combination with NA (b) in Caco-2 cells at the intracellular level. Increased and decreased metabolites are highlighted in red and green, respectively. Figure 5. Relevant metabolic pathways that were found signiﬁcantly altered after treatment with proinﬂammatory stimuli (LPS and IL-1β) (a) and after treatment with proinﬂammatory stimuli in combination with NA (b) in Caco-2 cells at the intracellular level. Increased and decreased metabolites are highlighted in red and green, respectively. Figure 5 Relevant metabolic pathways that were found significantly altered after treatment with Figure 5 Relevant metabolic pathways that were found signiﬁcantly altered after treatment with Figure 5. Relevant metabolic pathways that were found significantly altered after treatment with proinflammatory stimuli (LPS and IL-1β) (a) and after treatment with proinflammatory stimuli in combination with NA (b) in Caco-2 cells at the intracellular level. Increased and decreased metabolites are highlighted in red and green, respectively. Figure 5. Relevant metabolic pathways that were found signiﬁcantly altered after treatment with proinﬂammatory stimuli (LPS and IL-1β) (a) and after treatment with proinﬂammatory stimuli in combination with NA (b) in Caco-2 cells at the intracellular level. Increased and decreased metabolites are highlighted in red and green, respectively. 3. Discussion The metabolic proﬁle is altered in conditions of active inﬂammation, such as those associated with IBD and has become an area of signiﬁcant interest for research [8]. Our previous work pointed out several metabolites altered by the inﬂammation process in patients with IBD [9]. Among all altered metabolites, NA levels were found to be lower in the feces of these patients. NA, also known as niacin or vitamin B3, is a water-soluble vitamin whose derivatives, such as NADH, NAD+, NADPH, and NADP+, play essential roles in energy metabolism in the living cell [13]. The designation vitamin B3 also includes the amide form, nicotinamide, or niacinamide. Severe lack of niacin causes deﬁciency disease pellagra, whereas a mild deﬁciency slows down the metabolism decreasing cold tolerance [14]. Among all the eﬀects of NA, diﬀerent studies proved its role in ameliorating the inﬂammatory process [15,16]. Moreover, it has been demonstrated that nicotinic acid metabolites, such as nicotinamide, have an antioxidant and anti-inﬂammatory eﬀect, on human primary monocytes and monocyte-derived macrophages [17] and it has been evidenced that these metabolites could interfere with early events associated to the diﬀerentiation of monocytic cell line THP-1 and that they aﬀect LPS-induced biological responses of the cell line [18]. In the present study, an in vitro model was used to study NA eﬀects on intestinal inﬂammation. Diﬀerentiated Caco-2 cells were used and inﬂammation was induced with the two major players in intestinal inﬂammation, LPS and IL-1β. LPS is a toxic component of the outer membrane of Gram negative bacteria and it is a potent initiator of inﬂammation [5,6,19]. Indeed, LPS induces systemic inﬂammatory injury and various pathological changes [20]. As known, LPS can stimulate Caco-2 cells proliferation [21], but in our study we used a wide and common model of diﬀerentiated Caco-2 cells, in which cells are seeded and kept in culture for 15 days before each treatment. During these days, the cells diﬀerentiate, reach the conﬂuency and 7 of 11 Metabolites 2020, 10, 204 form a monolayer, so that their possible further growth is arrested. Among cytokines, IL-1β seems to play a particularly important role in intestinal inﬂammation as several clinical studies have reported high levels of IL-1β secretion by colon lamina propria monocytes from patients with active IBD [22,23]. In the present study, we used LPS and IL-1β as inﬂammation stimuli, assessing eﬀective, but not lethal, concentration by performing an MTT assay. 3. Discussion A high dose of LPS (50 µg/mL) was used to induce IL-8 production and to avoid statistically signiﬁcant cell death. Moreover, a concentration of 50 µg/mL or higher of LPS was used to induce an inﬂammatory response in Caco-2 cells in several studies [24,25]. Stimulation of inﬂammation was evaluated by measuring IL-8 production after treatment with LPS and IL-1β at 50 µg/mL and 25 ng/mL respectively. Indeed, one of the earliest reported chemokines, produced by intestinal epithelial cells, is the IL-8 [26]. Similar to our ﬁndings, IL-8 mRNA levels were signiﬁcantly higher in inﬂamed mucosa of IBD patients than in not inﬂamed mucosa [27]. Furthermore, our ﬁndings reported that the production of IL-8 is higher after IL-1β treatment compared to LPS. Our results revealed an altered metabolism as an eﬀect of inﬂammation. Both LPS and IL-1β had a similar eﬀect on a pattern of metabolites resulting in the alteration of energetic pathways, such as TCA cycle, glycolysis, urea cycle, and lipid metabolism. It has already been reported that ongoing inﬂammatory and immune responses are associated with dramatic shifts in tissue metabolism [1]. Particularly, a signiﬁcant metabolic change during inﬂammation and within the immune response involves the generation of lipid mediators and glycolysis. Indeed, impaired glycolysis is a hallmark of inﬂammatory cells [28] as shown also by our results. This conﬁrms the state of inﬂammation induced in these cells after been treated with both IL-1β and LPS, suggesting that this model may be reliable to study inﬂammatory mechanisms. The results indicate that NA can reduce inﬂammation in the enterocytes and this eﬀect is dose-dependent. Our data are in agreement with previous studies showing anti-inﬂammatory eﬀects of NA in in vitro and in vivo models [16,29]. In the present study, we also evaluated the eﬀect of NA on normal and inﬂammation related metabolism. NA seems to inﬂuence Caco-2 cell metabolism. Indeed, comparing NA non-treated and NA treated cells there was an increase of ATP, lactic acid, glycerol-phosphate, and glycerophosphocholine and a decrease of fumaric acid and pyruvic acid at the intracellular level and an increase of serine and tyrosine and a decrease of alanine at the extracellular side. The increase of ATP levels may indicate a higher energetic metabolism. These data may help to understand and clarify the eﬀects of NA on lipids and glucose metabolism described in the literature [30]. 3. Discussion As mentioned before, the eﬀects of NA on the inﬂammation metabolism were evaluated as well. Interestingly, from the results obtained in the present study, NA treatment seemed to have a restoring eﬀect on diﬀerent metabolites. For example, at the intracellular side, the level of glutamic acid, which was upregulated by IL-1β treatment, was then decreased comparing it with IL-1β plus NA treated cells. On the other hand, levels of glutamine, isoleucine, ornithine, and glycerophosphocholine, which were downregulated by IL-1β, were increased in the comparison between IL-1β and IL-1β plus NA treated cells. A similar trend was conﬁrmed at the extracellular level, where the levels of alanine and glutamine that were increased by IL-1β were then decreased by NA treatment, and the ornithine level that was downregulated by IL-1β was increased by NA. Looking at the eﬀect of NA on LPS induced inﬂammation, it is possible to observe again the restoring eﬀect of NA, as it was able to increase the level of glycerophosphocholine, which was decreased by LPS treatment at h i ll l id d d h l l f i hi h i d b LPS l 4.1. Cell Culture Caco-2 cells (ECACC Salisbury, Wiltshire UK) were cultured in Dulbecco’s modiﬁed Eagle’s medium low glucose with L-glutamine and sodium pyruvate (Euroclone, Milan, Italy) supplemented with 10% heat-inactivated bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were cultured in monolayers, at 37 ◦C, in a humidiﬁed atmosphere of 5% CO2, replacing the medium twice a week. 8 of 11 Metabolites 2020, 10, 204 4.2. MTT Viability Test The MTT assay was assessed on Caco-2 cells to evaluate the viability of the cells in the presence of the tested compounds. Cells were seeded in 96-well plates (5 × 104 cells/mL in 100 µL) and exposed to diﬀerent concentrations of the compounds alone: 10, 25, 50 and 100 µg/mL for LPS from Escherichia coli O111:B4 (Sigma-Aldrich, Milan, Italy), 10 and 25 ng/mL for IL-1β (Merck, Rome, Italy) and 100, 150, 200 and 300 µg/mL for NA (Sigma-Aldrich, Milan, Italy). NA was dissolved in the cell medium. After 48 h incubation, the medium was removed and 100 µL of MTT solution (2.5 mg/mL in fresh medium) were added and left for 2 h at 37 ◦C. Formazan crystals were then solubilized by adding 100 µL of dimethyl sulfoxide (DMSO). The absorbance was read at 570 nm using a microplate reader (Inﬁnite 200, Tecan, Salzburg, Austria). 4.3. Treatments For experimental studies, Caco-2 cells were plated in Petri dishes and used 15–17 days post-seeding, when fully diﬀerentiated [31]. To induce the inﬂammation process, cells were stimulated with two diﬀerent compounds: LPS and IL-1β. The eﬀects of NA were then studied by pretreating cells for 24 h with NA, and then stimulated with both NA plus LPS and NA. At the end of the experiment, cells and media were collected for further analysis. 4.4. Evaluation of IL-8 Protein Levels An aliquot of 150 µL of cell culture medium was collected from the Petri dishes and used for ELISA detection. Levels of IL-8 were quantiﬁed using the Human IL-8 ELISA kit (CliniScinces, Nanterre, France) and following the manufacturer’s instructions. Sample absorbance values were read at 450 nm in a microplate reader (Inﬁnite 200, Tecan, Salzburg, Austria). 4.9. Univariate Statistical Analysis GraphPad Prism software (version 7.01, GraphPad Software, Inc., CA, USA) was used to perform the univariate statistical analysis on the data. Speciﬁcally, a Student’s t-test was performed to test statistical signiﬁcance. All experimental data are the mean of three experiments. 4.7. Gas Chromatography–Mass Spectrometry Analysis Derivatized samples were injected splitless into a 7890A gas chromatograph coupled with a 5975C network mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a 30 m × 0.25 mm ID, fused silica capillary column, with a 0.25 µM TG-5MS stationary phase (Thermo Fisher Scientiﬁc, Waltham, MA, USA). The injector and transfer line temperatures were at 250 ◦C and 280 ◦C, respectively. The gas ﬂow rate through the column was 1 mL/min. The column initial temperature was kept at 60 ◦C for 3 min, then increased to 140 ◦C at 7 ◦C/min, held at 140 ◦C for 4 min, increased to 300 ◦C at 5 ◦C/min, and kept for 1 min. Identiﬁcation of metabolites was performed using the standard NIST 08 and GMD mass spectra libraries and, when available, by comparison with authentic standards. Peak detection and deconvolution, ﬁltering, and normalization were performed using a pipeline on Knime [32]. The total protein content together with the total area of chromatograms were used to normalize the metabolites measurements of each cell and medium sample. 4.5. Cell Culture Samples Preparation for Metabolomics Analysis 4.5. Cell Culture Samples Preparation for Metabolomics Analysis To perform metabolomics analysis, the growth medium was removed from the Petri dishes and aliquoted (500 µL) in eppendorf tubes to be treated similarly to the cells. Cells were washed with 3 mL of physiological solution, and intracellular metabolites were extracted with 1.2 mL of cold methanol/water (80:20) and shacked for 15 min at low temperature (4 ◦C). Then cells were harvested by scraping and transferred in eppendorf tubes. To ensure the complete lysis of the cells, the extraction was combined with 10 min of ultrasonic treatment at a controlled temperature (4 ◦C). The growth medium was extracted as described above. Brieﬂy, 500 µL of medium were aliquoted in eppendorf tubes and centrifuged at 5500× g, 10 min, 4 ◦C, to remove cell debris and dead cells. Then 1.2 mL of cold methanol/water with the internal standard (80:20) was added to the supernatant of the medium and the extraction procedure followed as described for the cells. Cell suspensions and growth medium were centrifuged at 5500× g for 30 min at 4 ◦C. For GC-MS analysis, 400 µL of supernatant was aliquoted and dried in an EppendorfTM Concentrator Plus overnight. Dried pellets were derivatized with 50 µL of a solution of methoxamine in pyridine (10 mg/mL; Sigma-Aldrich, St. Louis, MO, USA). After 1 h at 70 ◦C, 50 µL of MSTFA (Sigma-Aldrich, St. Louis, MO, USA) were added and left at room temperature for one hour. Successively, 50 µL of hexane were added and samples were transferred in a vial for the GC-MS analysis. A pool of all samples was created and used as quality control (QC.) For 1H-NMR analysis, 700 µL of supernatant was aliquoted and dried in an EppendorfTM Concentrator Plus overnight. Dried hydrophilic cells and medium extracts were redissolved in 690 µL of potassium phosphate buﬀer in D2O (100 mM, pH 7.4) and 10 µL of TSP (sodium 3-trimethylsilyl-propionate-2,2,3,3,-d4) as a chemical shift reference (δ 0.0; 98 atom % D, Sigma-Aldrich, St. Louis, MO, USA). An aliquot of 650 µL was analyzed by 1H-NMR. 9 of 11 Metabolites 2020, 10, 204 4.8. 1H-NMR Measurements 1H-NMR analysis was performed using a Varian UNITY INOVA 500 spectrometer operating at 499.839 MHz for proton and equipped with a 5 mm double resonance probe (Agilent Technologies, CA, USA). 1H-NMR spectra were acquired at 300 K with a spectral width of 6000 Hz, a 90◦pulse. The acquisition time was of 1.5 s, the relaxation delay was of 2 s, and for each sample, 512 FID were collected into 64 K data points. The residual water signal was suppressed by applying a presaturation technique with low power radiofrequency irradiation for 2 s. After Fourier transformation with 0.3 Hz line broadening and a zero-ﬁlling to 64 K, 1H-NMR spectra were manually phased and baseline corrected using ACD Lab Processor Academic Edition (Advanced Chemistry Development, 12.01, 2010). Spectral chemical shift referencing on the TSP CH3 signal at 0.00 ppm was performed on all spectra. Metabolites were identiﬁed and quantify of each NMR spectra of samples using the Chenomx NMR Suite 7.1 (Chenomx Inc., Edmonton, Alberta, Canada) [33]. The total protein content together with the total area of spectra was used to normalize the metabolites measurements of each cell and medium sample. 4.6. Protein Extraction and Quantiﬁcation Total proteins were extracted with the Bradford assay, with some modiﬁcations. Cell pellets were added with 500 µL Cell Lytic M lysis buﬀer (Sigma-Aldrich, Milan, Italy) for protein extraction, supplemented with mammalian protease and phosphatase inhibitor cocktail (1:100 v/v). Cells were scraped on ice, incubated for 15 min on ice, centrifuged at 12,500× g at 4 ◦C for 7 min and then stored at −20 ◦C prior to quantiﬁcation. The calibration curve was generated using bovine serum albumin (BSA) in Cell Lytic M reagent, at diﬀerent standard concentrations (0.1–5 mg/mL). 500 µL of Bradford Reagent solution (Sigma-Aldrich, St. Louis, MO, USA) were added and incubated with 10 µL of standard and protein sample at room temperature for 5 min. The absorbance was measured at 595 nm using a microplate reader (Inﬁnite 200, Tecan, Salzburg, Austria) at a controlled temperature of 37 ◦C. 5. Conclusions In conclusion, this in vitro study demonstrated that NA could exert a counteracting eﬀect in intestinal inﬂammation, by reducing IL-8 production after inﬂammatory stimuli. Furthermore, data presented here also showed how NA plays a role in metabolic rewiring, by restoring metabolites levels, altered in the inﬂammation process. Taken together, our data pinpointed a potential strategy 10 of 11 Metabolites 2020, 10, 204 to counteract intestinal inﬂammation, although, future studies are needed to clarify the mechanism behind the NA eﬀects in IBD. Surely, to test the real potentiality of NA as an anti-inﬂammatory compound in IBD it should be tested as well in in-vivo and human studies. Supplementary Materials: The following are available online at http://www.mdpi.com/2218-1989/10/5/204/s Author Contributions: Conceptualization, M.L.S. and L.A.; Methodology, M.L.S., M.D. and L.A.; Formal Analysis, M.L.S.; Investigation, M.L.S., C.P., F.M., M.S., L.T., and G.S.; Resources, L.A.; Data Curation, M.L.S.; Writing—Original Draft Preparation, M.L.S., F.M., L.T., C.P.; Writing—Review and Editing, V.P.L., M.D. and L.A.; Visualization, M.L.S.; Supervision, L.A.; Project Administration, L.A. All authors have read and agreed to the published version of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Kominsky, U.J.; Campbell, E.; Colgan, S.P. Metabolic shifts in immunity and inﬂammation. J. Immunol. 2010, 184, 4062–4068. [CrossRef] 2. Khan, I.; Ullah, N.; Zha, L.; Bai, Y.; Khan, A.; Zhao, T.; Che, T.; Zhang, C. Alteration of Gut Microbiota in Inﬂammatory Bowel Disease (IBD): Cause or Consequence? IBD Treatment Targeting the Gut Microbiome. Pathogens 2019, 8, 126. [CrossRef] [PubMed] 3. Medzhitov, R. Origin and physiological roles of inﬂammation. Nature 2008, 454, 428–435. [CrossRef] [PubMed] 4. Neurath, M.F. Cytokines in inﬂammatory bowel disease. Nat. Rev. Immunol. 2014, 14, 329–342. [CrossRef] [PubMed] 5. Pasternak, B.A.; D’mello, S.; Jurickova, I.I.; Han, X.; Willson, T.; Flick, L.; Petiniot, L.; Uozumi, N.; Divanovic, S.; Traurnicht, A.; et al. 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https://openalex.org/W4292103281	https://gjeta.com/sites/default/files/GJETA-2022-0133.pdf	English	null	Application of Newton’s laws using CD hovercraft	Global Journal of Engineering and Technology Advances	2,022	cc-by	2,947	Adesunloro Gbenga Michael * Department of Science Technology, Physics Unit, Federal Polytechnic, Ado-Ekiti, Nigeria. Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 Publication history: Received on 05 July 2022; revised on 10 August 2022; accepted on 12 August 2022 Department of Science Technology, Physics Unit, Federal Polytechnic, Ado-Ekiti, Nigeria. Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 Publication history: Received on 05 July 2022; revised on 10 August 2022; accepted on 12 August 20 Article DOI: https://doi.org/10.30574/gjeta.2022.12.2.0133 Article DOI: https://doi.org/10.30574/gjeta.2022.12.2.0133 Abstract Hovercrafts were constructed using a Compact Disk and Baloons to calculate linear and average displacement, speed and acceleration. The innovative concepts was used to promote learning of Astrophysics to 15 aspiring physics enthusiast to explain the Newton’s laws of Physics as it applies to the working principle of an hovercraft and training of astronauts Keywords: Hovercraft; Astrophysics; Speed; Acceleration p g g ; epartment of Science Technology, Physics Unit, Federal Polytechnic, Ado-Ekiti, Nigeria. * Corresponding author: Adesunloro Gbenga Michael; Email: Copyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons At Copyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. hor(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0 1. Introduction A Hovercraft is a vehicle that flies like a plane but can float like a boat, and can drive like a car but will traverse ditches and gullies as it is a flat terrain. A Hovercraft is also sometimes called an air cushion vehicle because it can hover over or move across land or water surfaces while being held off from the surfaces by a cushion of air [1]. A Hovercraft can travel over all types of surfaces including grass, mud, muskeg, sand, quicksand, water and ice. Hovercraft prefer gentle terrain although they are capable of climbing slopes up to 20%, depending upon surface characteristics. Modern Hovercrafts are used for many applications where people and equipment need to travel at speed over water but be able to load and unload on land. For example, they are used as passenger or freight carriers, as recreational machines and even use as warships. Hovercrafts are very exciting to fly and the feeling of effortlessly travelling from land to water and back again is unique Over the centuries there have been many efforts to reduce the element of friction between moving parts. A hovercraft is a relatively new means of transportation. The concept of the hovercraft was born when engineers came up with an experimental design to reduce drag on ships [2]. The revolutionary idea was to use a cushion of air between boats and the water that they ploughed through to reduce friction. This idea eventually led to what is known today as the hovercraft, basically a vehicle that uses 1 or more fans to float on a cushion of air. These fans serve a dual purpose, to push air below the craft and force it off ground, and to create a forward thrust by pushing air out the back of the craft [3]. This research article therefore, presents an innovative way to use a compact disk and balloon to construct a hovercraft that explains the Newton’s laws of motion in a way that motivates learning of the concepts. Group of students design hovercrafts and perform simple tasks to calculate displacement, speed, acceleration and their results were correlated using charts. Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 2.1. Materials Compact disks (CDs), Plastic Bottle, Round balloon (five-inch size), Meter rules, Nail, Super glue/hot glue gun, Air Pump, Scissors, Stopwatch Students were divided to three groups to produce one CD hovercraft each. The three hovercrafts were used together to perform tasks which were recorded. This report therefore, present a group finding of the experiments carried out 2.2. Design Considerations and Precautions Glue station with one or two glue guns was set up for the students. We use low-temperature glue guns. The heat from high temperature guns may warp the CD. There was a dish of cold water near the glue station for a good safety step. Peradventure if we get hot glue on their fingers, we will immerse the fingers in cold water will immediately “freeze” the glue and minimize any discomfort. It was preferred, by the supervisor to operate the glue gun. Eye protection was recommended and made available when we were working with glue guns. Pop-up spouts for water bottles was substituted for the PVC pipe and rubber stopper. We removed the cap from the bottle and attach it to the upper side of the CD with hot glue. We then fit the balloon over the pop-up spout. We inflated the balloon by blowing through the underside of the hovercraft and push the spout down to hold the air until ready. Caution was made that if there was any student with a latex allergy, so we wash the balloons before using them. Allergic students wore non-latex plastic gloves and inflate the balloons with a balloon pump Thus, the hovercraft was assembled by each group Thus, the hovercraft was assembled by each group Thus, the hovercraft was assembled by each group 2.3.2. Running the Hovercraft 2.3.1. Assembling the Hovercraft_ The Process 2.3.1. Assembling the Hovercraft_ The Process  We squeeze a bead of hot glue on the edge of one end of the PVC pipe and immediately press the glued end to the center of the CD (label side up). The pipe surround the hole in the center of the CD.  We squeeze a bead of hot glue on the edge of one end of the PVC pipe and immediately press the glued end to the center of the CD (label side up). The pipe surround the hole in the center of the CD.  When the glue has cooled and hardened (in about 1-2 minutes), we checked for any gaps betwee pipe. When a gap was noticed, we squeeze some glue in to fill the gaps. We stretch the latex balloon a couple of times to relax it for inflating.  We stretch the latex balloon a couple of times to relax it for inflating.  We Stretch the balloon nozzle over the wide end of the rubber stopper.  We used a hole punch and punch a hole through the center of the gummed paper dot. We app We used a hole punch and punch a hole through the center of the gummed paper dot. We applied th th d id f th CD t th h l the underside of the CD to cover the hole. the underside of the CD to cover the hole. 2.3.2. Running the Hovercraft 2.3.2. Running the Hovercraft  We inflated the balloon by blowing through the hole of the stopper or by inserting the nozzle of a balloon hand pump into the stopper hole.  We twist the balloon so that the nozzle is closed off and we pressed the small end of the stopper into the upper end of the PVC pipe in the hovercraft. The hovercraft was ready to launch.  We placed the craft on a smooth, level surface such as a tabletop. We then released the balloon. It untwist and start blowing air downward through the small hole in the center. The thin cushion of air lifted the CD and eliminate friction with the tabletop.  We tried pushing the hovercraft across a tabletop with the balloon inflated and again with it uninflated. We compared the craft’s movement in the two runs.  We experiment with the optimum size of the hole in the paper dot. The hole can be enlarged by pushing the point of a pencil into it. The hole size determined how fast the air runs out.  We recorded the data on the Hovercraft Record and Result Page 43 43 Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 Figure 1 Construction and demonstration of CD Hovercraft Figure 1 Construction and demonstration of CD Hovercraft Figure 1 Construction and demonstration of CD Hovercraft 3. Results and discussion 3. 3. Results and discussion Results and discussion The table below show how far hovercraft travels before it stop on its own Table 1 The distribution of distance covered by different balloons Balloon First Run Distance (cm) Second Run Distance (cm) Third Run Distance (cm) Average Distance (cm) Balloon 1 298 274 245 272.33 Balloon 2 286 226 99 203.67 Balloon 3 300 257 225 260.67 The table below shows the time the hovercraft covers the distance Table 2 The distribution of time for hovercraft to cover distance Balloon First Run in seconds Second Run in seconds Third Run in seconds Average Time in seconds Balloon 1 90 79 49 72.67 Balloon 2 111 54 46 70.33 Balloon 3 75 69 59 67.67 The table below shows the measurement of how fast the hovercraft covers The table below show how far hovercraft travels before it stop on its own Table 1 The distribution of distance covered by different balloons Balloon First Run Distance (cm) Second Run Distance (cm) Third Run Distance (cm) Average Distance (cm) Balloon 1 298 274 245 272.33 Balloon 2 286 226 99 203.67 Balloon 3 300 257 225 260.67 The table below show how far hovercraft travels before it stop on its own The table below shows the time the hovercraft covers the distance The table below shows the time the hovercraft covers the distance Table 2 The distribution of time for hovercraft to cover distance Balloon First Run in seconds Second Run in seconds Third Run in seconds Average Time in seconds Balloon 1 90 79 49 72.67 Balloon 2 111 54 46 70.33 Balloon 3 75 69 59 67.67 The table below shows the measurement of how fast the hovercraft covers The table below shows the measurement of how fast the hovercraft covers The table below shows the measurement of how fast the hovercraft covers The table below shows the measurement of how fast the hovercraft covers 44 44 Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 The table below shows the speed of the hovercraft The table below shows the speed of the hovercraft Table 3 The distribution shows the speed of the hovercraft covered by different balloons Balloon First Run speed in cm/sec Second Run speed in cm/sec Third Run speed in cm/sec Average Speed in cm/sec Balloon 1 3.31 3.47 5.00 3.75 Balloon 2 2.57 4.18 2.15 2.97 Balloon 3 4.00 3.72 3.81 3.84 able 3 The distribution shows the speed of the hovercraft covered by different balloons Figure 1 The average distribution of Distance covered by different balloons Figure 1 The average distribution of Distance covered by different balloons Figure 2 The distribution of Time for hovercraft to cover distance Figure 2 The distribution of Time for hovercraft to cover distance 45 45 Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 Figure 1 and 2 show a variation in the distance covered and time taken for each of the hovercraft contracted with different balloons. Balloons 1 which is the smallest of the three balloons covered the highest distance, while the biggest balloon (balloon 3) covered the least distance. Figure 3 The distribution of speed of the hovercraft covered by different balloons Figure 3 The distribution of speed of the hovercraft covered by different balloons From figure 3 it can be declared from the average speed tabulated that the average speed of each of the hovercraft (3.75cm/s, 2.97cm/s and 3.84cm/s respectively) was close to each other; almost the same which implies that the size of the balloon do not play a significant role in the speed of the hovercraft. he students use the result to answer the following questions in group. The response is tabulated blo The students use the result to answer the following questions in group. The response is tabulated blow: able 4 Discussion Feedback on Nexus of Hovercraft on Newton’s laws of motion Discussion Question Response from Group What causes the hovercraft to become frictionless? Air from the balloon escapes beneath the hovercraft. It forms a thin cushion that lifts the craft a few millimetres above the table. Without direct contact with the table top, friction is greatly reduced What happens to the hovercraft’s movement when the balloon runs out of air? Why? When the balloon runs out of air, the lifting cushion stops. The full surface of the CD bottom contacts the table top, friction is greatly increased, and the hovercraft stops How do different surfaces affect the hovercraft? Smooth surfaces permit a uniform cushion of air to lift the craft. Rough surfaces allow air to escape more in some directions than others and the craft is no longer level. Parts of the CD touch the surface and cause drag. How does the size of the paper dot hole affect the hovercraft? The hole controls the flow of air from the balloon. If the paper is removed, the hole is very large and the air escapes quickly. A tiny hole greatly slows the flow of air and may not provide enough lifting force. How does Newton’s Laws of Motion control the movement of the hovercraft? 4. Conclusion Hovercraft is a vehicle which can float in any lands also it is known by Air Cushion Vehicle due to its ability to move by cushion or skirt filled with air. The concept of the hovercraft is simplified using Compact Disks and balloons to explain the Newton’s laws to motivate young learners to Astrophysics Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 Acknowledgments The author acknowledge the project students whose honest feedbacks form the bedrock of the research and would like to thank the journal reviewers for their valuable and in time valid comments, which was useful in enhancing the technical quality and readability of this paper. 1. An unbalanced force is needed to lift the craft. Another force is needed to propel the craft along the table. able 4 Discussion Feedback on Nexus of Hovercraft on Newton’s laws of motion 46 Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 2. The lifting force is determined by how much air is released (its mass) and how fast it accelerates out of the hole. 3. The action force of the air released from the balloon creates a reaction force lifting the hovercraft. Pushing on the hovercraft to cause it to move along the tabletop is also an example of action and reaction. How can hovercraft technology be used to simulate microgravity when training astronauts? During Astronauts training, three air bearing pads, similar in size and identical in function to the CD hovercraft, produce great lifting force and nearly eliminate friction. The bearing pads are able to provide much greater lifting force than the CD hovercraft because high pressure air from compressors is used. When the two astronauts push on each other, they fly apart in a great demonstration of Newton’s Laws of Motion. . Conclusion Hovercraft is a vehicle which can float in any lands also it is known by Air Cushion Vehicle due to its ability to move by ushion or skirt filled with air. The concept of the hovercraft is simplified using Compact Disks and balloons to explain he Newton’s laws to motivate young learners to Astrophysics ompliance with ethical standards cknowledgments Global Journal of Engineering and Technology Advances, 2022, 12(02), 042–047 2. The lifting force is determined by how much air is released (its mass) and how fast it accelerates out of the hole. 3. The action force of the air released from the balloon creates a reaction force lifting the hovercraft. Pushing on the hovercraft to cause it to move along the tabletop is also an example of action and reaction. How can hovercraft technology be used to simulate microgravity when training astronauts? During Astronauts training, three air bearing pads, similar in size and identical in function to the CD hovercraft, produce great lifting force and nearly eliminate friction. The bearing pads are able to provide much greater lifting force than the CD hovercraft because high pressure air from compressors is used. When the two astronauts push on each other, they fly apart in a great demonstration of Newton’s Laws of Motion. References Author’s Biography Adesunloro Gbenga M. is the secretary of research meetings of the School of Science and Computer Studies; committee member, Centre for Research, Innovation and Development (CRID), member, E-Learning Committee of the School of Science and Computer Studies of the Federal Polytechnic, Ado-Ekiti and member of research seminar series of the department of Science Technology in the institution. He had supervised over 50 research students in groups and individually on various research areas. He is a professional member of the Nigeria Institute of Physics and the Society of Amateur Radio Astronomers. He is a mentor to many youth groups and organisations. Adesunloro Gbenga M. is the secretary of research meetings of the School of Science and Computer Studies; committee member, Centre for Research, Innovation and Development (CRID), member, E-Learning Committee of the School of Science and Computer Studies of the Federal Polytechnic, Ado-Ekiti and member of research seminar series of the department of Science Technology in the institution. He had supervised over 50 research students in groups and individually on various research areas. He is a professional member of the Nigeria Institute of Physics and the Society of Amateur Radio Astronomers. He is a mentor to many youth groups and organisations. 47
https://openalex.org/W2922180080	https://link.springer.com/content/pdf/10.1007/s00223-019-00539-8.pdf	English	null	Paget’s Disease of Long Bones: Microstructural Analyses of Historical Bone Samples	Calcified tissue international	2,019	cc-by	8,428	Calcified Tissue International (2019) 105:15–25 https://doi.org/10.1007/s00223-019-00539-8 Calcified Tissue International (2019) 105:15–25 https://doi.org/10.1007/s00223-019-00539-8 ORIGINAL RESEARCH Paget’s Disease of Long Bones: Microstructural Analyses of Historical Bone Samples Elena Nebot1,2,3 · Patrick Heimel4,5 · Stefan Tangl4 · Martin Dockner6 · Janina Patsch7 · Gerhard W. Weber6 · Michael Pretterklieber8 · Maria Teschler‑Nicola9 · Peter Pietschmann1 Elena Nebot1,2,3 · Patrick Heimel4,5 · Stefan Tangl4 · Martin Dockner6 · Janina Patsch7 · Gerhard W. Weber6 · Michael Pretterklieber8 · Maria Teschler‑Nicola9 · Peter Pietschmann1 Received: 14 November 2018 / Accepted: 3 February 2019 / Published online: 8 March 2019 © The Author(s) 2019 Received: 14 November 2018 / Accepted: 3 February 2019 / Published online: 8 March 2019 © The Author(s) 2019 * Peter Pietschmann peter.pietschmann@meduniwien.ac.at Elena Nebot enebot@ugr.es Abstract Although Paget’s disease of bone (PDB) is the second most common metabolic bone disease, there is only limited informa- tion about the microarchitecture of affected bones. Therefore, the aim of this study was to determine cortical and trabecular bone properties in clinically relevant locations by microcomputed tomography (µCT). Ten femora and ten tibiae affected by Paget’s disease taken from the Natural History Museum Vienna were compared to 13 femora and 10 tibiae of non-affected body donors. Digitization of the cortical and trabecular bone microarchitecture was performed with an X-ray-based µCT scanner. Additionally, semi-quantitative gradings of trabecular and cortical architectural parameters of the femora and the tibiae were generated. Microcomputed tomography images showed changes in the thickness of cortices, cortical porosity, and trabecularization of cortical structures. Moreover, severe disorganization of trabecular structures, trabecular defects, and thickening of (remaining) trabeculae were detected. Numerical cortical analyses showed lower total bone volume (BV) and lower BV in the outer region (66–100%) (− 36%, p = 0.004, and − 50%, p < 0.001, respectively), lower total volume (TV) in the outer region (66–100%) (− 42%, p < 0.001), lower total bone volume fraction (BV/TV) and BV/TV in the outer region (66–100%) (− 23%, and − 12%, p < 0.001, respectively), higher BV and TV in the middle region (33–66%) and higher BV/ TV in the inner region (0–33%) (123%, p = 0.011, 147%, p = 0.010, and 33%, p = 0.025, respectively) in Pagetic compared to non-affected bones. Trabecular analyses showed higher BV/TV (96%, p = 0.008) and Tb.Th (43%, p = 0.004) in Pagetic compared to non-affected bones. There is a major and consistent structural alteration of PDB at cortical and trabecular sites in weight-bearing long bones. Our findings are relevant for the differential diagnosis of PDB and for the pathogenesis of associated complications, since the disorder produces abnormalities in the structure that might lead to bone fragility. Acquisition of Pagetic and Control Bones We selected dry preparations of ten femora and ten tibiae affected by Paget’s disease from the Pathological-Anatomi- cal Collection at the Natural History Museum Vienna (most of them date to the first half of the twentieth century AD). It is important to mention that all the selected bones were macroscopically affected by the disease. We selected femora and tibiae with the clinicopathologic diagnosis of PDB. This diagnosis was macroscopically validated according to the description of Ortner and Putschar [17]. If weight-bearing long bones are concerned, the most characteristic diagnos- tic feature is bowing and thickening, which is a representa- tive criterion of the late phase of the disease (early stages implied, e.g., by terminated areas of lytic resorption, are non-diagnostic). This feature is interpreted as being the con- sequence of “complete or incomplete transverse pathological fractures” caused by the mechanical impairment of Paget bone. On the cut surface of a dry bone, a thickening and lamination of the cortical bone, a narrowing of the medul- lary canal, and irregularly formed coarsened cancellous bone become visible [17]. Because of the obvious macroscopic alterations of the Pagetic bones, blinding of the observers was not possible. Since this collection does not contain healthy bones, we collected 13 femora and 10 tibiae from the Division of Anatomy, Medical University of Vienna. No abnormalities could be detected during the macroscopic inspection of bones obtained from these body donors. Despite the fact that PDB is the second most common metabolic bone disease [9], analyses of bone structure in Paget’s disease on a quantitative level are rare, with the exception of histomorphometric analyses of biopsies of the iliac crest [4, 10] and the spine [11]. To date, there are no histomorphometric studies based on long bones, except one case report [12] of a femur fracture associated with PDB in an Asian patient. Over the past years—as an alternative to bone histomor- phometry—non-invasive radiological imaging and quanti- fication techniques for the study of bone microarchitecture have been developed [13–15]. Microcomputed tomography (µCT) produces high-resolution images of trabecular and cortical structures. However, the increasing availability of advanced imaging techniques of bone microstructure could be of hallmark importance for a better understanding of bone deformity and bone fragility in PDB. Up to now, there are no studies using µCT to investigate the micro- architecture of Pagetic long bones. Abstract Nebot et al. control group of femora and tibiae of non-affected body donors provided by the Division of Anatomy, Medical University of Vienna. Introduction Paget’s disease of bone (PDB), also known as Osteitis deformans, was originally described by Sir James Paget in 1877. His report has become a classic in the medical literature [1]. The disease is a chronic bone abnormal- ity, which may affect a single, several, or many bones but never involves the entire skeleton. The axial skeleton is frequently involved, and the bones most commonly affected include the pelvis (70%), femur (55%), lumbar spine (53%), skull (42%), and tibia (32%) [2, 3]. Neverthe- less, Pagetic bone lesions can occur at any site of the skel- eton [4]. The diagnosis of PDB is rare before the age of 50. The disease affects both sexes [5], but males have been reported to show a higher frequency. Moreover, a variation in age and regional (geographical) distribution has been observed: Schmorl [6] found a prevalence of 3% of PDB in a series over 4600 autopsies of individuals above 40 years of age. The prevalence of the disease is highest in West- ern Europe (in particular in Britain), North America, and Australia, whereas the disease is rare in Scandinavia, Asia, and Africa [7]. Most patients are asymptomatic [8], while others develop complications such as bone pain, osteoar- thritis, fracture, deformity, deafness, and nerve compres- sion syndromes [4]. Although most µCT scanners are rather limited with regard to sample size, a few scanners, like the one we used at the Vienna Micro-CT Lab, allow the non-destructive imaging of microstructures of large bones such as femora and tibiae. This offers a unique and novel chance to extend our knowledge and to make advances in identifying the metabolism of Pagetic bone disease—even in a less pro- gressed phase. To the best of our knowledge, this is the first prospective systematic study based on high-resolution µCT scans concomitantly analyzing the effects of the disease on trabecular and cortical structure in long bones. Abstract words Paget’s disease of bone · Microcomputed tomography · Microarchitecture · Pagetic long bon Vol.:(0123456789) 1 3 * Peter Pietschmann peter.pietschmann@meduniwien.ac.at Elena Nebot enebot@ugr.es 1 Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria 2 Department of Physiology, School of Pharmacy, and Institute of Nutrition and Food Technology, University of Granada, Granada, Spain 3 Department of Preventive Medicine and Public Health, School of Pharmacy, University of Granada, Granada, Spain 4 Karl Donath Laboratory for Hard Tissue and Biomaterial Research, Department of Oral Surgery, University Clinic of Dentistry, Medical University of Vienna, Vienna, Austria 5 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology ‑ Austrian Cluster for Tissue Regeneration, AUVA Research Center, Vienna, Austria 6 Department of Anthropology, and Core Facility for Micro‑Computed Tomography, University of Vienna, Vienna, Austria 7 Department of Biomedical Imaging and Image‑Guided Therapy, Medical University of Vienna, Vienna, Austria 8 Division of Anatomy, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria 9 Pathological‑Anatomical Collection in the Fool’s Tower, Department of Anthropology, Natural History Museum Vienna, Vienna, Austria Vol.:(0123456789) 1 3 * Peter Pietschmann peter.pietschmann@meduniwien.ac.at Elena Nebot enebot@ugr.es 1 Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria 2 Department of Physiology, School of Pharmacy, and Institute of Nutrition and Food Technology, University of Granada, Granada, Spain 3 Department of Preventive Medicine and Public Health, School of Pharmacy, University of Granada, Granada, Spain 4 Karl Donath Laboratory for Hard Tissue and Biomaterial Research, Department of Oral Surgery, University Clinic of Dentistry, Medical University of Vienna, Vienna, Austria 5 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology ‑ Austrian Cluster for Tissue Regeneration, AUVA Research Center, Vienna, Austria 6 Department of Anthropology, and Core Facility for Micro‑Computed Tomography, University of Vienna, Vienna, Austria 7 Department of Biomedical Imaging and Image‑Guided Therapy, Medical University of Vienna, Vienna, Austria 8 Division of Anatomy, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria 9 Pathological‑Anatomical Collection in the Fool’s Tower, Department of Anthropology, Natural History Museum Vienna, Vienna, Austria 5 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology ‑ Austrian Cluster for Tissue Regeneration, AUVA Research Center, Vienna, Austria 6 Department of Anthropology, and Core Facility for Micro‑Computed Tomography, University of Vienna, Vienna, Austria 7 Department of Biomedical Imaging and Image‑Guided Therapy, Medical University of Vienna, Vienna, Austria 8 Division of Anatomy, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria 9 Pathological‑Anatomical Collection in the Fool’s Tower, Department of Anthropology, Natural History Museum Vienna, Vienna, Austria 5 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology ‑ Austrian Cluster for Tissue Regeneration, AUVA Research Center, Vienna, Austria 6 Department of Anthropology, and Core Facility for Micro‑Computed Tomography, University of Vienna, Vienna, Austria 1 Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria 7 Department of Biomedical Imaging and Image‑Guided Therapy, Medical University of Vienna, Vienna, Austria 2 Department of Physiology, School of Pharmacy, and Institute of Nutrition and Food Technology, University of Granada, Granada, Spain 3 Department of Preventive Medicine and Public Health, School of Pharmacy, University of Granada, Granada, Spain 4 Karl Donath Laboratory for Hard Tissue and Biomaterial Research, Department of Oral Surgery, University Clinic of Dentistry, Medical University of Vienna, Vienna, Austria (0123 1 3456789) 3 16 E. Cortical and Trabecular Bone Microarchitecture We performed scanning of the cortical and trabecular bone microarchitecture with an X-ray based µCT scanner (Vis- com X-8060-II, Vienna Micro-CT Lab, Department of Anthropology, University of Vienna) using the following scan parameters: transmission tube, digital detector, 110 kV, 380 µA, filter: 0.50 mm copper. The zoom factor was in the range between × 3.0 and × 4.3 to achieve a spatial resolution of 40 µm. Since most of the compact bone is located at the diaphysis of long bones, but the cancellous bone is located in the metaphysis, we have selected the central region of the shafts of femora and tibiae as cortical bone sites, and the proximal metaphyses for analyzing the trabecular micro- architecture of those same bones. Since the incidence of the disease is higher in femora than in the tibiae [22, 23], we have selected the femora for further detailed investigation. Therefore, numerical analyses of bone microarchitecture in Pagetic and control femora were per- formed using (Fiji) [24], ImageJ [25], 1.51 h with the BoneJ [26] 1.3.11 plugin. Since the exact spatial boundaries of cortical bone are very difficult to determine, the bone was measured in differ- ent density brackets to get comparable results. The following procedure was performed using Definiens Developer XD 2.1 (Definiens AG, Munich, Germany). The original µCT scans were available at an isotropic resolution of 40 µm. These images were downscaled to 50% (80 µm isovoxel). The images were then thresholded and pores inside the seg- mented bone were filled by dilating the bone [27] by three voxels followed by erosion of three voxels. An approxi- mately 34 mm large region in the center of the scan was selected for measurement and adjusted for the orientation of the femur. This region was selected to maximize the area in the center of the femur while excluding artifacts present at the edge of the scans. The bone was divided into three subregions based on the local bone volume fraction (BV/ TV) which ranged from 0 to 33%, 33 to 66%, and 66 to 100% (Fig. 1a). The local density was measured for each voxel as the bone volume fraction of the surrounding volume up to a distance of 800 µm. The void volume between inside the bone was assigned to density brackets using surface tension- constrained region growing. Acquisition of Pagetic and Control Bones The goal of the present study was to examine the cor- tical and trabecular bone microstructure in historic and clinically untreated PDB cases stored at the Pathologi- cal-Anatomical Collection housed at the Natural His- tory Museum Vienna (PASiN-NHM) [16]. Some of its specimens exhibit deformities caused by Paget’s disease according to the bequeathed hand-written documentary evidence. For comparative purposes, we also include a The contemporary control bones were prepared in a way that resembled the conventional preparation technique used in the nineteenth century, warm water maceration at 35 °C. By this approach, possible preparation-related effects could be avoided. Preparation of bones involved bone disarticula- tion, soft tissue removal or bone cleaning, maceration, bone bleaching, and labeling [18]. There are a variety of methods 1 3 3 3 Paget’s Disease of Long Bones: Microstructural Analyses of Historical Bone Samples 17 or techniques used in bone preparation [19]. This includes maceration of dissected specimens through submersion in water for a period of time, burying in soil, as well as boiling in hot water [20]. Values refer either to an increase (+) or a decrease (−) of the parameter relative to normal (0). An expert bone researcher (EN) interpreted the qualitative grading of the µCT images and, in case of doubt, a second expert bone researcher (PP) was involved in the process. Cortical and Trabecular Bone Microarchitecture The resulting subregions were copied to the full resolution images (40 µm) and the bone Our measurements, terminology, and units used for the present analyses generally followed the recommendations of the Nomenclature Committee of the American Society of Bone and Mineral Research [21]. Semi‑quantitative Grading Semi-quantitative gradings of cortical bone (i.e., thickness of cortices, porosity, and trabecularization) and trabecular bone (i.e., trabecular defects, thickness of trabeculae, visual perception of the amount of trabeculae, separation of tra- beculae, and sclerosis) were performed. The scale consisted of the following four grades: 0 = within normal limits, 1/− 1 = slight alterations, 2/− 2 = moderate alterations, 3/− 3 = severe alterations. 1 3 Fig. 1 a Different regions within the midshaft of the femur (from inside to outside), considering the 100% value the outside part, and < 33% value the inner part of the midshaft. b Performance of trabecular measurements in the femurs for the head and neck regions 1 3 1 3 3 18 E. Nebot et al. regions of interest were copied to the full resolution images (40 µm). The bone inside the different regions was thres- holded and BV/TV was measured. The regions were then exported as image stacks. The original scans and regions of interest were imported into Fiji (Fiji [24] + ImageJ) [25] and trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), bone surface (BS), and con- nectivity density (Conn.D) were measured using the BoneJ plugin [26]. Due to the large size of the region of interest and excessive amount of processing power required, the images were downsized to 50% (80 µm isovoxel) of their original resolution for these measurements. was re-thresholded inside these subregions to measure the BV/TV. The subregions were then exported as image stacks. The original scans and subregions were imported into Fiji (Fiji [24] + ImageJ) [25] and the thickness of the brackets was measured using the thickness measurement imple- mented in the BoneJ plugin [26]. Due to the large size of the region of interest, the images were again downsized to 50% (80 µm isovoxel) of their original resolution for these thickness measurements. Trabecular measurements in the femora were performed separately for the head and neck regions (Fig. 1b). To avoid subjectivity, the exact size and position of the region was determined automatically according to the following proce- dure which was performed using Definiens Developer XD 2.1 (Definiens AG, Munich, Germany). The µCT scans were taken at an isotropic resolution of 40 µm. These images were initially downscaled to 12.5% (320 µm isovoxel) of their original size. Statistical Analysis Results are presented as mean and standard deviation. A Pearson Chi-square test was used for the semi-quantitative analyses to find association between various parameters with respect to PDB. Furthermore, a homogeneity test was employed to determine equal or unequal variance. Differ- ences between non-affected and Pagetic groups were ana- lyzed using one-way analysis of variance (ANOVA). All analyses were conducted using the Statistical Package for Social Sciences (IBM-SPSS, version 24.0 for Windows; SPSS Inc., Chicago, IL), and the level of statistical signifi- cance was set at 0.05. Semi‑quantitative Grading At this resolution, a threshold was set isoin- tense to bone and through erosion and dilation, and identi- fying volume enclosed by bone, the void inside the femur was filled to identify the position of the femur in the scan. A multistep procedure was used to find the regions of interest. As a first step, the coordinates of the estimated center of the femur head and center of the neck were determined manu- ally. Next, using these coordinates, the radius of the head was approximated as a percentile of the distance of voxels in the femoral head to the estimated center. For this measure- ment, only voxels which were located further from the neck than the approximated center were considered. Then, based on these initial measurements, the center of the head and neck were repositioned. From these new coordinates, the radius of the femoral head was measured and the final coor- dinates determined. Using these coordinates and the meas- ured radius, the final positions of head and neck were deter- mined. The neck and head regions were limited by planes perpendicular to the axis between head and neck center. Based on initial observation of the samples, the following dimensional parameters were selected to define the region of interest. The head was defined along this axis as a region from its most distal part to a plane 1.33 times the radius distant. The neck reached a plane 2.33 times the length of the radius. Segmentation between the cortical and trabecu- lar compartments is challenging because there is rarely a precise border between them [28]. Therefore, to exclude the cortical bone, parts of the head and neck which were < 10% of the diameter away from the outside of the femur were excluded. Additionally, if cortical bone, defined as the outer bone layer with at least a local bone volume fraction of 75%, still reached into the measurement region in the center, it was also excluded. Regions within the trabecular bone with a local bone volume fraction of more than 75% were identified and measured separately because the bone in these regions revealed to lack a regular trabecular structure. The resulting Demographic Characteristics of Bone Samples The demographic characteristics of our samples are pre- sented in Table 1. For femoral samples: mean (SD) age was 78.5 (13.3) and 65.6 (6.5) years in non-affected and Pagetic, respectively. In the non-affected group (n = 13) there were 9 males (69.2%) and 4 females (30.8%), and in the Pagetic group (n = 10) 3 males (30.0%), 6 females (60.0%), and 1 was unknown (10%). For tibial samples, mean (SD) age was 76.9 (14.9) and 69.2 (9.5) years in non-affected and Pagetic, respectively. The non-affected group (n = 10) consisted of 6 males (60%),and 4 females (40%), and the Pagetic group (n = 10) consisted of 3 males (30%), 2 females (20%), and 5 were unknown (50%). µCT Imaging Studies Microcomputed tomography images at the midshaft of the Pagetic femora showed cortical thickening, marked cortical porosity, and trabecularization of cortical struc- tures (Fig. 2a, c). Similar alterations were also present in the affected tibiae (Fig. 2b, d). At the trabecular 1 3 3 Paget’s Disease of Long Bones: Microstructural Analyses of Historical Bone Samples 19 Table 1 Demographic characteristics of bone samples Femora Non-affected (n = 13) Pagetic (n = 10) Age (years) (mean, SD) 78.5 (13.3) 65.6 (6.5) Sex (n, %) 9 male (69.2%) 4 female (30.8%) 3 male (30.0%) 6 female (60.0%) 1 unknown (10.0%) Tibiae Non-affected (n = 10) Pagetic (n = 10) Age (years) (mean, SD) 76.9 (14.9) 69.2 (9.5) Sex (n, %) 6 male (60.0%) 4 female (40.0%) 3 male (30.0%) 2 female (20.0%) 5 unknown (50.0%) Table 1 Demographic characteristics of bone samples Femora Non-affected (n = 13) Pagetic (n = 10) Age (years) (mean, SD) 78.5 (13.3) 65.6 (6.5) Sex (n, %) 9 male (69.2%) 4 female (30.8%) 3 male (30.0%) 6 female (60.0%) 1 unknown (10.0%) Tibiae Non-affected (n = 10) Pagetic (n = 10) Age (years) (mean, SD) 76.9 (14.9) 69.2 (9.5) Sex (n, %) 6 male (60.0%) 4 female (40.0%) 3 male (30.0%) 2 female (20.0%) 5 unknown (50.0%) of cortices (p = 0.011), higher porosity (p < 0.001), and increased trabecularization (p = 0.011) when comparing Pagetic to non-affected bones (Fig. 4). Semi-quantitative grading of cortical architectural parameters of the tibial midshaft showed higher thickness of cortices (p = 0.008), porosity and trabecularization (both, p < 0.001) when com- paring Pagetic to non-affected bones (Fig. 5). compartments, severe structural disorganization and trabecular defects were noted; the remaining trabeculae appeared thickened (Fig. 3). Semi‑quantitative Grading Semi-quantitative grading of cortical architectural param- eters of the femoral midshaft showed higher thickness 1 3 bia com- ages of Pagetic nd non- ic (d) tibia Fig. 2 Representative com- puted tomography images of non-affected (a) and Pagetic (c) femur midshafts, and non- affected (b) and Pagetic (d) tibia midshafts 1 3 1 3 3 20 E. Nebot et al. in the Pagetic compared to non-affected bones (Figs. 3, 4). Semi-quantitative grading of trabecular architectural parameters of the tibial condyle showed higher trabecular defects (p = 0.015), thickness of trabeculae (p = 0.019), and in the Pagetic compared to non-affected bones (Figs. 3, 4). Semi-quantitative grading of trabecular architectural parameters of the tibial condyle showed higher trabecular defects (p = 0.015), thickness of trabeculae (p = 0.019), and Semi-quantitative grading of trabecular architectural parameters of the proximal femur showed higher thick- ness of trabeculae (p < 0.001) and separation of trabeculae (p = 0.037), but lower amount of trabeculae (p = 0.023) Fig. 3 Representative com- puted tomography images of non-affected (a) and Pagetic (b) human femoral heads Fig. 4 a Semi-quantitative grading of femoral cortical midshaft archi- tecture. b Semi-quantitative grading of trabecular architecture in the proximal femur. Pearson Chi-square test. Semi-quantitative grading scale: 0 = within normal limits, 1/− 1 = slight, 2/− 2 = moderate, and 3/− 3 = severe alterations Fig. 3 Representative com- puted tomography images of non-affected (a) and Pagetic (b) human femoral heads Fig. 3 Representative com- puted tomography images of non-affected (a) and Pagetic (b) human femoral heads Fig. 3 Representative com- puted tomography images of non-affected (a) and Pagetic (b) human femoral heads Fig. 4 a Semi-quantitative grading of femoral cortical midshaft archi- tecture. b Semi-quantitative grading of trabecular architecture in the proximal femur. Pearson Chi-square test. Semi-quantitative grading scale: 0 = within normal limits, 1/− 1 = slight, 2/− 2 = moderate, and 3/− 3 = severe alterations idshaft archi- ecture in the ative grading scale: 0 = within normal limits, 1/− 1 = slight, 2/− 2 = moderate, and 3/− 3 = severe alterations Fig. 4 a Semi-quantitative grading of femoral cortical midshaft archi- tecture. b Semi-quantitative grading of trabecular architecture in the proximal femur. Pearson Chi-square test. Semi-quantitative grading scale: 0 = within normal limits, 1/− 1 = slight, 2/− 2 = moderate, and 3/− 3 = severe alterations Fig. Discussion Analysis of bone structure in Paget’s disease on a quantita- tive (histomorphometric) level is surprisingly rare. A histo- morphometric study [29], which was carried out in two his- toric skeletons with PDB, found evidence of an increased Tb.Th in the femoral shaft, the calcaneus, and in the sacrum, respectively. Histomorphometric results from Seitz et al. [4] showed a high bone turnover with a significant increase in bone resorption and bone formation indices (Tb.N, osteoid volume and osteoid surface, osteoblast number, and surface of osteoclasts) and an increased BV in Pagetic iliac crests. Fig. 6 Cortical bone microarchitecture in Pagetic versus non-affected femora. p < 0.05, p < 0.01, *p < 0.001. BV bone volume, TV total volume, BV/TV bone volume fraction. Percentages mean the dif- ferent regions within the midshaft of the bone, considering the 100% value the outside part, and < 33% value the inner part of the midshaft ferent regions within the midshaft of the bone, considering the 100% value the outside part, and < 33% value the inner part of the midshaft Fig. 6 Cortical bone microarchitecture in Pagetic versus non-affected femora. p < 0.05, p < 0.01, *p < 0.001. BV bone volume, TV total volume, BV/TV bone volume fraction. Percentages mean the dif- Table 2 Three-dimensional outcomes for trabecular bone microarchitecture in Pagetic versus non-affected femoral head and neck Mean (standard deviation). BV bone volume, TV total volume, BV/TV bone volume fraction; Tb.N trabecu- lar number, Tb.Th trabecular thickness, Tb.Sp trabecular separation, BS bone surface, Conn.D connectivity density. Semi‑quantitative Grading 5 a Semi-quantitative grading of tibial cortical midshaft archi- tecture. b Semi-quantitative grading of trabecular architecture of the tibial condyle. Pearson Chi-square test. Semi-quantitative grading scale: 0 = within normal limits, 1/− 1 = slight, 2/− 2 = moderate, and 3/− 3 = severe alterations Fig. 5 a Semi-quantitative grading of tibial cortical midshaft archi- tecture. b Semi-quantitative grading of trabecular architecture of the tibial condyle. Pearson Chi-square test. Semi-quantitative grading scale: 0 = within normal limits, 1/− 1 = slight, 2/− 2 = moderate, and 3/− 3 = severe alterations 3 21 Paget’s Disease of Long Bones: Microstructural Analyses of Historical Bone Samples separation of trabeculae (p < 0.001), but a lower amount of trabeculae (p = 0.012) in the Pagetic compared to non- affected bones (Fig. 5). Numerical analysis of trabecular regions of the femo- ral head and neck is presented in Table 2. Analyses of the femoral trabecular parameters showed higher BV/TV (96%, p = 0.008) and Tb.Th (43%, p = 0.004) in the femoral neck in Pagetic compared to non-affected bones. Cortical and Trabecular Bone Microarchitecture Numerical analysis of the cortical bone of the femurs is shown in Fig. 6. Analysis of the femoral cortical parameters showed lower total bone volume (BV) and lower bone vol- ume in the outer region (66–100%) (− 36%, p = 0.004, and − 50%, p < 0.001, respectively), lower total volume (TV) in the outer region (66–100%) (− 42%, p < 0.001), and lower total BV/TV and BV/TV in the outer region (66–100%) (− 23%, and − 12%, p < 0.001, respectively) in Pagetic compared to non-affected bones. However, both BV and TV in the middle region (33–66%) and BV/TV in the inner region (0–33%) were higher (123%, p = 0.011 for BV, 147%, p = 0.010 for TV, and 33%, p = 0.025 for BV/TV, respec- tively) in Pagetic compared to non-affected bones. a Percentage of difference between non-affected and Pagetic groups was computed as ((Pagetic − non- affected)/Pagetic) × 100 Table 2 Three-dimensional outcomes for trabecular bone microarchitecture in Pagetic versus non-affected femoral head and neck Mean (standard deviation). BV bone volume, TV total volume, BV/TV bone volume fraction; Tb.N trabecu- lar number, Tb.Th trabecular thickness, Tb.Sp trabecular separation, BS bone surface, Conn.D connectivity density. Discussion Since genetic factors inducing type 2 diabetes are very complex, even aDNA analyses of the bones would not give us suffi- cient information to decide whether these individuals were affected by both PDB and type 2 diabetes.f Likewise, Meunier et al. [10] reported an increased trabecu- lar BV, excessive resorption surfaces, and elevated osteoid surfaces in iliac bone biopsies of Pagetic patients. In agree- ment with these data, our µCT-based imaging analysis of the Pagetic trabecular architecture of both the femoral head and the tibial condyle shows thickening of the trabeculae and higher separation of trabeculae, but decreased amount of trabeculae (Fig. 3). Furthermore, in the histomorphometric study by Pestka et al. [11], affected vertebral body biopsies revealed a significant increase both in trabecular BV as well as osteoid parameters. In comparison to histomorphometric data obtained from extra-spinal skeletal locations affected by PDB (i.e., iliac crest), a similar bone microarchitecture of the vertebral bodies was observed. They concluded that vertebral body height and the spine BV together with bone density might play an important role in the manifestation of Pagetic bone alterations [11]. In another histopathology study,[30] based on temporal bone of only eight subjects, no quantitative results are presented. In addition, Cherian et al. [31] observed that cortical and trabecular bone density was increased in vertebrae affected by PDB. Nevertheless, cortical quantitative CT values were underestimated in PDB compared with physical measure- ments of density [31]. In contrast to the above-mentioned data on the lumbar spine, we have observed differences in the thickness of cortices as well as an increased porosity, and trabecularization of the compact bone in the midshaft of the Pagetic femurs and tibias (Fig. 2a–d). f The structural differences between cortical and cancellous bone are widely recognized [39]. However, less is known about the complex changes concerning either architecture or thickness of bone in patients suffering from skeleton-affect- ing diseases. Deeper insight into the regular distribution and architecture of cancellous bone within the human skeleton seems to be essential to better understand both, the role of bone cellular activity and also the diagnostic validity of BV measurements [40]. There are striking differences between peripheral and axial measurement sites and even between local areas within these sites. Generally, in healthy subjects, trabecular BV at the femoral neck (15.8%) is higher than at the lumbar spine (8.3%) or the iliac crest (11.5%). Discussion a Percentage of difference between non-affected and Pagetic groups was computed as ((Pagetic − non- affected)/Pagetic) × 100 Trabecular parameters femoral head and neck Non-affected (n = 13) Pagetic (n = 8) %a p Femoral head BV (mm3) 53068.6 (24316.4) 47053.5 (23411.5) − 11.3 0.581 TV (mm3) 171137.3 (86790.9) 145076.1 (47577.4) − 15.2 0.386 BV/TV (1) 0.33 (0.09) 0.35 (0.19) 6.1 0.814 Tb.N (1/mm) 0.0068 (0.0012) 0.0056 (0.0018) − 17.6 0.115 Tb.Th (mm) 0.32 (0.06) 0.37 (0.10) 15.6 0.285 Tb.Sp (mm) 0.73 (0.17) 1.00 (0.46) 37.0 0.069 Femoral neck BV (mm3) 19622.8 (13936.6) 28218.1 (9222.2) 43.8 0.106 TV (mm3) 89489.4 (45080.7) 66608.0 (24698.0) − 25.6 0.150 BV/TV (1) 0.23 (0.12) 0.45 (0.17) 95.7 0.008 Tb.N (1/mm) 0.0055 (0.0011) 0.0067 (0.0030) 21.8 0.322 Tb.Th (mm) 0.30 (0.08) 0.43 (0.08) 43.3 0.004 Tb.Sp (mm) 1.02 (0.21) 1.03 (1.01) 1.0 0.967 Total BS (mm2) 22992.2 (15780.4) 23308.5 (14252.7) 1.4 0.963 Conn.D (1/mm3) 2.6 (1.4) 1.9 (0.7) − 26.9 0.161 Trabecular parameters femoral head and neck Non-affected (n = 13) Pagetic (n = 8) %a p 1 1 3 3 22 E. Nebot et al. neck was increased [29]. In agreement with the studies by Seitz et al. [4] and Meunier et al. [10] performed at the iliac crests, and by Pestka et al. [11] in the spine, we have also noticed increased trabecular BV/TV at the Pagetic femo- ral neck (Table 2). When both trabecular regions (head and neck) were combined, a trend toward diminished Conn.D in Pagetic femurs was seen. Interestingly, osteoporotic patients are characterized by reduced Conn.D at the femoral neck [35]. In line with our data, and taking into account that corti- cal porosity and severe trabecular defects were found to be present in our series of specimens, we may assume that bone strength could be diminished despite the increased Tb.Th and BV/TV leading to an elevated fracture risk [36, 37]. However, the reliability of these opposed phenomena should be further analyzed in biomechanical studies to confirm the consequences. It is also well known that cortical porosity increases with diabetes [38], and it may be the case that these observed changes in our study are not specific of PDB but might also coexist with type 2 diabetes mellitus. Discussion The 1 3 3 Paget’s Disease of Long Bones: Microstructural Analyses of Historical Bone Samples 23 human remains was reported by Pales [47], which included a brief discussion of a femur from the Neolithic in France. There was considerable anterior curvature of the diaphysis with a marked expansion of the cortex. Moreover, five cases of PDB in prehistoric skeletons from the United States were published by Denninger [48]. In all cases, the long bones had a thickened cortex. However, neither X-ray inspection nor histology was used in any of these studies, making the diagnosis likely but not certain [49]. Wells and Woodhouse [50] investigated a skeleton recov- ered from a burial ground in England, which was char- acterized by bowed long bones. The authors’ diagnosis of PDB was based on the deformation of bones and the features observed in the X-rays of the femora and vertebrae in plain radiograms [49]. With regard to paleopathology, our findings—obtained by a non-invasive technique—will substantially aid to the differential diagnosis of PDB of historic skeletal remains. In contemporary clinical prac- tice, biopsies of long bones for suspected Paget’s disease are performed only in rare cases; nevertheless, our µCT findings also in this setting could contribute to establish a diagnosis. mean age and age variability of the non-affected subjects was larger (average age: 79 ± 13 years) than the Pagetic ones. It is important to note that, according to the records of the collection, in some Pagetic subjects, the age was not known. Even considering the fact that cortical porosity and Tb.Sp increase (and cortical thickness decreases) [43] with age, the Pagetic bones are extensively overshooting the age effect. Interestingly, in a longitudinal study, Shanbhogue et al. [44] stated that both in women and men, parameters of trabecular microarchitecture at the tibia did not change with age. Thus, we may assume that the alterations seen in the cancellous compartment are caused by the metabolic bone disorder. Since the age at which the diagnosis of PDB was established in our samples is not known, another limitation is that some of the observed microarchitectural changes may correlate with specific gene alterations which increase or decrease the susceptibility to develop the dis- ease [45]. [ ] High-resolution peripheral QCT (HR-pQCT) allows acquisition of images which enable the characterization of cortical and trabecular microarchitecture in unique detail [44]. Conclusions In conclusion, in weight-bearing long bones, PDB is responsible for major structural alterations both at cortical and trabecular sites. The main findings of this study were (i) femurs and tibias affected by PDB showed marked cor- tical porosity, and trabecularization of cortical structures. (ii) PDB was also associated with severe disorganization of trabecular structures, trabecular defects, and thickening of (remaining) trabeculae. These findings are relevant for the differential diagnosis of PDB and for the pathogenesis of associated complications, since the disorder produces abnormalities in the structure that might lead to bone fra- gility, increasing bone fracture risk, enhanced by low bone mass, and microarchitectural deterioration of bone tissue [36, 53]. According to the study by Smith et al. [46], which divided the spectrum of the pathologic alterations of the PDB into three stages: the lytic phase (incipient-active), in which osteoclasts predominate; the mixed phase (active), in which osteoblasts cause repair superimposed on the resorption; and the blastic phase (late-inactive) in which osteoblasts predominate and areas of sclerosis may develop. Due to a lack of clinical data, it was not possible to determine in which phase of the disease the affected subjects were at the time of death. However, we could assume that Pagetic subjects were in a mixed phase of the disease, since sclerosis was infrequently observed. The cause of death was unknown; nevertheless, we did not observe Paget sarcoma in any of our specimens.f Acknowledgements Open access funding provided by Medical Uni- versity of Vienna. Discussion Of note, there is a systematic variation in trabecular microarchitec- ture of the iliac crest, showing highest bone mass within the anterior part and lower values for the medial and dorsal part [40]. g g Consistent with the imaging findings, numerical analyses in this study have shown that BV/TV was lower in Pagetic femoral cortical midshafts than in the non-affected bones. For additional analyses, the cortex of the midshaft was subdivided (from inside to outside) into three subregions (i.e., inner: 0–33%, middle: 33–66%, and outer: 66–100%) (Fig. 1a) considering the 100% value in the outside part, and < 33% value in the inner part of the midshaft. Interestingly, in Pagetic femora, the BV/TV of the outer—but not of the inner cortex—was significantly decreased (Fig. 6). Moreo- ver, we have also observed decreased BV and TV of the outside part of the cortex. This could suggest a higher risk of fractures since cortical porosity and trabecularization of cor- tical structures have been associated with an increased frac- ture risk [32, 33]. In this context, Van Staa et al. [34] evalu- ated epidemiologic and clinical aspects of PDB in England and Wales using the General Practice Research Database. They described that patients with PDB had an increased risk of fractures (relative risk: 1.2) when compared to matched controls; remarkably, the risk of hip fractures was increased by 44%. The structural alterations observed in long bones with Paget’s disease—in particular, those seen at the corti- ces—could thus be indicative of impaired bone strength. On the other hand, regarding the trabecular compartment of the Pagetic femurs, we have observed that Tb.Th of the femoral Several potential limitations of the present study should be mentioned. There are differences in age-related varia- tions of cortical and trabecular bone for both women and men. Many studies have evaluated the age- and sex-related changes in the cortical and trabecular bone microarchitec- ture [41–44]. There is increasing clinical interest in assess- ing bone microstructure, with the ultimate goal of improv- ing the prediction of fracture risk [41]. It is well known that cortical porosity as well as Tb.Sp increase with age, and it might be the case that these observed changes in our study are not specific of PDB. However, we have noticed quite severe alterations in the specimens of this study obtained from Pagetic subjects aged 66 ± 6 years. Discussion Moreover, this technology can provide novel and con- temporary clinical information about the differential diag- nosis of the disease. Several studies [41, 43, 44] have dem- onstrated sexual dimorphisms in bone microstructure, and particularly different patterns of bone loss associated with increasing age which leads to weaker bones in postmeno- pausal women [44]. There seems to be, however, agreement on a dominance of cortical over trabecular bone loss in both sexes. Given the small sample sizes in our study, sexes were not balanced in the Pagetic and control group which lim- its the conclusions with regard to sexual dimorphisms. The magnitude of structural alterations in Pagetic bone samples was nevertheless found to be similar in both sexes (Figs. 4, 5; Table 1). Finally, the current investigation could be useful and support several studies related to the bone microstructure analyses in experimental models (i.e., transgenic animal models) of Paget’s disease [51, 52]. Acknowledgements Open access funding provided by Medical Uni- versity of Vienna. References 1. Paget J (1877) On a form of chronic inflammation of bones (ostei- tis deformans). Med Chir Trans 60:37–64.9 22. 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https://openalex.org/W4225713361	https://link.springer.com/content/pdf/10.3758/s13428-021-01722-2.pdf	English	null	REPP: A robust cross-platform solution for online sensorimotor synchronization experiments	Behavior research methods	2,022	cc-by	13,084	REPP: A robust cross-platform solution for online sensorimotor synchronization experiments Manuel Anglada‑Tort1 · Peter M. C. Harrison1,2 · Nori Jacoby1 Accepted: 6 October 2021 © The Author(s) 2022 / Published online: 11 February 2022 https://doi.org/10.3758/s13428-021-01722-2 Behavior Research Methods (2022) 54:2271–2285 https://doi.org/10.3758/s13428-021-01722-2 Behavior Research Methods (2022) 54:2271–2285 2 Centre for Music and Science, Faculty of Music, University of Cambridge, Cambridge, UK * Manuel Anglada‑Tort manuel.anglada-tort@ae.mpg.de Peter M. C. Harrison pmch2@cam.ac.uk Nori Jacoby nori.jacoby@ae.mpg.de 1 Computational Auditory Perception Group, Max Planck Institute for Empirical Aesthetics, Frankfurt am Main, Germany 2 Centre for Music and Science, Faculty of Music, University of Cambridge, Cambridge, UK 1 Computational Auditory Perception Group, Max Planck Institute for Empirical Aesthetics, Frankfurt am Main, Germany Abstract Sensorimotor synchronization (SMS), the rhythmic coordination of perception and action, is a fundamental human skill that supports many behaviors, including music and dance (Repp, 2005; Repp & Su, 2013). Traditionally, SMS experiments have been performed in the laboratory using finger tapping paradigms, and have required equipment with high temporal fidelity to capture the asynchronies between the time of the tap and the corresponding cue event. Thus, SMS is particularly challenging to study with online research, where variability in participants’ hardware and software can introduce uncontrolled latency and jitter into recordings. Here we present REPP (Rhythm ExPeriment Platform), a novel technology for measuring SMS in online experiments that can work efficiently using the built-in microphone and speakers of standard laptop computers. In a series of calibration and behavioral experiments, we demonstrate that REPP achieves high temporal accuracy (latency and jitter within 2 ms on average), high test-retest reliability both in the laboratory (r = .87) and online (r = .80), and high concurrent validity (r = .94). We also show that REPP is fully automated and customizable, enabling researchers to monitor experiments in real time and to implement a wide variety of SMS paradigms. We discuss online methods for ensuring high recruiting efficiency and data quality, including pre-screening tests and automatic procedures for quality monitoring. REPP can therefore open new avenues for research on SMS that would be nearly impossible in the laboratory, reducing experimental costs while massively increasing the reach, scalability, and speed of data collection. Keywords Sensorimotor synchronization · Rhythm · Movement · Timing · Online experiments For example, some studies have used external hardware to record responses and control auditory feedback (e.g., a MIDI percussion pad or keyboard connected to com- puter software), such as FTAP (Finney, 2001) and Max-MSP (Patel et al., 2005; Repp et al., 2005). Researchers have also proposed solutions that use the low-level timing hardware of Arduino microcontrollers, including TapArduino (Schultz & van Vugt, 2016) and TeensyTap (van Vugt, 2020). Another popular solution is MatTAP, a MATLAB-based toolbox for dedicated data acquisition hardware (Elliot et al., 2009). Others have developed an iOS application for tapping exper- iments that takes advantage of specific hardware in mobile Apple devices (Tap-It, Kim et al., 2012). In previous work, we have used a simple, low cost, in-lab method that achieves high temporal fidelity by simultaneously recording the audio stimulus and tapping responses using a standard sound card with an audio loopback cable (Elliott et al., 2018; Jacoby & McDermott, 2017; see Experiment 2 for a description of this method). experiments consist of a relatively simple procedure: par- ticipants tap with their index finger to a rhythmic sequence of auditory stimuli. This procedure presents a methodologi- cal challenge: how to measure the asynchrony (or synchro- nization error) between the time of the tap and the corre- sponding cue event with high millisecond-level precision. To meet this challenge, previous studies have used various laboratory-based methods that rely on specialized software and hardware. For example, some studies have used external hardware to record responses and control auditory feedback (e.g., a MIDI percussion pad or keyboard connected to com- puter software), such as FTAP (Finney, 2001) and Max-MSP (Patel et al., 2005; Repp et al., 2005). Researchers have also proposed solutions that use the low-level timing hardware of Arduino microcontrollers, including TapArduino (Schultz & van Vugt, 2016) and TeensyTap (van Vugt, 2020). Another popular solution is MatTAP, a MATLAB-based toolbox for dedicated data acquisition hardware (Elliot et al., 2009). Others have developed an iOS application for tapping exper- iments that takes advantage of specific hardware in mobile Apple devices (Tap-It, Kim et al., 2012). In previous work, we have used a simple, low cost, in-lab method that achieves high temporal fidelity by simultaneously recording the audio stimulus and tapping responses using a standard sound card with an audio loopback cable (Elliott et al., 2018; Jacoby & McDermott, 2017; see Experiment 2 for a description of this method). Keywords Sensorimotor synchronization · Rhythm · Movement · Timing · Online experiments Another important source of noise in online experiments is altered participant behavior compared to laboratory set- tings (e.g., Clifford & Jerit, 2014). This can be challenging in SMS tasks because they usually require participants to pay close attention to the task and take a large number of trials per session. There is also a higher risk of fraudulent responders (Ahler et al., 2019; Crump et al., 2013), includ- ing both computer “bots” and non-serious respondents, such as participants who do not tap at all or tap at a regular rate irrespective of the external auditory cue. When performing online research on SMS, it is therefore important to analyze experimental trials and monitor performance in real time, providing feedback to participants and excluding fraudulent responders. p Here we present REPP (Rhythm ExPeriment Platform), a novel technology for measuring SMS in online experiments that can work efficiently using hardware and software avail- able to most participants online, specifically standard laptops with working speakers and microphones. To address core issues related to latency and jitter, REPP uses a free-field recording approach: the audio stimulus is played through the laptop speakers and the original signal is simultane- ously recorded with participants’ tapping response using the built-in microphone (Fig. 1a). The success of this method relies on a simple observation: although the initial onset in a recording is hard to control due to the interplay between the sound card and operating system, once a sound card starts recording, it registers all subsequent sound events as audio samples encoded with high precision with respect to the beginning of the recording. Thus, by using a single audio recording to simultaneously capture the stimulus and tap- ping onsets, we can remove the most significant sources of delay in both response and presentation latencies. We then apply audio filtering and other signal processing techniques to the resulting audio recording to split the different compo- nents of the recording into separate channels and therefore extract the stimulus and tapping onsets with reliable timing. Finally, we use custom markers with known temporal loca- tions to unambiguously identify the position of the tapping and stimulus onsets in the audio recording, allowing a precise alignment to measure participants' asynchronies. REPP can be executed rapidly in real time and is fully customizable, enabling researchers to adapt the code to support a wide range of SMS paradigms in online and laboratory settings. Keywords Sensorimotor synchronization · Rhythm · Movement · Timing · Online experiments Keywords Sensorimotor synchronization · Rhythm · Movement · Timing · Online experiments Sensorimotor synchronization (SMS) is a fundamental human skill that involves the temporal coordination of rhythmic movement with a predictable external event (Repp, 2005; Repp & Su, 2013). SMS requires individuals to pre- cisely integrate visual or auditory perception with motor production, supporting a wide range of human behaviors. For example, the ability to entrain to an external auditory cue plays a key role in musical experiences across human cultures (Savage et al., 2015; Jacoby et al., 2021) and has been linked to specific genotypes, suggesting an innate human sensitivity to rhythm (Niarchou et al., 2021). SMS has also been associated with the development of literacy skills, such as reading and speech (Carr et al., 2014; Flaug- nacco et al., 2014; Ladányi et al., 2020; Tierney & Kraus, 2013), and various neurodevelopmental disorders, including attention deficit hyperactivity disorder (Noreika et al., 2013) and Parkinson’s disease (Bieńkiewicz & Craig, 2015). Quantitative research on SMS dates back at least to 1886 (Stevens, 1886), but its popularity has increased consider- ably in recent decades (see Repp, 2005; Repp & Su, 2013, for reviews). SMS experiments can differ substantially in their implementation, using different production modes (e.g., finger tapping, clapping, or speaking), different stimulus domains (e.g., visual or auditory), and different experimental designs, including rate limits (London, 2002), perturbation studies (Repp, 2002a), simulated partners (Repp & Keller, 2008), and transmission chains (Jacoby & McDermott, 2017; Ravignani et al., 2016). However, at their core, most SMS * Manuel Anglada‑Tort manuel.anglada-tort@ae.mpg.de Peter M. C. Harrison pmch2@cam.ac.uk Nori Jacoby nori.jacoby@ae.mpg.de 1 Computational Auditory Perception Group, Max Planck Institute for Empirical Aesthetics, Frankfurt am Main, Germany 2 Centre for Music and Science, Faculty of Music, University of Cambridge, Cambridge, UK (0121 3456789) 3 2272 Behavior Research Methods (2022) 54:2271–2285 experiments consist of a relatively simple procedure: par- ticipants tap with their index finger to a rhythmic sequence of auditory stimuli. This procedure presents a methodologi- cal challenge: how to measure the asynchrony (or synchro- nization error) between the time of the tap and the corre- sponding cue event with high millisecond-level precision. To meet this challenge, previous studies have used various laboratory-based methods that rely on specialized software and hardware. Keywords Sensorimotor synchronization · Rhythm · Movement · Timing · Online experiments In this paper, we aim to validate REPP in a series of experiments while demonstrating how to best implement it in online stud- ies to ensure high data quality. Nevertheless, none of these methods are viable for per- forming SMS experiments over the internet, where research- ers have very limited control over participants’ hardware and software. This lack of experimental control combined with the technical demands of SMS tasks makes studying SMS with online research a true challenge. In particular, SMS experiments performed online, such as tapping on the spacebar or mouse in synchronization to an external beat, can introduce all kinds of delay in latency and jitter into the recorded timestamps (Anwyl-Irvine et al., 2020; Bridges et al., 2020). Latency refers both to the time gap between a participant pressing a key and the device registering the key- press, and the time interval between initiation of audio play- back and the physical start of the sound. It is often related to issues concerning scan rates, device drivers, internet connec- tion, operating system variability, and sound card start-up latencies. Jitter is closely related and refers to the variation in latency. It can be either introduced in each tapping onset or across tapping trials (e.g., operating systems usually pro- cess each keyboard stroke with different temporal latencies). These inaccuracies can be in the order of 60 to 100 ms and can vary considerably between platforms, browsers, and devices (Anwyl-Irvine et al., 2020). Thus, measuring par- ticipants' asynchronies in online settings with high precision is currently unfeasible. This paper continues with an overview of REPP. We then present a series of calibration and behavioral experi- ments demonstrating key aspects of this technology: tem- poral accuracy (Experiment 1), test-retest reliability and concurrent validity in the laboratory (Experiment 2), and test-retest reliability in a larger-scale online experiment, as 1 3 3 Behavior Research Methods (2022) 54:2271–2285 2273 1 3 Fig. 1 REPP: A robust cross-platform solution for online SMS exper- iments. a REPP uses a free-field recording approach that can work efficiently using standard hardware and software available to most online participants. b REPP comprises five main steps. c Example of a recording using REPP in a trial of beat synchronization to music. REPP uses a unique frequency range for each audio element in the recording: metronome (blue), tapping (green), markers (yellow), and test channel (red). Overview: REPP REPP can be organized around five main steps: (i) stimulus preparation, (ii) recording phase, (iii) onset extraction, (iv) onset alignment, and (v) performance analysis (see Fig. 1b).i REPP takes two inputs: an audio file with the stimulus (e.g., a metronome or a music clip) and a list of the cor- responding stimulus onsets. Prior to performing an experi- ment, the stimulus must be prepared to be used efficiently in the subsequent steps. First, we filter the stimulus to avoid any overlap with the frequency range that will be occupied by participants' tapping responses (e.g., 50–500 Hz). Sec- ond, we add custom markers with known temporal locations to the beginning and end of the stimulus, shifting the cor- responding stimulus onsets accordingly (see Fig. 1c; yel- low boxes). These markers are critical to unambiguously identify the position of the tapping and stimulus onsets in the audio recording, enabling their precise alignment. Thus, we designed the markers to be robustly detected across par- ticipants’ hardware and software, including cases of noise- cancellation technologies and noisy recordings. In particular, we generate the markers’ sound by combining a short burst of filtered white noise (50%, filtered to 200–340 Hz) and pure tones (50%, also filtered to 200–340 Hz), and play the resulting sound at nearly maximum volume (see Custom Markers in supplementary information for technical details on the markers’ generation procedure).i The last challenge consists of aligning the extracted taps to their position in the audio stimulus. Since the sound card guarantees that all events are recorded with high precision with respect to the beginning of the recording, we can use the first detected marker as a single frame of reference to align the stimulus and tapping response. We use the other markers to assess REPP’s timing performance in each trial and exclude participants with incompatible hardware or soft- ware (see Failing Criteria in supplementary information). For example, we calculate the markers’ timing error by sub- tracting the known markers’ locations against the detected markers’ location. This metric is critical to ensure that the timing accuracy of our technology remains high in all trials. Importantly, by relying on the markers’ accurate timing, we do not need to extract the stimulus onsets from the recorded signal, which can be challenging in online studies due to noise-cancellation technologies and interference from other audio elements (e.g., participants' tapping response and background noise). Keywords Sensorimotor synchronization · Rhythm · Movement · Timing · Online experiments d Output of the performance analysis after the sig- nal processing steps, including the number of detected tapping onsets, detected markers, and mean and SD of asynchrony Fig. 1 REPP: A robust cross-platform solution for online SMS exper- iments. a REPP uses a free-field recording approach that can work efficiently using standard hardware and software available to most online participants. b REPP comprises five main steps. c Example of a recording using REPP in a trial of beat synchronization to music. REPP uses a unique frequency range for each audio element in the recording: metronome (blue), tapping (green), markers (yellow), and test channel (red). d Output of the performance analysis after the sig- nal processing steps, including the number of detected tapping onsets, detected markers, and mean and SD of asynchrony 1 3 Behavior Research Methods (2022) 54:2271–2285 2274 extract the tapping signal from the raw recording by using bandpass filters with cutoff frequencies set to these ranges.ii well as methods for ensuring high data quality while mini- mizing costs (Experiment 3). Finally, we discuss the limita- tions of REPP and implications for future work on SMS. We release REPP as a free and open-source framework along- side the publication of this paper. Code available in https:// gitlab.com/computational.audition/repp Similarly, we use a specific range to filter and identify the marker locations (i.e., the same range used to generate the marker sound, i.e., 200–340 Hz). In addition, to enhance the markers’ extraction procedure, we compare the amplitude of the filtered markers’ channel against a test channel set to be one octave below the markers’ range, i.e., 100–1070 Hz (Fig. 1c; orange boxes). The test channel is used to compute an estimate of the temporal locations that contain the mark- ers. We then use these estimates to boost the markers’ signal exactly in temporal locations where we found more energy in the markers’ frequency range than in the test frequency range. The rationale here is that tapping sounds will have similar energy within the markers and test channels, whereas marker sounds will have all energy in the markers channel and nearly no energy in the test channel. Thus, by using this principle, we can be sure that the detected signal corre- sponds to the markers and not to other sources of noise (e.g., tapping signal or background noise). Keywords Sensorimotor synchronization · Rhythm · Movement · Timing · Online experiments In particular, the result- ing signal has enhanced the markers’ amplitude, which helps combat signal attenuation as a result of noise cancelation and noisy backgrounds, effectively increasing the chance of detection only in those areas containing the markers (see Custom Markers in supplementary information for techni- cal details on the markers’ extraction procedure, including Figure S7 with examples of successful and failed trials). Next, REPP applies a simple onset extraction algorithm to the filtered markers and tapping channels to detect all samples exceeding a relative threshold (Elliott et al., 2018), returning a vector of extracted tapping onsets and extracted marker onsets. Validation experiments Three experiments were conducted to validate REPP and show how it can be implemented in online experiments to produce high-quality data. In Experiment 1, we assess the timing accuracy of REPP using an independent calibration system. In Experiment 2, we assess REPP’s test-retest reli- ability and concurrent validity when measuring individual differences in SMS in the laboratory. Finally, in Experiment 3, we assess the test-retest reliability of REPP with a larger sample of participants recruited online and also provide sug- gestions to reach high data quality while minimizing recruit- ing costs. See supplementary information for additional methods and demographic information. All datasets are available in an OSF repository: https://osf.io/r2pxd/ REPP’s timing performance was tested in the laboratory against an independent calibration system. This allowed us to measure the cue events (either tapping or stimulus onsets) separately in the two systems, providing an upper bound on inaccuracies of both REPP and the independent calibration system. Based on an established method previously used in our work (Jacoby & McDermott, 2017), we used a calibra- tion system that offers a simple solution for measuring the ground-truth recording of REPP (see Fig. 2a). We tested two variants of this system: one where participants tap on a tapping sensor (part 2) and another where participants tap on the surface of the laptop (part 3). In general, the independent calibration system uses two external synchronized devices to record the stimulus and tapping signals as soon as they are produced by the laptop speakers and finger tap, respectively. Since both the stimulus and tapping signals produce a highly precise sound wave, we can then apply a simple onset extrac- tion algorithm to precisely identify the location of the onsets at the earliest possible moment. To record the audio stimulus (both the markers and stimulus onsets), we directed a Shure SM58 microphone to the laptop speakers being tested. To record the tapping onsets, we used a custom-made tapping sensor device. The sensor consisted of a soft pad with ear- buds installed inside (Apple EarPods). The earbuds offer a low-sensitivity microphone that is well-suited to precisely detect touch on the surface of the sensor while being insen- sitive to external noises and minimizing auditory feedback. The tapping sensor was placed next to the laptops’ built- in microphone to capture the sound of the finger tapping. Overview: REPP Instead, we use the list of stimulus onsets provided in the stimulus preparation step and, therefore, do not need to extract the onsets from the recorded audio In the recording phase, REPP uses a free-field recording approach: the prepared stimulus is played through the laptop speakers and the resulting audio signal is simultaneously recorded along with the participant’s tapping response using the built-in microphone (see Fig. 1a). This method returns an audio file where both the audio stimulus and tapping onsets are mixed in the same channel. The next step (i.e., onset extraction) therefore applies signal processing techniques to split the mono recording into separate channels. REPP uses a unique frequency range for each relevant audio element in the recording, including a marker range and a tapping range (see Fig. 1c). The tapping range is determined by the acous- tic spectrum of the sound produced by participants’ mode of tapping. In our procedure, participants tap with their index finger on the surface of their laptop, producing a crisp sound with a significant part of its energy between 80 and 500 Hz. Since we have previously filtered the audio stimulus to avoid any overlap with this tapping range, we can efficiently 3 3 3 Behavior Research Methods (2022) 54:2271–2285 2275 stimulus, minimizing any interference with other elements in the signal processing pipeline. This method allows us to support SMS experiments using “virtual” onsets that are not clearly defined in the audio signal, such as when working with music (Colley et al., 2018; Dannenberg & Wasserman, 2009; Patel et al., 2005; Repp, 2002b). Namely, we can use the list of preregistered onset lists even in the absence of any physical onset in the stimuli. The output of this step is a list of realigned stimulus onsets and realigned tapping onsets. Finally, we calculate several metrics to assess the perfor- mance of REPP and measure participants' tapping accuracy, including the mean and standard deviation of the tap-stim- ulus asynchrony, percentage of detected taps, percentage of detected markers, and markers’ timing error (see Fig. 1d). Wing (Vorberg & Wing, 1996; Vorberg & Schulze, 2002), including the three hypothesized components (i.e., time- keeper noise, motor noise, and error correction), and lag-1 autocorrelation of the asynchrony and inter-tap interval as alternative measures of error correction. Experiment 1: Timing accuracy This experiment assessed the timing accuracy of REPP by comparing its performance with a ground-truth recording obtained from an independent calibration system. The exper- iment was divided into three parts. Part 1 and 2 were large- scale validation experiments aimed to extensively test the timing accuracy of the audio stimulus and tapping response, respectively. Part 3 was smaller in terms of the number of data points but aimed to test all components of REPP together (i.e., markers, stimulus, and tapping response). Validation experiments Both the microphone and tapping sensor were connected to a Focusrite Scarlett 2i2 USB sound card to record the signal on a separate MacBook computer running Ableton Live 10 Software, saving the resulting recording as a wave file. To measure tapping performance, we followed common practices established in previous tapping studies (Repp, 2005). Asynchronies were defined as An = Rn – Sn, where Rn denotes a response onset and Sn denotes a stimulus onset in a given tapping trial. We then computed the mean asynchrony and standard deviation (SD) of asynchrony. Throughout the paper, we report the SD of the asynchrony, as it provides a more consistent measurement of tapping accuracy than mean asynchrony. Typically, the mean asynchrony is nega- tive due to a human tendency to anticipate taps by a few tens of milliseconds when synchronizing to an external cue event (Repp, 2005). Mean asynchrony is also more influenced by tapping tasks, production modality, and auditory feedback biases compared to the SD of asynchrony. However, we rep- licated the main results reported in the behavioral experi- ments (Experiment 2 and 3) when using mean asynchrony (see Figure S8 in supplementary information). Furthermore, we repeated the same analyses using alternative (and more sophisticated) measures of SMS (see Trialling Alternative Measures of SMS in supplementary information). These included vector length using circular statistics (Fisher, 1993), a leading model of SMS proposed by Vorberg and To validate the timing accuracy of the audio stimulus (part 1), REPP was programmed to produce 100 isochronous 1 3 2276 Behavior Research Methods (2022) 54:2271–2285 Fig. 2 Results of Experiment 1: Timing accuracy. a External cali- bration system used to measure REPP’s timing accuracy. b Example of the beginning of a 500 ms IOI trial recorded in the two systems, showing the three marker sounds placed at the beginning of the stimulus and the two first metronome clicks. The figure illustrates the two alternative measures to assess timing accuracy: the differ- ence between the first marker and the stimulus (marker-to-stimulus), and the inter-onset interval. Note that the calibration system has two input channels (external microphone and tapping sensor) but we com- bine them in the figure for simplicity. c Distribution of the difference between the time the stimulus onsets (metronome clicks) were pro- duced and the time they were detected by REPP. N refers to the total number of tested onsets. Validation experiments d Distribution of the difference between the time the physical taps were produced and the time they were detected by REPP. N refers to the total number of tested onsets input channels (external microphone and tapping sensor) but we com- bine them in the figure for simplicity. c Distribution of the difference between the time the stimulus onsets (metronome clicks) were pro- duced and the time they were detected by REPP. N refers to the total number of tested onsets. d Distribution of the difference between the time the physical taps were produced and the time they were detected by REPP. N refers to the total number of tested onsets Fig. 2 Results of Experiment 1: Timing accuracy. a External cali- bration system used to measure REPP’s timing accuracy. b Example of the beginning of a 500 ms IOI trial recorded in the two systems, showing the three marker sounds placed at the beginning of the stimulus and the two first metronome clicks. The figure illustrates the two alternative measures to assess timing accuracy: the differ- ence between the first marker and the stimulus (marker-to-stimulus), and the inter-onset interval. Note that the calibration system has two input channels (external microphone and tapping sensor) but we com- bine them in the figure for simplicity. c Distribution of the difference between the time the stimulus onsets (metronome clicks) were pro- duced and the time they were detected by REPP. N refers to the total number of tested onsets. d Distribution of the difference between the time the physical taps were produced and the time they were detected by REPP. N refers to the total number of tested onsets metronome clicks at four different inter-onset intervals (IOIs): 250 ms, 500 ms, 750 ms, and 1000 ms. No finger taps were produced for this part and only the audio stimulus was recorded using the external microphone (Fig. 2a). To validate the timing accuracy of the tapping response (part 2), the same trials were produced and a researcher tapped in time to the clicks, resulting in four trials of 100 taps at four different IOIs: 250 ms, 500 ms, 750 ms, and 1000 ms. The tapping response was recorded using the tapping sensor and the audio stimulus was recorded using the external micro- phone (Fig. 2a). Validation experiments In the third part, to validate the timing accu- racy of all components of the system together (i.e., markers, stimulus, and tapping response), REPP was programmed to produce 20 isochronous metronome clicks at two IOIs: 500 ms and 1000 ms. This time, the researcher tapped on the surface of the laptop in anti-phase, so the stimulus and tap- ping onsets could be unambiguously distinguished in the recording. Both stimulus and tapping onsets were recorded with the same external microphone. The recording was then separated into three channels (i.e., markers, stimulus, and tapping response) and manually cleaned to only contain the corresponding elements in each channel (e.g., stimulus onsets in the stimulus channel, tapping onsets in the tapping channel). The results of the timing accuracy analysis in all valida- tion parts are reported in Table 1. The average latency and jitter of REPP was within 2 ms and similarly accurate for all components of the system: markers, audio stimulus, and tapping response. Timing accuracy was computed as the difference between the time the stimulus or tapping onsets were produced (measured using the external calibration system) and the time they were detected by REPP, using the first detected marker as the single frame of reference (Fig. 2b). We used two alternative measurements to calcu- late timing accuracy: marker-to-stimulus or marker-to-tap (i.e., the interval between the first marker onset and each subsequent onset in the audio or tapping signal), and inter- onset interval (i.e., the interval between onsets). Figure 2c, d shows the distribution of the time difference between the stimulus onsets (part 1) and tapping onsets (part 2) measured in the two systems, confirming that REPP’s timing accuracy is high and consistent. 3 3 Behavior Research Methods (2022) 54:2271–2285 2277 Table 1 Timing accuracy results “Part” refers to each validation experiment: part 1 (only stimulus), part 2 (only tapping response), and part 3 (stimulus and tapping response together). N refers to the total number of tested onsets. Experiment 2: Reliability and concurrent validity USB. The tapping tasks were implemented using MATLAB, mirroring the experimental procedure used in REPP. To detect the stimulus and tapping onsets at the earliest pos- sible moment, we used the same onset extraction algorithm described in Experiment 1 (Jacoby & McDermott, 2017). Experiment 1 showed that REPP can measure tapping and stimulus onsets with high temporal accuracy. In Experiment 2, we aimed to examine whether REPP can reliably meas- ure derived psychological quantities such as a particular individual’s tapping accuracy. Specifically, we assessed the test-retest reliability of REPP and compared its performance against a completely independent method: a well-established method previously used in the laboratory to measure SMS with high precision (Elliot et al., 2018). The materials and experimental procedure were identi- cal in the two methods (see Instructions in supplementary information). Before starting the main tapping task, partici- pants performed a practice phase to get familiar with each method (see Practice Phase in supplementary information). The main tapping tasks consisted of a short battery of tap- ping trials (approximately 8–10 minutes long) using two common paradigms in the tapping literature (Repp, 2005; Repp & Su, 2013): isochronous tapping and beat synchro- nization to music. The isochronous tapping consisted of four 30-second-long trials of isochronous tapping to a metronome sound (two with IOIs of 800 ms and two with the IOIs of 600 ms). The presentation order was fixed, using the fol- lowing sequence: 800 ms, 600 ms, 800 ms, and 600 ms. The beat synchronization task consisted of four 30-second-long excerpts of music from two distinct music genres with dif- ferent style, tempo, and tapping difficulty, also with fixed order of presentation (see Beat Synchronization Task in sup- plementary information). To assess test-retest reliability, the same group of partici- pants (N = 20) performed a short battery of tapping tasks two times in each method, using the following sequence: method 1 (pre), method 2 (pre), method 1 (post), method 2 (post). Half of the participants started the experiment using REPP, whereas the other half started using the independent in-lab method. To assess concurrent validity, we correlated the participants’ overall tapping performance in the two methods. Participants completed the experiment in a quiet testing room with two tables, one for each method. The independent in-lab method consisted of a loop-back setup to measure participants’ asynchronies with high tem- poral fidelity (see Fig. 3a). Validation experiments Part N Min-max Latency (SD) Min-max Latency (SD) Relative to first marker Inter-onset interval 1 + 2 Markers 40 −.08 to 4.9 1.85 (1.76) - - 1 Stimuli 400 −1.3 to 4.9 1.15 (1.61) −4 to 1 .02 (.39) 2 Tapping 400 −2.8 to 5.9 −.2 (1.37) −5 to 8 .03 (.13) 3 Markers 10 0 to 1 .58 (.48) - - 3 Stimuli 40 −.3 to .9 −.03 (.23) 0 to 1 .04 (.17) 3 Tapping 40 −3.1 to 1 −1.04 (.89) −4 to 2 −.02 (1.13) “Part” refers to each validation experiment: part 1 (only stimulus), part 2 (only tapping response), and part 3 (stimulus and tapping response together). N refers to the total number of tested onsets. tion experiment: part 1 (only stimulus), part 2 (only tapping response), and part 3 (stimulus and tapping response al number of tested onsets. Experiment 3: Online demonstration the recommendations of Koo and Li (2016), ICC estimates and their 95% confidence intervals were calculated based on single-rating, absolute-agreement, 2-way mixed-effects models (ICC3). We found a good ICC in both REPP (ICC = .86, 95% [.72, .93]) and the loop-back setup (ICC = .89, 95% [.77, .95]). This ICC is comparable to the values reported in previous work assessing the test-retest reli- ability of similar rhythmic production tasks (Bégel et al., 2018). Moreover, an ANOVA confirmed that participants’ mean tapping performances were similar across test-retest conditions and tapping tasks (all p-values > .05). Finally, we found that REPP has a high concurrent validity (r = .94 and ρ = .89), as indicated by the correlation between the overall tapping performances (averaging over both test and retest) measured by the two methods (Fig. 3b). In con- clusion, the converging evidence of these analyses is that REPP produces reliable estimates to measure individual differences on SMS in a way that is consistent with the results produced by a completely independent method. the recommendations of Koo and Li (2016), ICC estimates and their 95% confidence intervals were calculated based on single-rating, absolute-agreement, 2-way mixed-effects models (ICC3). We found a good ICC in both REPP (ICC = .86, 95% [.72, .93]) and the loop-back setup (ICC = .89, 95% [.77, .95]). This ICC is comparable to the values reported in previous work assessing the test-retest reli- ability of similar rhythmic production tasks (Bégel et al., 2018). Moreover, an ANOVA confirmed that participants’ mean tapping performances were similar across test-retest conditions and tapping tasks (all p-values > .05). Finally, we found that REPP has a high concurrent validity (r = .94 and ρ = .89), as indicated by the correlation between the overall tapping performances (averaging over both test and retest) measured by the two methods (Fig. 3b). In con- clusion, the converging evidence of these analyses is that REPP produces reliable estimates to measure individual differences on SMS in a way that is consistent with the results produced by a completely independent method. Having demonstrated that REPP achieves high temporal accuracy (Experiment 1) and test-retest reliability in the laboratory (Experiment 2), this experiment aimed to show that the technology can work in practice in an online setup that is similar to a large-scale data collection process. Experiment 2: Reliability and concurrent validity Finally, we found that REPP has a high concurrent validity (r = .94 and ρ = .89), as indicated by the correlation between the overall tapping performances (averaging over both test and retest) measured by the two methods (Fig 3b) In con Experiment 3: Online demonstration Having demonstrated that REPP achieves high temporal accuracy (Experiment 1) and test-retest reliability in the laboratory (Experiment 2), this experiment aimed to show that the technology can work in practice in an online setup that is similar to a large-scale data collection process. We also provide suggestions to ensure high data quality while enabling realistic data collection, in particular concerning pre-screening tasks and feedback based on recording quality and tapping performance. Participants were recruited from Amazon Mechanical Turk (see Participants in supplemen- tary information) and performed the same battery of tapping tasks used in Experiment 2. In a total of six experimental b t h (8 t 10 h h) ll t d lid t i d t Fig. 3 Results of Experiment 2: reliability and concurrent validity. a Loop-back setup: independent in-lab method using a loop-back cable to measure participants’ tapping asynchronies with high temporal fidelity (Elliot et al., 2018). b Test-retest reliability in the two meth- ods. c Concurrent validity: correlation between the overall tapping performance measured in the two methods 2278 Behavior Research Methods (2022) 54:2271–2285 Behavior Research Methods (2022) 54:2271–2285 2278 Fig. 3 Results of Experiment 2: reliability and concurrent validity. a Loop-back setup: independent in-lab method using a loop-back cable to measure participants’ tapping asynchronies with high temporal fidelity (Elliot et al., 2018). b Test-retest reliability in the two meth- ods. c Concurrent validity: correlation between the overall tapping performance measured in the two methods fidelity (Elliot et al., 2018). b Test-retest reliability in the two meth- ods. c Concurrent validity: correlation between the overall tapping performance measured in the two methods Fig. 3 Results of Experiment 2: reliability and concurrent validity. a Loop-back setup: independent in-lab method using a loop-back cable to measure participants’ tapping asynchronies with high temporal Fig. 3 Results of Experiment 2: reliability and concurrent validity. a Loop-back setup: independent in-lab method using a loop-back cable to measure participants’ tapping asynchronies with high temporal fidelity (Elliot et al., 2018). b Test-retest reliability in the two meth- ods. Experiment 2: Reliability and concurrent validity c Concurrent validity: correlation between the overall tapping performance measured in the two methods fidelity (Elliot et al., 2018). b Test-retest reliability in the two meth- ods. c Concurrent validity: correlation between the overall tapping performance measured in the two methods Experiment 2: Reliability and concurrent validity This method has been extensively used in the laboratory and field research on SMS (Elliot et al., 2018; Jacoby & McDermott, 2017; Jacoby et al., 2021). The loop-back setup consists of a cable connected to a sound card to simultaneously record the input signal from the headphones and the output signal from a tapping sensor with nearly zero latency. We used professional headphones (i.e., Sennheiser HD 280 Pro headphones) to deliver the stimulus, and the custom-made tapping sensor described in Experiment 1 to record participants’ tapping response with high precision. Both the headphones and tapping sensor were connected to an external sound card (Focusrite Scar- lett 2i2 USB) using the loop-back setup described above (Fig. 3a). The sound card was connected to a MacBook via The results of Experiment 2 are plotted in Fig. 3. Tap- ping performance was measured using the SD of the tap- stimulus asynchrony (see supplementary information for the results of the same analysis using alternative measures of SMS). To examine test-retest reliability, an aggregated performance score was calculated for each participant in each test (pre and post) and method by averaging their tapping performance in the two tapping tasks (i.e., isoch- ronous tapping and beat synchronization to music). The test-retest correlation in REPP was high (r = .87; ρ = .81) and similar to the one achieved by the independent loop- back setup (r = .89; ρ = .83; Fig. 3a). We further examined test-retest reliability by calculating the intraclass correla- tion coefficient (ICC; Shrout & Fleiss, 1979). Following 1 3 3 the recommendations of Koo and Li (2016), ICC estimates and their 95% confidence intervals were calculated based on single-rating, absolute-agreement, 2-way mixed-effects models (ICC3). We found a good ICC in both REPP (ICC = .86, 95% [.72, .93]) and the loop-back setup (ICC = .89, 95% [.77, .95]). This ICC is comparable to the values reported in previous work assessing the test-retest reli- ability of similar rhythmic production tasks (Bégel et al., 2018). Moreover, an ANOVA confirmed that participants’ mean tapping performances were similar across test-retest conditions and tapping tasks (all p-values > .05). Experiment 3: Online demonstration We also provide suggestions to ensure high data quality while enabling realistic data collection, in particular concerning pre-screening tasks and feedback based on recording quality and tapping performance. Participants were recruited from Amazon Mechanical Turk (see Participants in supplemen- tary information) and performed the same battery of tapping tasks used in Experiment 2. In a total of six experimental batches (8 to 10 hours each), we collected valid tapping data for 226 participants. We used two pre-screening tasks to ensure high data quality while minimizing recruiting costs (see Pre-screen- ing Tests in supplementary information). First, we used an attention test to determine whether participants were pay- ing attention to the instructions. Participants who failed the attention test were excluded from the experiment. Second, 1 3 Behavior Research Methods (2022) 54:2271–2285 2279 Fig. 4 Results of Experiment 3: Online demonstration. a Test-retest reliability of REPP when measuring participants’ tapping perfor- mance online. b Convergent validity: correlation between overall tapping performance and participants’ musical training. c Estimated markers’ error in Windows and macOS computers. N indicates the number of participants using each operating system, but we plot the data in all tapping trials (2814 in total) Fig. 4 Results of Experiment 3: Online demonstration. a Test-retest reliability of REPP when measuring participants’ tapping perfor- mance online. b Convergent validity: correlation between overall tapping performance and participants’ musical training. c Estimated markers’ error in Windows and macOS computers. N indicates the number of participants using each operating system, but we plot the data in all tapping trials (2814 in total) the practice phase, the four audio recordings were analyzed in real time using a failing criteria designed to identify and fail trials where participants used incompatible hardware and software, or where participants did not tap as indicated in the instructions (see Failing Criteria in supplementary information). Those participants who failed two or more trials were excluded from the experiment. After the prac- tice phase, participants started with the main experimental task, which consisted of the same battery of tapping tasks employed in Experiment 2 (i.e., four trials of isochronous tapping and four trials of beat synchronization to music, 30 seconds long each). Participants repeated the same battery of tapping tasks a second time in order to measure test-retest reliability. Experiment 3: Online demonstration Moreover, participants’ tapping performance was similar across test-retest conditions in the two tapping tasks, as indicated by two paired-samples t-tests with test condition as the independent variable and tap- ping performance in each tapping task as dependent variables (all p-values > .05). As a measure of convergent validity, we further examined whether participants’ tapping performance was related with their self-reported levels of musical training. We calculated an aggregated tapping performance score (aver- aging over test and retest in the two tapping tasks) for all partici- pants who provided good tapping (N = 226). Musical training was measured using a reduced version of the Gold-MSI musical training factor (Müllensiefen et al., 2014). Replicating a recur- ring finding in the literature (e.g., Niarchou et al., 2021; Repp, 2010; Thompson et al., 2015), we found a significant negative correlation (r = -.32, p < .001) between tapping variability and self-reported musical training (Fig. 4b), indicating that partici- pants with more musical training were better at synchronizing to an external beat. To explore the robustness of our technology across operating systems and laptop models, we compared the markers’ detection accuracy (i.e., the delay between the known marker locations and the detected marker onsets) across the two most common operating systems, Windows and macOS (Fig. 4c). Overall, trials recorded in macOS computers achieved slightly better temporal accuracy (M = 1.48, SD = .68) than tri- als recorded in Windows (M = 1.74, SD = .85), t(2812) = 9.36, p < .001. A small difference in this direction is not surprising: macOS computers are typically better equipped for delivering sound and recording audio than Windows computers, which also tend to exhibit greater variability in hardware. on the ground-truth recording, we estimated the average latency and jitter of REPP to be within 2 ms and similarly accurate for all elements in the system (i.e., markers, audio stimulus, and tapping response). In a laboratory experiment (Experiment 2), we then compared the test-retest reliability of REPP with a completely independent method that uses specialized equipment (i.e., a loop-back setup) to measure SMS with optimal temporal fidelity. We found that our tech- nology achieves a high test-retest reliability (r = .87, ρ = .81, ICC = .86) that is equivalent to the reliability obtained by the independent in-lab method (r = .89, ρ = .83, ICC = .89). Experiment 3: Online demonstration By correlating the overall tapping performance measured in the two methods, we also found that REPP has a high concurrent validity (r = .94 and ρ = .80). Finally, we showed that REPP can work in practice in an online setup that is similar to a large-scale data collection process (Experiment 3). In particular, we confirmed that REPP has a high test-retest reliability using a larger sample of par- ticipants recruited online (N = 166; r = .80, ρ = .81, ICC = .82). We also provided suggestions of tapping instruc- tions and pre-screening tests to ensure high data quality in online experiments while minimizing recruitment costs (see supplementary information). Together, these experiments demonstrate that our technology is well-equipped to support a wide variety of SMS experiments using standard hardware and software available to most online participants. p p A core aspect of REPP is the ability to analyze partici- pants’ recordings in real time, making it possible to monitor experiments online and provide feedback almost instantly. We used a software testing tool1 to reliably estimate the speed of processing of this technology under different analy- sis conditions (for each condition, we repeated the same analysis a thousand times)2. We found that running the analysis when plotting is disabled leads to the fastest per- formance. For example, the average running time (Q1–Q3) to analyze a 15-second-long recording is 0.66 seconds (0.64–0.67), whereas the average running time (Q1–Q3) for a 30-second-long recording is 1.46 seconds (1.42–1.49). When plotting is enabled (generating and saving plots for each analysis), the average running time (Q1–Q3) is 3.84 seconds (3.75–3.89) for a 15-second-long recording and 5.55 seconds (5.29–5.81) for a 30-second-long record- ing. This is mainly due to the slow implementation of the python package Matplotlib3 that we used for plotting. In case this is important, experimenters can avoid plotting (as it is only used for debugging) or consider implementing a more 1 https://pypi.org/project/pytest-benchmark/ 2 We used a MacBook Pro (macOS Catalina version 10.15.7), with an Intel processor 2.3 GHz 8-Core Intel Core i9, and Python 3.9.4. 3 https://matplotlib.org/ Experiment 3: Online demonstration 4a; r = .80 and ρ = .81), also confirmed by an intraclass correlation analysis (ICC = .82, 95% [.77, .86]). Moreover, participants’ tapping performance was similar across test-retest conditions in the two tapping tasks, as indicated by two paired-samples t-tests with test condition as the independent variable and tap- ping performance in each tapping task as dependent variables (all p-values > .05). As a measure of convergent validity, we further examined whether participants’ tapping performance was related with their self-reported levels of musical training. We calculated an aggregated tapping performance score (aver- aging over test and retest in the two tapping tasks) for all partici- pants who provided good tapping (N = 226). Musical training was measured using a reduced version of the Gold-MSI musical training factor (Müllensiefen et al., 2014). Replicating a recur- ring finding in the literature (e.g., Niarchou et al., 2021; Repp, 2010; Thompson et al., 2015), we found a significant negative correlation (r = -.32, p < .001) between tapping variability and self-reported musical training (Fig. 4b), indicating that partici- pants with more musical training were better at synchronizing to an external beat. To explore the robustness of our technology across operating systems and laptop models, we compared the markers’ detection accuracy (i.e., the delay between the known marker locations and the detected marker onsets) across the two most common operating systems, Windows and macOS (Fig. 4c). Overall, trials recorded in macOS computers achieved slightly better temporal accuracy (M = 1.48, SD = .68) than tri- als recorded in Windows (M = 1.74, SD = .85), t(2812) = 9.36, p < .001. A small difference in this direction is not surprising: macOS computers are typically better equipped for delivering sound and recording audio than Windows computers, which also tend to exhibit greater variability in hardware. we only consider participants who provided at least one valid tapping trial for each stimulus in each tapping task and test- retest condition (N = 166). Test-retest analyses were performed using the same procedure described in Experiment 2. Results indicated a high test-retest correlation when using REPP to measure participants’ tapping performance online (Fig. 4a; r = .80 and ρ = .81), also confirmed by an intraclass correlation analysis (ICC = .82, 95% [.77, .86]). Experiment 3: Online demonstration we used a recording test to determine whether participants were using hardware and software that were not compat- ible with REPP, such as malfunctioning speakers or micro- phones, or the use of strong noise-cancellation technologies. Participants who did not pass the recording test were also excluded from the experiment. To assess the efficacy of the recording test in comparison to the attention test on its own, we only used the recording test in half of the participants. we used a recording test to determine whether participants were using hardware and software that were not compat- ible with REPP, such as malfunctioning speakers or micro- phones, or the use of strong noise-cancellation technologies. Participants who did not pass the recording test were also excluded from the experiment. To assess the efficacy of the recording test in comparison to the attention test on its own, we only used the recording test in half of the participants. Before the main tapping tasks, participants were instructed on several key aspects concerning the proper functioning of REPP, including instructions about the tech- nical requirements and tapping procedure for the experi- ment, a volume calibration test, and a tapping calibration test (see Instructions in supplementary information). Next, participants undertook a practice phase consisting of four tri- als of isochronous tapping to a metronome sound (see Prac- tice Phase in supplementary information). After completing Before the main tapping tasks, participants were instructed on several key aspects concerning the proper functioning of REPP, including instructions about the tech- nical requirements and tapping procedure for the experi- ment, a volume calibration test, and a tapping calibration test (see Instructions in supplementary information). Next, participants undertook a practice phase consisting of four tri- als of isochronous tapping to a metronome sound (see Prac- tice Phase in supplementary information). After completing The results of Experiment 3 are visible in Fig. 4 (see sup- plementary information for the same analysis using alterna- tive measures of SMS). For measuring test-retest reliability, 1 3 Behavior Research Methods (2022) 54:2271–2285 2280 we only consider participants who provided at least one valid tapping trial for each stimulus in each tapping task and test- retest condition (N = 166). Test-retest analyses were performed using the same procedure described in Experiment 2. Results indicated a high test-retest correlation when using REPP to measure participants’ tapping performance online (Fig. 3 Discussion SMS is an active area of research with a long history of tapping experiments performed in the laboratory (Repp, 2005; Repp & Su, 2013). However, it currently lacks a robust method to precisely measure participants’ asynchronies in online experi- ments. In this paper, we presented REPP, a cross-platform solution for online SMS experiments that achieves high tempo- ral accuracy and reliability while also being practical in terms of large-scale data collection. We release this technology as a free and open-source framework alongside this paper.i We validated REPP in three experiments. We first dem- onstrated that it achieves high temporal accuracy using an independent calibration system (Experiment 1). Based 1 3 3 Behavior Research Methods (2022) 54:2271–2285 2281 efficient plotting library (such as VisPy4 or PyQtGraph5). Overall, these results show that REPP is suitable to process participants’ recordings in real time, enabling researchers to filter participants based on performance or provide trial- to-trial feedback. The ability to rapidly process participants’ performance is also crucial to enable more complex SMS paradigms in online settings, such as iterated tapping exper- iments where new stimuli are generated on the fly based on previous tapping responses. In Jacoby et al. (2021), for example, we successfully adapted an iterated tapping para- digm using REPP in a large-scale online tapping experiment conducted in the US, India, and Brazil. an unfiltered metronome sound (Experiment 2, N = 166, SD of asynchrony = 40.68; SD = 11.98), also supported by a large correlation between participants’ tapping performance in the two tasks (r = .58, ρ = .59, all p-values < .001). This suggests that the filtering step does not drastically impair participants ability to synchronize to a musical beat when using REPP in online SMS experiments. Moreover, we learned that collecting good tapping data over the internet can necessitate a high exclusion rate, at least when using a large-scale recruiting strategy via Ama- zon Mechanical Turk. In Experiment 3, for example, a total of 727 participants began the online task. This includes any- one who accepted the experiment regardless of their inten- tions to take the task seriously or whether they met the tech- nical requirements to provide good tapping data. Thus, we used a practice phase to familiarize participants with the task and exclude cases who could not provide good tapping data in the majority of trials. Discussion Note that we used relatively strict failing criteria to exclude trials based on whether the signal could be correctly recorded and whether participants pro- duced a minimally acceptable number of tapping responses (see Failing Criteria in supplementary information). A total of 226 participants (31%) passed the practice phase and were able to provide good tapping data. The remaining 483 par- ticipants (69%) were excluded from the experiment and com- prised a mix of fraudulent participants (e.g., computer bots or non-serious responders) and participants that did not meet the technical requirements of REPP, such as poor internet connection or incompatible hardware and software. Com- mon sources of failure included participants’ behavior, such as not tapping at all, using desktop computers without built- in microphones, or performing the experiment with head- phones instead of the laptop speakers. We also noticed that many participants did not follow the instructions to eliminate background noise, such as music or speaking, resulting in noisy recordings. An additional problem was the usage of remote desktops, which may be employed by some partici- pants to alter their geographical reported location. Since the remote desktop will open a microphone that is not physically connected to the computer of the participant, the technology is not able to record any signal. Furthermore, there were several cases where the technology failed due to laptops with low quality or malfunctioning speakers. The same occurred in laptops with strong noise-cancelling technologies, where the marker sounds are suppressed and cannot be detected in the signal. This last issue requires further investigation, as in theory it is possible to turn off noise cancellation manually in most devices, but the way to do so changes in different computer models and brands. , , REPP currently has some limitations. First, it does not support all functionalities that are offered in many in-lab methods (e.g., FTAP, Max-MSP, or MatTAP). For exam- ple, REPP currently does not support real-time response feedback (Mates & Aschersleben, 2000; Finney & Warren, 2002). A possible solution would be to play the real time feedback with a JavaScript audio process; this feedback may have compromised accuracy, but at least the feedback signal could be recorded and monitored with a variant of our technology. Developing this approach would however require significant additional work. Second, REPP currently only works with laptop speakers. We see great potential in extending our technology to mobile devices and tablets as well. 4 https://vispy.org/ 5 https://www.pyqtgraph.org/ Discussion In language production experiments that require participants to record themselves using a microphone to extract voice onset latencies, the exclusion rate can be around 60% (Vogt et al., 2021). Thus, the exclusion rate of REPP (~60–70%) is not unexpected when using a large- scale recruiting strategy via Amazon Mechanical Turk, as it is technically more demanding than previous paradigms. In particular, REPP can only work in SMS experiments when the marker sounds can be detected with high millisecond- level precision and participants take the task seriously (i.e., tapping with their index finger on the surface of their laptop in time to an auditory stimulus). A high exclusion rate is not particularly problematic when using online recruitment sys- tems with large pools of active participants, but other modes of recruiting may require different strategies, such as when recruiting participants from special populations or using internal university systems. In these cases researchers can significantly reduce exclusion rates by using more relaxed failing criteria and taking more time to support participants and ensure they follow the instructions and meet the techni- cal requirements. REPP can also be used in laboratory stud- ies and field research with an exclusion rate of effectively 0%, as shown in Experiment 2. It is worth noting that REPP can be easily extended to support online experiments requiring precise timing of tap- ping response without any synchronization to an external stimulus. For example, to perform unconstrained finger tap- ping paradigms where participants are not given an external stimulus but instead are asked to tap at their preferred rate (e.g., Collyer et al., 1994), or imitation experiments in which participants replicate a rhythm from memory (e.g., Ravig- nani et al., 2016). In these cases, researchers can skip the stimulus preparation and onset alignment steps, and simply use the parts of the pipeline that are directly related to the onset extraction procedure. We have successfully explored these options using a simplified method for several experi- ments that do not require stimulus-response synchronization, and support this variant in the code package published with this paper. Another simple extension of REPP is from finger tapping to other modes of production, including clapping, tapping on a table, or speech. Discussion This would not only make REPP more accessible, but it would also open the possibility to run SMS experiments while in motion, such as walking or dancing. Combining REPP with features available in most mobile devices, such as multi-touch screens or accelerometers, can also enable new ways to collect data and modes of interaction. Third, REPP relies on a stimulus preparation step that filters the audio stimulus to remove lower frequencies that would oth- erwise interfere with other aspects of the signal process- ing pipeline, such as the analysis of participants’ tapping response. This procedure decreases the perceived quality of complex auditory stimuli, such as music, and it could there- fore compromise its ecological validity. We encourage future research to test this in the lab, for example, by comparing participants’ tapping accuracy in a beat synchronization task using music stimuli filtered at different frequency ranges. Nevertheless, it is worth noting that in the two behavioral experiments conducted here we did not receive any com- plaints from participants regarding the quality of the music stimuli. More importantly, participants’ tapping performance in the online beat synchronization task (using filtered music) was within an expected range (Experiment 2, N = 166, SD of asynchrony = 42.68; SD = 15.82) and similar to the their performance in the online isochronous task tapping using Naturally, the more demanding the online tasks, the higher the exclusion rate. In previous work, we found exclu- sion rates of only about 10% in Amazon Mechanical Turk experiments with minimal technical requirements, such as 1 Behavior Research Methods (2022) 54:2271–2285 2282 when using visual rating scales in the browser (Harrison et al. 2020). However, the exclusion rate increases when the experiment becomes technically more demanding. For example, a pre-screening test that requires participants to wear headphones to perform an auditory perception task produces an estimated exclusion rate of 36% (Wood et al., 2017), whereas performing online research with computer webcams can necessitate an exclusion rate of about 40% (Tran et al., 2017). In language production experiments that require participants to record themselves using a microphone to extract voice onset latencies, the exclusion rate can be around 60% (Vogt et al., 2021). Thus, the exclusion rate of REPP (~60–70%) is not unexpected when using a large- scale recruiting strategy via Amazon Mechanical Turk, as it is technically more demanding than previous paradigms. Discussion In particular, REPP can only work in SMS experiments when the marker sounds can be detected with high millisecond- level precision and participants take the task seriously (i.e., tapping with their index finger on the surface of their laptop in time to an auditory stimulus). A high exclusion rate is not particularly problematic when using online recruitment sys- tems with large pools of active participants, but other modes of recruiting may require different strategies, such as when recruiting participants from special populations or using internal university systems. In these cases researchers can significantly reduce exclusion rates by using more relaxed failing criteria and taking more time to support participants and ensure they follow the instructions and meet the techni- cal requirements. REPP can also be used in laboratory stud- ies and field research with an exclusion rate of effectively 0%, as shown in Experiment 2. non-serious respondents (Crump et al., 2013). In addition to pre-screening tests, we encourage the use of data quality checks to monitor participants’ performance throughout the experiment. REPP computes several metrics to check the quality of a given recording, such as the number of detected markers or the time error between the known locations of the markers and the detected onsets, which provides a reliable measure of timing accuracy at the trial level. We can also know how well participants are tapping by computing the ratio between the number of detected onsets and the num- ber of stimulus onsets. Using these metrics, we can provide feedback each time a participant completes a tapping trial, indicating whether their recording quality is sufficiently good and if it is not, suggesting ways to improve it for the subsequent trials (we apply this strategy in the practice phase of Experiment 3). We encourage future research to explore these options further in order to increase recruit- ment efficiency, such as providing more detailed feedback after tapping trials or nudging participants online to meet the technical requirements. when using visual rating scales in the browser (Harrison et al. 2020). However, the exclusion rate increases when the experiment becomes technically more demanding. For example, a pre-screening test that requires participants to wear headphones to perform an auditory perception task produces an estimated exclusion rate of 36% (Wood et al., 2017), whereas performing online research with computer webcams can necessitate an exclusion rate of about 40% (Tran et al., 2017). Discussion We noticed that our technology works well for clapping or tapping on a table, but adapting it to spoken utterances may require a modification to the parameters of the signal processing pipeline (see Experiment 6 in Jacoby & McDermott, 2017). Potentially, our technol- ogy could also be used to support online experiments requir- ing precise timing in domains other than rhythm perception and production. This includes any experiment measuring reaction times to auditory stimuli, such as auditory lexi- cal decision tasks (e.g., Blumstein et al., 1982; Goldinger, 1996), priming paradigms using spoken words (e.g., Radeau et al., 1998), sounds (e.g., Schön et al., 2010), or music (e.g., Bharucha & Stoeckig, 1986, 1987), and experiments on tem- poral processing using time interval production tasks (e.g., Jazayeri & Shadlen, 2010). p Since exclusion rates may be high when using technical demanding tasks in online recruiting systems, such as Ama- zon Mechanical Turk or Prolific, we strongly recommend the use of pre-screening tests to determine whether participants will take the experiment seriously and meet the technical requirements to provide good tapping data. In Experiment 3, we analyzed the efficacy of two pre-screening tests, an atten- tion test and a recording test (see supplementary information for a full description). We defined the exclusion rate of the pre-screening tests in terms of the proportion of participants who successfully passed the practice phase. Accordingly, we found that when using both pre-screening tests, 68% of the participants were able to pass the practice phase and deliver good tapping data in the experiment. In contrast, when only using an attention test without the recording test, the percentage was 31%. Thus, adding a recording test at the beginning of the experiment reduces the costs of recruiting participants online by nearly half. Such practices can also help maintain a good reputation in the online community; for example, both the attention test and recording test help eliminate a large proportion of the failure rate that comes from fraudulent participants, including computer bots and 3 3 Behavior Research Methods (2022) 54:2271–2285 2283 Funding Open Access funding enabled and organized by Projekt DEAL. The authors did not receive support from any organization for the submitted work. We hope REPP plays a major role in improving the effi- ciency, scalability, and reach of SMS research. Discussion Finding new ways to allow online data collection has become particu- larly important during the COVID-19 pandemic, with many researchers unable to run experiments in the laboratory. Sup- porting online experiments on SMS will also significantly reduce the time and resources that researchers usually spend to recruit and test participants in the laboratory. For instance, recruiting 20 participants in the laboratory (Experiment 2) took us approximately one week of work, whereas in the online version of the same experiment (Experiment 3) we recruited 20 valid participants in about 2 hours. Importantly, online experiments allow for the collection of significantly larger and more diverse samples of participants, both demo- graphically and culturally. This is crucial for moving away from the relatively restricted and small samples of univer- sity students that laboratory studies tend to rely on (Hen- rich et al., 2010). Moreover, since online SMS experiments are more accessible and easier to share, they can improve research diversity and collaboration worldwide, an important challenge in today’s cognitive science (Barret, 2020; Savage et al., 2021). Finally, by increasing the reach, scalability, and speed of data collection, online experiments open new avenues for research on SMS that would be nearly impos- sible in the laboratory. For example, very large-scale studies with hundreds of participants to study individual differences on SMS in the general population (Niarchou et al., 2021), or cross-cultural experiments that include a diverse sample of participants from around the world (Jacoby et al., 2021). Online tapping experiments can also allow for large scale simulated experiments on cultural transmission that were previously conducted only with small cohorts of lab par- ticipants (Raviginani et al. 2016). Similarly, the ability to collect large tapping datasets online can also help increase our understanding of the role of SMS in the context of vari- ous neurodevelopmental disorders, including attention defi- cit hyperactivity disorder (Noreika et al., 2013), dyslexia (Colling et al., 2017; Thomson & Goswami, 2008), and Par- kinson’s disease (Bieńkiewicz & Craig, 2015). Declarations Conflicts of interest The authors have no conflicts of interest to de- clare. Ethics approval Approval was granted by the Max Planck Society Eth- ics Council (application 2018-38). Ethics approval Approval was granted by the Max Planck Society Eth- ics Council (application 2018-38). Consent to participate Informed consent was obtained from all indi- vidual participants included in the study. Consent for publication Not applicable. Code availability We release REPP as a free and open-source frame- work alongside the publication of this paper. Code available in https:// gitlab.com/computational.audition/repp Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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https://openalex.org/W4300887996	https://zenodo.org/records/996927/files/11.%20DEFINING%20THE%20MORPHO-FUNCTIONAL%20INDICATORS%20-%20Hadjar%20Mohamed%20Kherfane.pdf	English	null	DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	6,243	DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES Chachou Seddaoui1, Houar Abdelatif 2, Hadjar Mohamed Kherfane3i 1OPAPS laboratory of the IEPS, Abdelhamid Ibn badis University, Mostaganem, Algeria 2Dr., OPAPS laboratory of the IEPS, Faculty of Human and Social Sciences, University of Ghardaia, Algeria OPAPS laboratory of the IEPS, Abdelhamid Ibn badis University, Mostaganem, Algeria 3Dr., OPAPS laboratory of the IEPS, Abdelhamid Ibn badis University, Mostaganem, Algeria Volume 3 │ Issue 10 │ 2017 Volume 3 │ Issue 10 │ 2017 Abstract: This study aims at defining the morphological and functional indicators for the selection of footballers according to the three play lines (defence, midfield, attack) for the category of under 18 years old. We will work on finding the differences in these morpho-functional indicators and their relation with the play lines and their requirements. For this, we made the morphological measurements, namely : height, weight, fat-weight, muscle weight, bone weight, and the functional tests, namely : Luc Leger’ Test to measure the maximal oxygen consumption (Vo2max), the retrieval capacity via Ruffier Dickson Test and Vitality Capacity Test V.C, for 10 first level clubs in the West of Algeria; as we selected 18 players from each team with the average of 6 players of each play line (180 players), making a rate of 32% of the original society as a sample. We also used the descriptive and statistical comparative methods among those indicators and each play line in order to know to what extent those morpho-functional variables cause differences among players according to their play lines, while defining those indicators as a reference to be adopted in the players selection process for this age category in order to support the selection process with observation. The results revealed Copyright © The Author(s). All Rights Reserved. © 2015 – 2017 Open Access Publishing Group 142 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES clear differences in the morph-functional indicators among the target category of players according to the differences in their playing lines at the level of the height indicator, as the significance value of selection reached 0.015 which is less than the significance level of the null hypotheses, while we did not find significant differences between the other morphological indicators. Meanwhile, there was a difference in the functional indicator at the level of the retrieval capacity, as the significance value reached 0.011 which is less than the test significance level 0.05; there was no difference at the level the other significances. Abstract: Thence, the researcher recommends the necessity of consolidating the selection process by the inclusion of observation through the use of some morphological measurements such as height, in addition to functional tests such as the retrieval capacity as important indicators in the players selection process according to the requirements of the play lines for this age category of players, in order to select the best talents that can be beneficial to Algerian football in the future. Keywords: morpho-functional indicators, player’s selection, play lines European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 1. Introduction Modern scientific research in the domain of sport training in general and football in particular, is interested in giving data and scientific truths in order to promote the quality of sport results and achievements existing in all the competitions and championships, as the training and form of the athletes has got an important share of researchers and specialists’ interest in order to know the main criteria of the athlete’s body, including morphological criteria and anthropometrical measurements that are meant to enable him to excel among his peers and contribute in a direct way in making him successful in his specialty or in his play centre, and thus to make him more effective in the team. That is why research in defining these specific criteria varied, (Bengoua Ali, 2004) mentioned that football is one of the sports that attract the majority of spectators, and that it is worth the competition among its coaches and interested people to get benefit from the tests and measurements at all levels, such as skill and physiological measurements of players, while making physical measurement in order to select the most talented youngsters, add to that the fact that football is one of those mass games which attract spectators throughout the world in the different international competitions and championships, and that is why it could get as much interest of science and research in order to arrive to suggestions and recommendations that lead to better achievements, and this requires specific criteria either at the morphological level or the functional and physical one which are related to the skill side and which is not opean Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 2017 143 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES enough for excelling or achieving the highest levels, (Samir Chiban, 2010) confirms that there is a difference in the morphological structure between the elite athlete and the championship’s one due to the interest in skills as well as the physical aspects while neglecting puberty and its influence on the selection process. 1. Introduction Morphological requirements are also considered to be the requirements of modern football, as the distinction within one team is dictated by the quality of the playing lines and centres which are: the goalkeeper, the centre of defense, the midfield and the attack. (Houar Abdelatif, 2015) showed that there were statistically significant differences between play lines and body measurements such as body weight, height, body mass index, and maximum oxygen consumption. This is an indication that the indicators for body building and formation and all that is related to measurements of dimensions and characteristics of body shape in general, has a direct relationship and positively affects the sport results through outstanding performance. Besides, the functional and physiological capabilities have an important role, as they took an important part of scientific research due to the strong relationship between high performance and readiness of the physiological functions and their efficiency, as there are individual differences between players in this aspect on one hand and the requirements of play centres of physical effort on the other. (Akbobi Habib, 2017) says that experience is not always enough for the coach, as he is in dire need of precise parameters or indicators of physical activity, tactic, physical condition and even biological aspect of the players for the play centres. It is also possible to predict on the basis of the capabilities and functional capabilities of the player such as the efficiency of the periodic and respiratory capabilities of the retrieval capabilities in the selection process at the high level, and this accelerates the achievement of success with economy in time, effort and money and this is consistent with the study that we are conducting, since the selection process by observation is not enough scientifically, and this fact led us to search other determinants which are more precise and objective to be used in the selection process in the under 18 years old category of footballers, as the study is considered to be an important and scientific dimension that is meant to support the observation-based selection process at the higher level in the national championship through the use of the database as a scientific reference European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 2. Problematic Modern football has known a number of changes in the methods of training and preparation, as it has become dependent on the results of scientific research and the application of supervisors of the training process considering those scientific methods European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 144 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES as a guiding platform in their training programs. Modern football is characterized by speed, strength and endurance at the higher levels, It is impossible to focus on some aspects and neglect others, as achievement is interdependent and integrated from the stages of selection and direction to the elite and high level stage. After the elimination of the national team from the first round in the African Cup, which was held in Gabon in 2017, and which was such a shock to the followers of the team and its supporters, the National Technical Directorate decided to reconsider its policy, which was dependent on the foreign product instead of the local as this was an obstacle in the African participation of the team because of the difference in environment and climate compared to Europe, thus, going back to reliance on local talent, as the case used to be in the eighties became the most prominent interest through the right scientific training in clubs and schools in terms of framing and how selection ways in accordance with the scientific methods studied in the medium and long terms as the best means, with the formation of national teams of the Federation similar to the international teams, and this to prepare them more to the high level and to build the national team as a harmonious team having gone across all stages of the smaller categories of the team. European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 2. Problematic (www.radioalgerie.dz, 2017) The problem lies not in the lack of talent, but in the lack of methods of discovery and selection of talents, relying on random and self-selection ways, away from the use of modern scientific and codified methods, which is nowadays adopted in selection processes at the high level, away from subjectivity in order to avoid wasting time, effort and money while giving opportunities for all. The players are almost equally skilled at all levels, thus, players can only be chosen by subjecting them to other parameters and criteria imposed by the game's requirements, and these are the roles and duties required by each of the three playing lines (defense, midfield and attack) and according the playing centers of each line . Among these determinants are the individual differences in the form and structure of the body, ie physical measurements and physical aspects required in the game according to lines and playing centers. (Samir Chiban, 2010), citing Carter (1985), says that with some physical measurements the talented player can be identified, as the morphological structure is an important and influential factor in the performance of the player in playing line according to the requirements of the positions and roles of the playing centers, the defender’s body measurements are different from those of midfielders and attackers, and each center has the physical and functional requirements that make them distinguished from each other in the roles and their peers at the high level and this is what Wahid Halilozitch pointed out reporting from (Alexnder Dellal, 2008) that each center has its own physiological needs. 2. Problematic The problem of research is that European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 2017 145 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES a ou e aoui, oua A e a i , a ja o a e e a e DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES the researcher is interested in the field of sports training in general and soccer in particular in the absence of real scientific indicators derived from the terrain in the process of selection of the players and even the studies and research that dealt with this subject found physical, morphological and functional determinants of selection but did not take into account the specificities and requirements of the playing lines and centers, as these determinants were collective and inclusive of different age groups, except the study of (Houar Abdelatif, 2015) Which led to the proposal of a computer program that directs the players according to the play centers based on physical, skill and morphological determinants. Based on these scientific data, it was necessary to think about setting some determinants on which the selection process in football can be based on and from real data taken from the real terrain of Algerian football. 2.1 Research Goals Determination of morpho-functional indicators for under 18 years category according to the playing lines. 2. Problematic The chosen determinants for the selection are the morphological and functional ones because they have a direct impact on the effectiveness of the player’s performance, especially in accordance with the requirements of defensive and offensive duties, and the bilateral and aerial conflicts in the midfield, and the length of the match and the intensity of competitions, as for the choice of the category of under 18 years it was based on the fact that this category is the one that separates between the youngsters and adults categories, and this makes the selection more accurate and important because of its of sensitive aspect in the selection of players who are able to represent the team among adults and provide a group of players superior to their peers skillfully, physically, morphology And functionally according to the requirements of the playing lines as a homogenous and integrated team; and to help solve the research problematic we ask the following question: “Do the morpho-functional variables contribute to the variation between football players under 18 years according to the lines of play?” European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 C. The human field The study was conducted on the players of the Algerian West teams for the under-18 category. We chose 18 players with 6 players in each line of defense (defense, midfield and attack). 3.1 Research Methodology The researcher used the descriptive method in the survey method to suit the nature of the research. 3.4 Measurements and tests applied in the research 3.4.1 Morphological measurements 3.4.1 Morphological measurements 2.2 Search Hypothesis  Yes, some morphological variables contribute to the difference between football players according to the lines of play under 18 years.  Yes, some functional variables contribute to the variation between football players according to the lines of play under 18 years. European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 146 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES 3.4.2 Functional tests  Luc Legér test to measure the maximum consumption ratio of oxygen VO2max;  Rufierer Dickson test for measuring retrieval capacity;  Luc Legér test to measure the maximum consumption ratio of oxygen VO2max;  Rufierer Dickson test for measuring retrieval capacity;  Luc Legér test to measure the maximum consumption ratio of oxygen VO2max;  Rufierer Dickson test for measuring retrieval capacity;  Measurement of the vital capacity.  Measurement of the vital capacity. 3.3 Research fields A. Spatial field Measurements and tests were carried out at the teams' stadiums, as we took the morphological measurements in the cloakroom rooms and functional tests in the field. B. Time domain dy was conducted between 05/10/2016 and 25/03/2017. This study was conducted between 05/10/2016 and 25/03/2017. European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES 3.4.1 Morphological measurements  Height  Weight - fat-weight - bone weight - muscle weight 3.2 Sample of the study The study was conducted on a sample of 10 teams from the west of Algeria under 18 years (180 players), which is more than 32% of the registered ones for the sports season 2016/2017. European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 4. Presentation and discussion of the results For the weight index the mean value is 0.551, which is greater than the significance of 0.05, and therefore we reject the alternative hypothesis and accept the null hypothesis i.e. X1 = X2 = X3 and conclude that there are no significant differences between the averages of the players in weight according to the lines of play in the weight index. For the fat mass index, the value of significance was 0.528, which is greater than the level of significance which is 0.05, and therefore we reject the alternative hypothesis and accept the null hypothesis i.e. X1 = X2 = X3, and we conclude that there are no statistically significant differences between the averages of the players according to their lines in the fat mass index. In the bone mass index, the value of significance is 0.550, which is higher than 0.05, therefore we reject the alternative hypothesis and accept the null hypothesis i.e. X1 = X2 = X3 and conclude that there are no statistically significant differences between the averages of the players according to their lines in the bone mass index. For the index of muscle mass, the value of significance was 0.921 which is higher than the level of significance 0.05, therefore, we reject the alternative hypothesis and accept the null hypothesis i.e. X1 = X2 = X3, and conclude that there is no statistically significant differences between the averages of the players according to their lines in the muscle mass index. The results shown in Table (04) show the extent of the difference between players according to their lines in the functional indicators. Table 3: descriptive statistics for functional indicators for football players as playing lines functional N mean std deviation Std. 4. Presentation and discussion of the results 4. Presentation and discussion of the results European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 147 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane , , j DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES Table 1: Descriptive statistics for indicators of morphological football players as playing line Sum of squares df Mean squares F Sig Height Between groups .033 2 .017 4.388 .015 Within groups .328 87 .004 Total .361 89 Weight Between groups 119.868 2 59.934 .600 .551 Within groups 8696.445 87 99.959 Total 8816.313 89 Fat-Weight Between groups 82.464 2 41.232 .643 .528 Within groups 5580.824 87 64.147 Total 5663.288 89 Bone Weight Inter-groups .434 2 .217 .602 .550 Within groups 31.342 87 .360 Total 31.776 89 Muscle Weight Between groups .562 2 .281 .082 .921 Within groups 296.971 87 3.413 Total 297.533 89 Table 2: Comparison between football players as playing lines in morphological indicators using variance analysis Sum of squares df Mean squares F Sig Height Between groups .033 2 .017 4.388 .015 Within groups .328 87 .004 Total .361 89 Weight Between groups 119.868 2 59.934 .600 .551 Within groups 8696.445 87 99.959 Total 8816.313 89 Fat-Weight Between groups 82.464 2 41.232 .643 .528 Within groups 5580.824 87 64.147 Total 5663.288 89 Bone Weight Inter-groupes .434 2 .217 .602 .550 Within groups 31.342 87 .360 Total 31.776 89 Muscle Weight Between groups .562 2 .281 .082 .921 Within groups 296.971 87 3.413 Total 297.533 89 By analyzing the results of Table No. 2, we find that in the height index, the value of significance is 0.015, which is below the significance level of 0.05. Therefore we accept the alternative hypothesis and we reject the null hypotheses i.e. X1 ≠ X2 ≠ X3 and opean Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 148 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES , , j DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES conclude that there are statistically significant differences between the averages of the players according to their lines in the height index. European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 4. Presentation and discussion of the results error 95% confidence interval for mean Minimum Maximum Lower bound Upper bound Vital capacité Defense 30 4.1560 .60419 .11031 3.9304 4.3816 3.25 6.10 Midfield 30 4.0550 .81160 .14818 3.7519 4.3581 2.88 6.77 Attack 30 4.4367 1.02575 .18727 4.0536 4.8197 2.71 7.87 Total 90 4.2159 .83832 .08837 4.0403 4.3915 2.71 7.87 VO2max Defense 30 56.6100 3.98656 .72784 55.1214 58.0986 50.60 62.60 Midfield 30 55.6533 2.81532 .51400 54.6021 56.7046 50.60 60.40 Attack 30 54.6067 4.30733 .78641 52.9983 56.2150 50.60 62.60 Total 90 55.6233 3.80570 .40116 54.8262 56.4204 50.60 62.60 Ruffier Dickson Defense 30 5.8400 3.08652 .56352 4.6875 6.9925 1.20 12.40 Midfield 30 6.9467 2.94029 .53682 5.8487 8.0446 3.60 14.00 Attack 30 8.1500 2.66623 .48679 7.1544 9.1456 4.40 13.20 Total 90 6.9789 3.02282 .31863 6.3458 7.6120 1.20 14.00 Table 3: descriptive statistics for functional indicators for football players as playing lines functional 149 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES Table 4: comparison between football players by playing functional indicators lines using single contrast analysis Anova Sum of squares df Mean squares F Sig Vital capacity Between groups 2.346 2 1.173 1.695 .190 Within groups 60.201 87 .692 Total 62.547 89 VO2max Between groups 60.241 2 30.120 2.133 .125 Within groups 1228.780 87 14.124 Total 1289.021 89 Ruffier Dickson Between groups 80.088 2 40.044 4.752 .011 Within groups 733.142 87 8.427 Total 813.230 89 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES In the biomass index, the value of significance is 0.190, which is higher than the significance level of 0.05; therefore, we reject the alternative hypothesis and accept the null hypothesis i.e. X1 = X2 = X3, therefore, we conclude that there are no statistically significant differences between the averages of the players according to their lines in the biomass indicator. The maximum consumption index of oxygen (VO2max) was 125, which is higher than 0.05. Therefore, we reject the alternative hypothesis and accept the null hypothesis i.e. X1 = X2 = X3, and conclude that there are no statistically significant differences between the averages of the players according to their lines in the maximum oxygen consumption index (VO2max). 4. Presentation and discussion of the results For the retrieval capacity index through Ruffier test, the value of significance was 0.011, which is higher than the significance level of 0.05. Therefore, we accept the alternative hypothesis and reject the null hypothesis i.e. X1 ≠ X2 ≠ X3; and conclude that there are statistically significant differences between the averages of the players according to their lines in the index of the retrieval capacity. European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 5. Conclusions 1. There was a difference in the height indicator between the players according to the three lines of play (defense, midfield and attack) which was in favor of the attackers and then defenders and then midfielders. 2. There is no difference in level in the fat mass index between the players according to the three lines of play. 2. There is no difference in level in the fat mass index between the players according to the three lines of play. 3. There is no difference in level in the bone mass index between players according to the three lines of play. 4. There is no difference in level in the muscle mass index between the players according to the three lines of play. opean Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 150 , , j DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES 5. There is a difference in the retrieval capacity index between the players according to the three lines of play. 5. There is a difference in the retrieval capacity index between the players according to the three lines of play. 5. There is a difference in the retrieval capacity index between the players according to the three lines of play. 6. There is no difference in the level of the maximum consumption of oxygen among the players according to the three lines of play. 6. There is no difference in the level of the maximum consumption of oxygen among the players according to the three lines of play. 7. There is no difference in level in the vital capacity indicator between players by the three playing lines. 7. There is no difference in level in the vital capacity indicator between players by the three playing lines. , , j DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane 6.1 Discussion of the first hypothesis We conclude from our analysis of Table (02) that there is a difference between football players according to the three lines of play (defense, midfield and attack) in the height index where the value of the test indication level is 0.015 which is less than the null hypothesis significance level 0.05, therefore we accept the null hypothesis and reject the alternative hypothesis and conclude that there are statistically significant differences between the averages of the players according to their lines in the height indicator. This indicates that there are differences between players in height according to the requirements of the three lines and play centers, as the attackers over performed in height and then the defenders and then midfielders due to the requirements of the offensive roles and the imposition of winning air conflicts with the defenders, and the same imposed by the defensive roles with the attackers, as (Houar Abdelatif, 2015, p. 157) states that defenders and attackers are distinguished from their peers in height, because of the nature and role of the central defender and the attacker in winning the air balls, as confirmed by (Samir Chiban, 2010, p. 10) and (Duffour AB.RA, 1987) who argue that there is a difference in the morphological structure between the elite players and amateur players and that is part of the requirements of modern football and that every play center has its own Morphological profile. As for the other morphological indicators of fat, bone and muscle mass there is no difference in the level of players according to the lines of play, and this is due to the nature of the modern game, which has become comprehensive and requires the player to play defensive and offensive roles throughout the match, besides, the players execute the same training program, especially in the period of physical preparation, leading to the equality of the players in those indicators of fat and bone with superiority in the index of muscle mass, as indicated by (Mohamed Sobhi Hassanein, 1996) and (Derbal Fathi, 2014), as the players are characterized by a balanced muscle model that fits the game and its requirements. European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 151 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES 6.2 Discuss the second hypothesis We conclude from our analysis of Table (04) that there are statistically significant differences in the Ruffier test between players according to the three lines of defense (defense, midfield and attack). The test value is 0.011, therefore, we accept the null hypothesis and reject the alternative hypothesis, and conclude that there are statistically significant differences between the averages of the players according to their lines in the retrieval capacity index. This indicates that this aspect must be taken into account in the selection process, as attack and defense lines require physical readiness throughout the match time, especially the speed of retrieval in order to maintain the same performance, especially for the midfield players who over performed attackers and defenders in this index, as (Derbal Fathi, 2014) pointed out that the center of the game requires a constant move forward and backward and on both sides, which affects the physical and functional aspect of the player and which is reflected positively on the players’ performance. As for the index of maximum consumption of oxygen and the indicator of the dynamic capacity between the players according to their lines, there were no differences, and this shows that the requirements of the modern game has forced the players in the defense, midfield and attack to play defensive and offensive roles throughout the whole match, and the player should have sufficient functional presence in the maximum oxygen consumption ratio and in critical capacity no matter what type of position they occupy, because they are considered the direct source of energy during performance, as (Bishop et Edge, 2006) notes reporting from (Alexnder Dellal, 2008) that Vo2max affects the performance of all players directly, especially to stabilize the performance at short speeds, that is why we did not notice any differences in level between players according to the lines of play in these indicators, although the requirements of the role of midfield players require a significant presence in the vital capacity and in Vo2max according to what is indicated by (Cazorla G., 1996) and (Houar Abdelatif, 2015) 7. Recommendations  Selection of players according to the lines of play by the adoption of the height indicator in the morphological side.  Selection of players according to the lines of play by the adoption of the height indicator in the morphological side.  Selection of players according to the lines of play by the adoption of the retrieval indicator in the functional aspect.  Use other morpho-functional parameters with another sample when selecting according to play lines.  Use other morpho-functional parameters with another sample when selecting according to play lines. European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 152 Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES References 1. AKbobi Habib (2017), Physiological and morphological indicators when Algerian football players according to the level of their play stations. Algéria: Abdelhamid ibn Badis University. Mostaganem. 2. Alexnder Dellal (2008), Le football de l'entrainement à la performance. France: Amphora. Paris. 2. Alexnder Dellal (2008), Le football de l'entrainement à la performance. France: Amphora. Paris. 3. Ben Goua Ali (2004), Select standard levels for some basic skills when football players Youth. Algeria: Scientific journal for the science and techniques of physical and sports activities, page 1 Mostaganem. 3. Ben Goua Ali (2004), Select standard levels for some basic skills when football players Youth. Algeria: Scientific journal for the science and techniques of physical and sports activities, page 1 Mostaganem. 4. Cazorla G. (1996), Evaluation physique et physiologique du footballeurs et oriontation de sa preparation physique. France: France: Paris. 4. Cazorla G. (1996), Evaluation physique et physiologique du footballeurs et oriontation de sa preparation physique. France: France: Paris. 5. Derbal Fathi (2014), physical configuration and construction standards and its relationship with physical and functional requirements for football players as play centers. Algéria: Université Abdelhamid ibn Badis. Mostaganem. Algéria. 6. Duffour AB.RA. (1987). Profil morphologique des hand balleurs francais de haut niveau. France: Science et motricité. Paris. 6. Duffour AB.RA. (1987). Profil morphologique des hand balleurs francais de haut niveau. France: Science et motricité. Paris. 7. Houar Abdelatif (2015), Elaboration d'un programme informatique pour orienter les jeunes footballeur vers des compartiments de jeu à base de profile morphologique et des attributs de l'aptitude physique et technique. Algeria: Université Abdelhamide ibn badis, Mostaganem. 7. Houar Abdelatif (2015), Elaboration d'un programme informatique pour orienter les jeunes footballeur vers des compartiments de jeu à base de profile morphologique et des attributs de l'aptitude physique et technique. Algeria: Université Abdelhamide ibn badis, Mostaganem. 8. Mohamed Sobhi Hassanein (1996), Factor analysis of physical abilities in the areas of physical education and sports. Egypt: the Arab thought House-I 2. 9. Samir Chiban (2010), Les dimensions corporelles en tant que critére de sélection les jeunes footballeurs algériens de 15-16 ans. France: France: Université Claude Bernard, Lyon 1. 9. Samir Chiban (2010), Les dimensions corporelles en tant que critére de sélection les jeunes footballeurs algériens de 15-16 ans. France: France: Université Claude Bernard, Lyon 1. 10. www.radioalgerie.dz, 2017. (s.d.). 10. www.radioalgerie.dz, 2017. (s.d.). Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane 10. www.radioalgerie.dz, 2017. (s.d.). Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS Creative Commons licensing terms Authors will retain the copyright of their published articles agreeing that a Creative Commons Attribution 4.0 International License (CC BY 4.0) terms will be applied to their work. Under the terms of this license, no permission is required from the author(s) or publisher for members of the community to copy, distribute, transmit or adapt the article content, providing a proper, prominent and unambiguous attribution to the authors in a manner that makes clear that the materials are being reused under permission of a Creative Commons License. Views, opinions and conclusions expressed in this research article are views, opinions and conclusions of the author(s). Open Access Publishing Group and European Journal of Physical Education and Sport Science shall not be responsible or answerable for any loss, damage or liability caused in relation to/arising out of conflict of interests, copyright violations and inappropriate or inaccurate use of any kind content related or integrated on the research work. All the published works are meeting the Open Access Publishing requirements and can be freely accessed, shared, modified, distributed and used in educational, commercial and non- commercial purposes under a Creative Commons attribution 4.0 International License (CC BY 4.0). Chachou Seddaoui, Houar Abdelatif, Hadjar Mohamed Kherfane DEFINING THE MORPHO-FUNCTIONAL INDICATORS FOR THE SELECTION OF FOOTBALLERS ACCORDING TO THEIR PLAY LINES References European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 153 Creative Commons licensing terms Authors will retain the copyright of their published articles agreeing that a Creative Commons Attribution 4.0 International License (CC BY 4.0) terms will be applied to their work. Under the terms of this license, no permission is required from the author(s) or publisher for members of the community to copy, distribute, transmit or adapt the article content, providing a proper, prominent and unambiguous attribution to the authors in a manner that makes clear that the materials are being reused under permission of a Creative Commons License. Views, opinions and conclusions expressed in this research article are views, opinions and conclusions of the author(s). Open Access Publishing Group and European Journal of Physical Education and Sport Science shall not be responsible or answerable for any loss, damage or liability caused in relation to/arising out of conflict of interests, copyright violations and inappropriate or inaccurate use of any kind content related or integrated on the research work. All the published works are meeting the Open Access Publishing requirements and can be freely accessed, shared, modified, distributed and used in educational, commercial and non- commercial purposes under a Creative Commons attribution 4.0 International License (CC BY 4.0). Creative Commons licensing terms Creative Commons licensing terms Authors will retain the copyright of their published articles agreeing that a Creative Commons Attribution 4.0 International License (CC BY 4.0) terms will be applied to their work. Under the terms of this license, no permission is required from the author(s) or publisher for members of the community to copy, distribute, transmit or adapt the article content, providing a proper, prominent and unambiguous attribution to the authors in a manner that makes clear that the materials are being reused under permission of a Creative Commons License. Views, opinions and conclusions expressed in this research article are views, opinions and conclusions of the author(s). Open Access Publishing Group and European Journal of Physical Education and Sport Science shall not be responsible or answerable for any loss, damage or liability caused in relation to/arising out of conflict of interests, copyright violations and inappropriate or inaccurate use of any kind content related or integrated on the research work. References All the published works are meeting the Open Access Publishing requirements and can be freely accessed, shared, modified, distributed and used in educational, commercial and non- commercial purposes under a Creative Commons attribution 4.0 International License (CC BY 4.0). European Journal of Physical Education and Sport Science - Volume 3 │ Issue 10 │ 2017 154
https://openalex.org/W2017326446	https://sajs.co.za/article/download/3952/5623	English	null	Observed and modelled trends in rainfall and temperature for South Africa: 1960–2010	South African journal of science	2,014	cc-by	9,884	Climate trends in South Africa Climate trends in South Africa Research Article Page 1 of 13 KEYWORDS: rainfall; temperature; historical trends; station observations; climate models HOW TO CITE: MacKellar N, New M, Jack C. Observed and modelled trends in rainfall and temperature for South Africa: 1960–2010. S Afr J Sci. 2014;110(7/8), Art. #2013-0353, 13 pages. http://dx.doi.org/10.1590/ sajs.2014/20130353 Process-based global climate models (GCMs) are the primary tools used to make projections of future climate under scenarios of anthropogenic greenhouse gas forcing. Although these models generally have skill in replicating large-scale climate features, their coarse spatial resolution does not provide adequate information at local scales. Hence a variety of downscaling procedures, both empirical and dynamical, have been developed in order to represent finer spatial details.17-19 A question that is relevant in the context of historical trend analysis is whether or not the climate models (or downscaled products derived from these models) are able to replicate local trends in rainfall and temperature. When simulating the climatic effects of anthropogenic emissions, the only persistent temporal forcing applied to a GCM is an increasing concentration of greenhouse gases. If an observed historical trend in a climate variable, for example rainfall, is not replicated by such a GCM simulation, it could imply that the change is not a result of an enhanced greenhouse effect. This subject of detection and attribution of anthropogenic climate change has received considerable attention of late.20-22 Alternatively, disagreement between observed and modelled trends could be the result of either (1) weaknesses in the models’ representation of key processes at the regional or local scale or (2) uncertainties in the observational record. Both possibilities have implications for the interpretation of both past and future climate projections. In this paper, we present an analysis of climatic trends in rainfall and temperature indices for South Africa for the period 1960–2010 and thus provide an update that will complement previous trend analyses. We first present a review of previous climate trend studies in South Africa to provide a context for our analysis. We then summarise the important modes of variability that are associated with intraseasonal and interannual variations in South Africa’s climate and which can have a marked influence on trends calculated over a 50-year period. Finally, we describe the methodology for our trend analysis and present the results. Included in the analysis is a comparison of the observed trends with statistically downscaled climate model simulations. Observed and modelled trends in rainfall and temperature for South Africa: 1960–2010 AUTHORS: Neil MacKellar1 Mark New1 Chris Jack2 AUTHORS: Neil MacKellar1 Mark New1 Chris Jack2 Mark New1 Chris Jack2 AFFILIATIONS: 1African Climate and Development Initiative, University of Cape Town, Cape Town, South Africa 2Climate System Analysis Group, University of Cape Town, Cape Town, South Africa Observed trends in seasonal and annual total rainfall, number of rain days and daily maximum and minimum temperature were calculated for a number of stations in South Africa for the period 1960–2010. Statistically significant decreases in rainfall and the number of rain days are shown over the central and northeastern parts of the country in the autumn months and significant increases in the number of rain days around the southern Drakensberg are evident in spring and summer. Maximum temperatures have increased significantly throughout the country for all seasons and increases in minimum temperatures are shown for most of the country. A notable exception is the central interior, where minimum temperatures have decreased significantly. Regionally aggregated trends for six water management zones covering the entire country are not evident for total rainfall, but there are some significant trends for the number of rain days. Temperature in these zones has increased significantly for most seasons, with the exception of the central interior. Comparison of the observed trends with statistically downscaled global climate model simulations reveals that the models do not represent the observed rainfall changes nor the cooling trend of minimum temperature in the central interior. Although this result does not rule out the possibility of attributing observed local changes in rainfall to anthropogenically forced global change, it does have major implications for attribution studies. It also raises the question of whether an alternative statistical downscaling method or dynamical downscaling through the use of a regional climate model might better represent regional and local climatic processes and their links to global change. AFFILIATIONS: 1African Climate and Development Initiative, University of Cape Town, Cape Town, South Africa AFFILIATIONS: 1African Climate and Development Initiative, University of Cape Town, Cape Town, South Africa 2Climate System Analysis Group, University of Cape Town, Cape Town, South Africa 2Climate System Analysis Group, University of Cape Town, Cape Town, South Africa © 2014. The Authors. Published under a Creative Commons Attribution Licence. EMAIL: mark.new@acdi.uct.ac.za EMAIL: mark.new@acdi.uct.ac.za POSTAL ADDRESS: African Climate and Development Initiative, University of Cape Town, Private Bag X3, Rondebosch 7701, South Africa POSTAL ADDRESS: African Climate and Development Initiative, University of Cape Town, Private Bag X3, Rondebosch 7701, South Africa HOW TO CITE: MacKellar N, New M, Jack C. Observed and modelled trends in rainfall and temperature for South Africa: 1960–2010. S Afr J Sci. 2014;110(7/8), Art. #2013-0353, 13 pages. http://dx.doi.org/10.1590/ sajs.2014/20130353 Introduction Historical trends in climatic variables are of interest in a variety of academic disciplines and economic sectors, such as ecology, agriculture and water resource management. As a consequence, numerous studies1-16 have investigated climatic trends in South Africa, mostly focusing on station records of temperature and rainfall as well as various indices derived from these quantities. The greatest restriction to such studies of historical climate is the availability of long-term meteorological station observations that have sufficient coverage to give an adequate representation of a region’s climate. South Africa has a relatively good network of rainfall and temperature recording stations compared to the rest of Africa and much of the southern hemisphere,1-3 which makes it possible to investigate trends and variability over multiple decades. However, it is often difficult to detect clear signals of long-term change given large variability across a range of spatial and temporal scales. This difficulty is particularly relevant for rainfall, which is highly variable in both space and time, thus making any trend calculation sensitive to the specific location and period of the observations. Re-evaluation of trends as new and updated observational data sets become available is therefore necessary and allows for an assessment of how representative a trend over a given record is of underlying long-term climatic changes. DATES: DATES: Received: 14 Nov. 2013 Revised: 15 Jan. 2014 Accepted: 21 Jan. 2014 KEYWORDS: KEYWORDS: rainfall; temperature; historical trends; station observations; climate models KEYWORDS: rainfall; temperature; historical trends; station observations; climate models Timescales of climate variability and teleconnections Kruger and Shongwe15 examined the period 1960–2003 and found that, with a few exceptions, stations in South Africa have reported increases in annual mean temperatures, with strongest warming having occurred in the interior of the country and during autumn months. The stations showed mixed results with respect to dtr, with no clear regional pattern of change. New et al.6 also indicate varied results for changes in dtr over a similar period, but they do show a tendency for the cold extremes of tmin to change more strongly than the cold extremes of tmax. They also reveal a general increase in hot extremes over South Africa. The most recent published work on South African temperature trends was done by Kruger and Sekele16 for the period 1962–2009. They focused on extreme temperature indices for 28 stations and found significant changes in the exceedances of the extreme percentile values for tmin and tmax at many of the stations. More specifically, increases in daily measurements in excess of the 90th percentile of tmax and tmin have occurred along with decreases in exceedances of the 10th percentile of tmin and tmax. These occurrences are indicative of an overall increase in hot extremes and decrease in cold extremes, with the strongest changes tending to occur in the western and northern interior of the country. There is also a general tendency for stronger increases in the tmax indices than for those related to tmin.16 Complexities are also introduced through the influence of other climatic modes, and interactions between these modes. The second prominent interannual mode relevant for southern Africa is a dipole pattern in SST anomalies between the southwestern and southeastern Indian Ocean. A positive phase of this subtropical Indian Ocean dipole, characterised by anomalously warm SSTs in the western part of the basin, has been linked to increased summer rainfall over parts of southern Africa29-31 as well as extreme rainfall events in the region32. Increased SSTs in the southwest Indian Ocean have also been associated with an enhancement of the El Niño effect over South Africa.26 At an intraseasonal timescale, the Madden–Julian Oscillation (MJO) has a noticeable impact on South African convection and rainfall.33 The MJO is an eastward propagation of large-scale convective clusters in the tropics with a period of 30–60 days. Timescales of climate variability and teleconnections Climate exhibits numerous modes of variability in global and hemispheric circulation patterns at intraseasonal (of the order of 1 or 2 months) and interannual (year-to-year) timescales. The El Niño-Southern Oscillation (ENSO) is recognised as the leading mode of interannual variability in the tropics and is driven by variations in sea-surface temperatures (SSTs) in the equatorial Pacific Ocean. Links between ENSO and southern Africa’s rainfall have been established, such that warm ENSO events (El Niño) are commonly associated with below-average summer rainfall over much of South Africa and cold events (La Niña) are typified by above-average rainfall in this region. It has indeed been shown that severe summer drought in South Africa tends to occur under El Niño conditions24,25 – a relationship which seems to have strengthened since the 1970s26. Furthermore, seasonal prediction of summer rainfall in South Africa shows more skill during strong ENSO phases.27 However, the relationship between ENSO and South Africa's rainfall is far more complex than a simple linear association and there are many more factors that influence the region’s climate. For example, the frequency of synoptic-scale patterns of convection over South Africa are modulated by ENSO events, but different synoptic regimes under the same ENSO phase can result in substantially different rainfall responses.28 Long-term trends in temperature-related indices tend to manifest themselves more strongly than changes in rainfall indices. As global mean temperature has been observed to increase over the last century, which is largely attributed to the warming effects of anthropogenic greenhouse gas emissions,23 so different regions have experienced changes of varying magnitude. In South Africa between 1950 and 1993, Easterling13 found an increase in annual mean daily maximum temperature (tmax) and widespread increases – although also some decreases – in annual mean daily minimum temperature (tmin). Despite a global tendency for a reduction in the diurnal temperature range (dtr), which is the difference between tmax and tmin, the results of Easterling13 show much of South Africa experienced an increase in dtr. In contrast, however, Hulme et al.14 show decreased dtr over South Africa during the 1950s and 1960s. They also show very strong warming in the central interior of southern Africa and in fact a cooling over the coastal regions of South Africa for the 1901–1995 period. It is not clear what the cause of this cooling may be. Review of climate trend studies in South Africa Increased dry spell duration is also evident for much of the Free State and Eastern Cape, and decreases in wet spell duration have been observed for parts of the Eastern Cape and the northeastern parts of South Africa during 1910–2004.8 studies. This discord can to a large extent be attributed to two factors: the period over which the data were analysed and the locations of the stations from which data were obtained. Large, naturally occurring variations in climate at yearly and decadal timescales can greatly affect the calculation of trends, so it is important to consider the length of record when evaluating any trend analysis. Regional inferences based on individual stations are reliant on how representative a station or group of stations is of that area and should also be treated with caution. Some studies also rely on gridded products where station records have been interpolated in space onto a continuous surface. Such products should closely match the raw station data in places where the observational record is good, but in data-sparse regions this information is less reliable, especially where strong environmental gradients exist. Details of the methods used to calculate trends also differ between studies, but these should rarely result in substantially different results. Further factors that can influence results are data quality and quality-control measures. Review of climate trend studies in South Africa © 2014. The Authors. Published under a Creative Commons Attribution Licence. Because South Africa’s mean annual precipitation (MAP) is highly variable from year to year4,5, few spatially coherent or statistically significant trends in this quantity have been observed6-8. However, of more relevance than MAP are the characteristics of how rainfall is distributed throughout the year. These characteristics include the timing of the Volume 110 \| Number 7/8 July/August 2014 Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za South African Journal of Science http://www.sajs.co.za 1 Climate trends in South Africa Research Article Page 2 of 13 onset and end of the rainy season, the typical durations of wet and dry periods and the occurrence of extreme heavy rainfall events. A review by Easterling et al.2 indicates a tendency for increased extreme precipitation in the southwestern and eastern parts of South Africa during most of the 20th century. In agreement with these observations are results from Groisman et al.9 who show a significant increase in the annual frequency of very heavy rainfall events over eastern South Africa from 1906 to 1997. Furthermore, Mason et al.10 demonstrate increases in the intensity of high rainfall events in the 1961–1990 period relative to 1931–1960 over much of South Africa. Kruger8 shows increases in extreme rainfall indices over the southern Free State and parts of the Eastern Cape from 1910 to 2004. New et al.6 also show some evidence for increased rainfall extremes over parts of South Africa for the 1961–2000 period. Nel7 demonstrated a shift in seasonality for stations in the KwaZulu-Natal (KZN) Drakensberg for 1955–2000. Here MAP showed no significant trend, but an increase in summer rainfall, accompanied by decreased autumn and winter rainfall, resulted in a shorter wet season and a more pronounced seasonal cycle. These findings are consistent with results from Thomas et al.11 for northwest KZN, which show an increase in early-season rainfall along with a decrease in late-season rainfall between 1950 and 2000. Seasonal shifts were also observed in Limpopo for the same period, where there has been a tendency for a later seasonal rainfall onset accompanied by increased dry spells and fewer rain days.11 A trend toward later onset of rainfall in Limpopo between 1979 and 1997 was also identified by Tadross et al.12, but they note that this trend is likely part of low-frequency variability rather than long-term change. South African Journal of Science http://www.sajs.co.za Data and methods The probability of this trend occurring by chance is also estimated, from which a measure of statistical significance can be assigned. We used a 5% level of significance, such that if the probability estimate was less than 0.05, the trend was deemed to be significant. Because tau does not provide an estimate of the absolute magnitude of the trend, we also calculated the slope of the trend using Sen’s slope estimator, which is the median of the slopes calculated between all pairs of data points in the series. Like the Mann–Kendall test, this method is also statistically robust and insensitive to outliers in the data. The above trend estimates were calculated for seasonal and annual means for all indices. The results are presented as follows. Firstly, annual and seasonal maps of the Mann–Kendall tau for each station and each variable are shown. These maps depict the relative strength of trends over the historical period at each station and give an indication of any coherent spatial patterns of change. Secondly, annual and seasonal time series plots were generated for six water management zones covering all of South Africa. The zones are those used in the LTAS programme for the assessment of climate change on water resources.40 To calculate these regional time series, an anomaly time series was computed for each station falling within the region by subtracting the 1960–2010 station mean from each value in the series. The resulting series was then summed and divided by the number of stations in that region, after which trend statistics were calculated for the regional mean series. A smoothed curve was added to the figures using a Loess filter with a bandwidth of 0.25. The resulting figures give an indication of the direction and magnitude of long-term trends as well as illustrate interannual and interdecadal variability in the time series. Two important points should be noted here. Firstly, although the definition of the zones has value in a hydro-climatic context, the zones do not necessarily coincide with homogeneous climates and climatic trends. Opposing trends at stations within the same zone will hence weaken any regional signal. Secondly, the stations falling within a particular zone may not necessarily be a good representation of spatial heterogeneity within that zone. This point is particularly true for indices related to rainfall and for zones in which station coverage is sparse. Data and methods Station observations were obtained though the Climate Information Portal (CIP) hosted by the University of Cape Town’s Climate System Analysis Group (http://cip.csag.uct.ac.za). The data originate from two main sources – the Computing Centre for Water Resources and the South African Weather Service – and were collated and quality controlled prior to being uploaded to CIP. Stations were selected based on their coverage of monthly data for the period 1960–2010 such that any station with 20% or more missing values was excluded from the analysis. Based on this condition, data from 73, 30 and 27 stations were available for rainfall (ppt), tmax and tmin, respectively. The indices presented in this paper are limited to ppt, number of rain days, tmax and tmin, but analysis was also performed for extreme values of rainfall and temperature. Results for the full set of indices are presented in the Long-Term Adaptation Scenarios Flagship Research Programme (LTAS) technical report on climate trends and scenarios for South Africa.39 Table 1: Global climate models used in the study Table 1: Global climate models used in the study Model name Institute BCC-CSM1 Beijing Climate Center, China Meteorological Administration BNU-ESM Beijing Normal University CanESM2 Canadian Centre for Climate Modelling and Analysis CNRM-CM5 Centre National de Recherches Météorologiques / Centre Européen de Recherches et Formation Avancées en Calcul Scientifique FGOALS-s1 Institute of Atmospheric Physics, Chinese Academy of Sciences GFDL-ESM2G Geophysical Fluid Dynamics Laboratory GFDL-ESM2M Geophysical Fluid Dynamics Laboratory MIROC5 Atmosphere and Ocean Research Institute (University of Tokyo), National Institute for Environmental Studies and Japan Agency for Marine-Earth Science and Technology MIROC-ESM-CHEM Atmosphere and Ocean Research Institute (University of Tokyo), National Institute for Environmental Studies and Japan Agency for Marine-Earth Science and Technology MIROC-ESM Atmosphere and Ocean Research Institute (University of Tokyo), National Institute for Environmental Studies and Japan Agency for Marine-Earth Science and Technology MRI-CGCM3 Meteorological Research Institute, Japan Trends in the indices for the 1960–2010 period were evaluated using a number of approaches. Firstly, the non-parametric Mann–Kendall trend test was applied. This method has the advantages of making no assumptions about the distribution of the underlying data and being relatively insensitive to outliers. In this method, a correlation coefficient, tau, is computed, which has a value between -1 and 1 and denotes the relative strength of the trend in a time series. Climate trends in South Africa Climate trends in South Africa Research Article Page 3 of 13 proxy records extending back as far as 600 years.36 The cause of this oscillation is not clear, but an ‘ENSO-like’ multidecadal pattern of variation has been identified at multiple periodicities.37 Interaction between phases of the multidecadal and interannual variations act to enhance or mitigate regional responses.37,38 In the context of the trend analysis presented in this study, it is very important to consider the possible influences of low- frequency variations on the calculation of long-term trends. To compare the observed trends for the six water management zones to climate model simulations for the same period, results from 11 different GCM simulations were used. The GCM simulations were obtained from the Coupled Model Intercomparison Project Phase 5 (CMIP5) and have been statistically downscaled to the same stations used in the trend analysis. Although more model simulations exist in the CMIP5 archive, we have limited our analysis to those models that are currently available on CIP and have been downscaled to the relevant stations. The GCMs used are listed in Table 1. The method of downscaling is described in Hewitson and Crane17 and the data are publicly available on CIP. Trends for 1960–2010 were computed using Sen’s slope estimator for each downscaled GCM, aggregated over the six zones. Data and methods Nevertheless, some useful information can be extracted from these spatially averaged indices. Observed trends for 1960–2010 The results for ppt, rain days, tmax and tmin are presented in Figures 1 to 8. The main findings that emerged from the analysis are summarised below for each of the six water management zones. Zone 1: Limpopo and parts of northern Mpumalanga South African Journal of Science http://www.sajs.co.za Timescales of climate variability and teleconnections It has been shown that convection over South Africa tends to be more strongly affected by the MJO during warm phases of ENSO and warm tropical Indian Ocean temperatures, such that intraseasonal variability is higher and convection is less active during El Niño events.33 A further low-frequency mode that is present in the mid-latitudes is the Antarctic Oscillation (AAO), which is defined by pressure anomalies between Antarctica and the southern hemisphere mid-latitudes. There is some indication of a link between a positive phase of the AAO and enhanced rainfall over central South Africa, which tends to be stronger during La Niña years.34 It has also been shown for the winter rainfall region of the southwestern Cape that particularly wet winters are associated with a negative phase of the AAO and vice versa.35 Beyond the interannual timescale are decadal-scale variations in climate which provide a slowly evolving background around which higher- frequency modes oscillate. For example, an approximately 18-year cycle in southern African rainfall has been identified in instrumental and Although some general tendencies are apparent for trends in both rainfall and temperature indices, there is some disagreement between Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 2 Climate trends in South Africa Zone 2: Majority of KwaZulu-Natal and part of southern Mpumalanga All but one station show significant increases in tmax (Figure 3), with the strongest warming signal occurring in June–August (JJA) and the weakest in DJF. The strongest warming of tmin occurs in DJF and JJA (Figure 4). Regionally aggregated ppt time series for this zone (Figure 5, top row) show large interannual and decadal-scale variability and no significant trends, but significant reductions occur in the number of rain days in DJF and MAM and in the annual mean (Figure 6, top row). The change in annual mean rain days translates to nearly 16 days over the 50-year period. Significant increases occur in tmax in JJA (0.022 °C/ year) and in the annual mean (0.018 °C/year) (Figure 7, top row), which represent approximately a 1 °C increase over the 50-year period. southern Mpumalanga A consistent spatial pattern of decreased ppt is shown for MAM (Figure 1), but the trends are significant at only three stations. In the southern part of the region in SON, a cluster of stations show increased ppt, but none of these trends is significant. Many stations indicate significant changes in rain days, but spatial coherence is weak (Figure 2). There is a suggestion, however, that coastal stations have experienced decreased rain days, whereas inland locations have experienced increases. A cluster of stations in the southern Drakensberg area, which also extends into Zone 5, shows significant increases in rain days in SON and DJF. 4 Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za Figure 1: Trends in annual and seasonal mean rainfall (ppt) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. Annual mean DJF MAM 1960-2010 filled symbol indicates trend is significant at 5% level ppt JJA SON Annual mean 1960-2010 filled symbol indicates trend is significant at 5% level ppt Annual mean Annual mean Figure 1: Trends in annual and seasonal mean rainfall (ppt) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. DJF MAM JJA SON DJF JJA MAM SON DJF DJF JJA SON SON JJA Figure 1: Trends in annual and seasonal mean rainfall (ppt) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. South African Journal of Science http://www.sajs.co.za Zone 1: Limpopo and parts of northern Mpumalanga There is a mixed signal in the spatial distribution of changes in rainfall indices in most seasons, but a tendency for reductions in ppt (Figure 1) at most stations in March–May (MAM), although this reduction is significant at only two stations. A cluster of stations in the Lowveld indicate increased ppt for MAM, but although there is some spatial coherence, the trends are small and not statistically significant. More pronounced trends are seen in the number of rain days (Figure 2), for which a number of stations show significant decreases in the September–November (SON), December–February (DJF) and MAM seasons. Two pairs of nearby stations exhibit contrasting significant trends in rain days. This South African Journal of Science http://www.sajs.co.za South African Journal of Science http://www.sajs.co.za 3 Volume 110 \| Number 7/8 July/August 2014 Volume 110 \| Number 7/8 July/August 2014 Climate trends in South Africa Research Article Page 4 of 13 Changes in tmin are generally smaller, with a 0.011 °C/year increase in annual mean (Figure 8, top row). Changes in tmin are generally smaller, with a 0.011 °C/year increase in annual mean (Figure 8, top row). result could be because of localised environmental conditions interacting in different ways with larger-scale rainfall processes, but a more detailed study of these locations is required to investigate this result further. southern Mpumalanga 4 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 Volume 110 \| Number 7/8 July/August 2014 Climate trends in South Africa Research Article Page 5 of 13 Figure 2: Trends in annual and seasonal rain days (days) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. Annual mean DJF MAM 1960-2010 filled symbol indicates trend is significant at 5% level rain_days JJA SON Annual mean 1960-2010 filled symbol indicates trend is significant at 5% level rain_days Annual mean DJF Annual mean DJF JJA MAM SON MAM SON DJF MAM SON JJA SON JJA JJA Figure 2: Trends in annual and seasonal rain days (days) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. Figure 2: Trends in annual and seasonal rain days (days) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. Data from only three temperature stations were available for this zone, which all show positive trends for tmax (Figure 3) and tmin (Figure 4), but not all of these trends are significant. No regionally averaged trends are seen in ppt (Figure 5, second row), but overall decreases in rain days are shown in DJF and MAM and in the annual mean (Figure 6, second row). It must be noted, however, that spatial variability in the number of rain day trends for this zone is high, which the regional average does not take into account. southern Mpumalanga The average increase in tmax for the three stations in this zone is highest for the MAM season (0.02 °C/year) and is 0.012 °C/ year for the annual mean (Figure 7, second row). The increase in annual mean tmin is 0.014 °C/year (Figure 8, second row). South African Journal of Science http://www.sajs.co.za Zone 3: Northern and central interior Opposing signals are shown at individual stations for ppt (Figure 1), with a rough distinction between increased rainfall in the west and decreased rainfall in the east in DJF. There is, however, a general tendency for South African Journal of Science http://www.sajs.co.za South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 Volume 110 \| Number 7/8 July/August 2014 5 Climate trends in South Africa Research Article Page 6 of 13 Annual mean DJF MAM 1960-2010 filled symbol indicates trend is significant at 5% level tmax JJA SON Annual mean 1960-2010 filled symbol indicates trend is significant at 5% level tmax Figure 3: Trends in annual and seasonal mean daily maximum temperature (tmax, °C) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. DJF MAM JJA SON DJF MAM DJF MAM SON JJA JJA Figure 3: Trends in annual and seasonal mean daily maximum temperature (tmax, °C) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. 6 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 Volume 110 \| Number 7/8 July/August 2014 Climate trends in South Africa Research Article Page 7 of 13 Annual mean DJF MAM 1960-2010 filled symbol indicates trend is significant at 5% level tmin JJA SON Annual mean 1960-2010 filled symbol indicates trend is significant at 5% level tmin Annual mean Figure 4: Trends in annual and seasonal mean daily minimum temperature (tmin, °C) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Zone 3: Northern and central interior Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. DJF MAM 5% level JJA SON DJF MAM DJF MAM SON JJA JJA Figure 4: Trends in annual and seasonal mean daily minimum temperature (tmin, °C) for each station according to the Mann–Kendall test. The value of tau represents the direction and relative strength of the trend. Shaded symbols denote trends that are significant at the 5% level. Seasons are summer (DJF), autumn (MAM), winter (JJA) and spring (SON). Grey borders represent boundaries of the six water management regions, which are identified by number (1–6) in the annual mean map. South African Journal of Science http://www.sajs.co.za 7 7 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 Volume 110 \| Number 7/8 July/August 2014 Research Article Page 8 of 13 Climate trends in South Africa Figure 5: Regional mean time series and trends in total rainfall (ppt) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. Figure 5: Regional mean time series and trends in total rainfall (ppt) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. Figure 5: Regional mean time series and trends in total rainfall (ppt) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. decreased ppt in MAM. The pattern is similar for the number of rain days (Figure 2), but more stations show significant decreases for MAM. this range is sparse. Regional means should therefore be interpreted with caution. Nevertheless, there are no trends in the aggregated time series of rainfall indices (Figures 5 and 6, fourth row). Increases in tmax occurred for all seasons, with the strongest trends seen in JJA (Figure 3). There is, however, a general tendency for reductions in tmin in all seasons (Figure 4). 7 Trends in regional mean ppt are not evident (Figure 5, third row), but decreases in rain days in MAM and in the annual mean of about 6 and 13 days, respectively, are seen over the 50-year period (Figure 6, third row). Strong warming trends in tmax of almost 2 °C (0.034 °C/year) in MAM and almost 1.5 °C (0.029 °C/year) in JJA are evident (Figure 7, third row). The decreases in tmin as seen at individual stations are not reflected in the aggregated results (Figure 8, third row). Tmax shows large significant increases of between 0.025 °C/year and 0.039 °C/year in all seasons, largely as a result of persistently above- average temperatures in the last 10 years of the record (Figure 7, fourth row). Increases in tmin are weaker than those for tmax, ranging from 0.007 °C/year to 0.019 °C/year (Figure 8, fourth row). Zone 5: Majority of Eastern Cape and southern part of South African Journal of Science http://www.sajs.co.za KwaZulu-Natal Changes in ppt are weak (Figure 1), but there are some significant increases in rain days across the region (southern Drakensberg and southern coastal areas) in all seasons (Figure 2). A single station on the northern coast, however, shows a significant reduction in rain days. Zone 4: Northern Cape, southern Free State and parts of Zone 4: Northern Cape, southern Free State and parts of Eastern Cape Trends in rainfall indices are generally not significant and show little consistency across the region (Figures 1 and 2). The number of rain days do, however, indicate drier conditions along the southern coastal regions, although the stations near the west coast show a tendency for increased rain days. Tmax and tmin have increased significantly at almost all stations in all seasons (Figures 3 and 4). The regionally aggregated ppt time series show no significant changes (Figure 5, bottom row), but rain days have decreased significantly in DJF (2.5 days) and MAM (3.5 days) and the annual mean has decreased significantly by 11.3 days over the 50-year record (Figure 6, bottom row). Significant increases in tmax are seen for all seasons and range from 0.015 °C/year to 0.027 °C/year, with strong warming occurring in the last 10–12 years of the record (Figure 7, bottom row). Increases in tmin are mostly smaller (0.011 °C/year to 0.021 °C/year) and are not significant for JJA (Fugure 8, bottom row). Zone 4: Northern Cape, southern Free State and parts of Figure 6: Regional mean time series and trends in number of rain days (days) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. Figure 6: Zone 4: Northern Cape, southern Free State and parts of Trends in ppt are weak for all stations (Figure 1), but some stations show significant increases in rain days in SON, DJF and JJA and one station shows significant decreases in rain days in MAM and SON (Figure 2). Data available from four stations for tmin indicate mostly significant positive trends (Figure 3) but three of the stations show weaker trends, particularly in JJA and SON (Figure 4). This zone is somewhat problematic as it spans a large climatic range and station coverage within Stations with temperature data are confined to the southern part of the region, where tmax and tmin have generally increased in all seasons (Figures 3 and 4). Regional means show no significant rainfall changes (Figures 5 and 6, fifth row). Significant increases in tmax (from 0.017 °C/ year to 0.03 °C/year) have occurred in all seasons except DJF (Figure 7, fifth row). Aggregated increases in tmin are generally weaker than for tmax, ranging from 0.008 °C/year to 0.014 °C/year (Figure 8, fifth row). Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 8 Climate trends in South Africa Research Article Page 9 of 13 Figure 6: Regional mean time series and trends in number of rain days (days) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. Figure 6: Regional mean time series and trends in number of rain days (days) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. Zone 6: Western Cape and parts of Northern and Eastern Cape from the analysis of observations. Both observed and modelled results represent the same stations and same period, so a direct comparison can be made between the two. Some interesting points can be made. The reductions in ppt for MAM that are evident in the observed trends for almost all regions is not captured by the models. In fact, the models tend to show an opposite trend. Similarly for rain days, the negative trends in Zones 1, 2, 3 and 6 fall outside the range of model simulations. In contrast, for SON, where observed trends are weak, the models show a tendency for reduced ppt in all regions. Strong trends in observed annual rain days lie well outside the model ranges. Overall, the observed trends in rainfall indices are poorly represented by the model simulations. Observed temperature trends generally fall within the range of model simulations, with the notable exception of tmin in Zone 3. Zone 3 is the region for which decreases in tmin were observed and it is interesting that none of the models is able to simulate this decrease. Tendencies for the models to over- or underestimate trends in tmin and tmax vary according to season, region and variable, but some consistencies are apparent – for example, a general underestimation of tmax in MAM and JJA, but an overestimation for both tmin and tmax in DJF. Comparison of model and observed trends for 1960–2010 Figure 9 shows a comparison of observed versus modelled trends aggregated for each of the six zones. This comparison gives an indication of whether or not the models captured the long-term trends that emerged Rainfall indices are particularly influenced by multi-year variations and therefore are highly sensitive to the temporal coverage of observations used. As expected, we demonstrated weak or non- 9 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 Climate trends in South Africa Research Article Page 10 of 13 Figure 7: Regional mean time series and trends in mean daily maximum temperature (tmax, °C) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. Figure 7: Regional mean time series and trends in mean daily maximum temperature (tmax, °C) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. existent trends in regionally aggregated ppt, which is consistent with previous analyses.6-8 More pronounced trends are seen at individual stations, with the most spatially coherent result being an overall tendency for decreased ppt in MAM and a reduction in rain days over the central and northeastern parts of the country. Our results also show a strong and cohesive tendency toward increased rain days, and to a lesser degree ppt, around the southern Drakensberg in DJF and SON. This summer increase is consistent with results previously shown for this location,7 and the springtime increase is suggestive of an earlier seasonal onset. South African Journal of Science http://www.sajs.co.za Comparison of model and observed trends for 1960–2010 Although other authors9,10,13 have reported increases in rainfall extremes during the 20th century for eastern South Africa, our analysis of the 90th percentile rainfall events (not shown in this paper) do not show much spatial coherence in trends, except for MAM, for which widespread decreases have occurred in line with the decreases shown for ppt and rain days. that it experienced a cooling trend in tmin, thus resulting in an increased diurnal temperature range (dtr). The reasons for this finding have not been rigorously explored in this study, but it is possibly related to a reduction in nocturnal cloud cover, more stable ambient atmospheric conditions, or both. Decreases in the number of rain days for much of this region suggest that cloud cover may indeed have been reduced, which would allow for greater radiative cooling of the surface at night. The cause of a reduction in cloud cover could in turn be an enhancement of the mid-tropospheric high pressure system as demonstrated by Engelbrecht et al.41 for a warming climate. Even in the absence of a change in cloud cover, an increase in atmospheric subsidence can promote stable conditions and the formation of nocturnal temperature inversions42 which trap cold air near the surface. We extended our analysis by comparing the observed trends to simulated trends for the same stations and same period from 11 downscaled GCMs. This analysis revealed stark differences in the modelled and observed trends in rainfall indices, but closer agreement for temperature indices. One possible reason for the disagreement in rainfall trends is that the observed trends are not attributable to the effects of increased radiative forcing from greenhouse gases. Although we did not set out to make any robust statements regarding attribution, our findings are consistent For the temperature indices, a significant warming trend in tmax is shown for almost all stations, which is in line with recent global and regional warming trends.23 The strongest regionally averaged increase in tmax over the 50-year analysis period reached close to 2 °C in the central interior in autumn, whereas the weakest increase (0.35 °C) occurred in the same region in summer. An interesting result for the central interior is One possible reason for the disagreement in rainfall trends is that the observed trends are not attributable to the effects of increased radiative forcing from greenhouse gases. Comparison of model and observed trends for 1960–2010 Although we did not set out to make any robust statements regarding attribution, our findings are consistent 10 Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 Climate trends in South Africa Research Article Page 11 of 13 Figure 8: Regional mean time series and trends in mean daily minimum temperature (tmin, °C) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. Figure 8: Regional mean time series and trends in mean daily minimum temperature (tmin, °C) for stations in the six water management zones for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Grey bars represent departures from the 1960–2010 mean for each year. Black curves are a Loess smoothing of the yearly data with a bandwidth of 0.25. Trend lines are shown for the Sen’s slope estimate. Solid trend lines indicate the trend is significant at the 5% level and dashed lines are not significant at this level. with those of Hoerling et al.20 who found similar inconsistencies between observed and GCM-simulated rainfall in southern Africa. Other studies, however, do find a detectable anthropogenic signal in rainfall at larger scales.21,22 Other potential causes of the discrepancies lie in either the GCM formulations themselves, or in the method that has been used to downscale the GCM results to the individual stations. As the downscaling method does not directly use the modelled rainfall, but rather a selection of variables representing regional atmospheric circulation (eg. wind fields and temperature lapse rate),17 these poor results cannot be blamed on the GCM formulation of cloud and rainfall processes. Likely causes are rather (1) poorly replicated atmospheric circulation patterns by the GCMs, (2) an inadequate empirical model linking the circulation fields to local-scale rainfall, or (3) differences in the interannual and decadal- scale temporal variations in climate. Comparison of model and observed trends for 1960–2010 The latter point can be interpreted as either the models’ inability to adequately represent the regional impact of global climate phenomena such as ENSO, or simply that the temporal evolution of low-frequency climate variations differs markedly from observed (in a freely evolving long-term climate simulation, such variability should not be expected to match observed). In any case, this disagreement between models and observations has implications for assessing projections of future climate over South Africa. Despite the challenges of identifying (and modelling) long-term trends in rainfall over South Africa, there is nevertheless a clear signal of increased temperatures since 1950. This finding has important implications for the functioning of natural systems and related societal impacts and sets a precedent for likely future changes resulting from further anthropogenic global warming. Acknowledgements The work presented in this paper forms part of the Long-Term Adaptation Scenarios Flagship Research Programme which is supported by the Department of Environmental Affairs of South Africa, the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety and the South African National Biodiversity Institute. We also thank two anonymous reviewers for their suggestions to improve the manuscript. South African Journal of Science http://www.sajs.co.za Authors’ contributions N.M. wrote the manuscript, performed the analysis and produced the figures; M.N. provided conceptual oversight; and C.J. provided conceptual input and technical support. Volume 110 \| Number 7/8 July/August 2014 Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za 11 Climate trends in South Africa Research Article Page 12 of 13 Figure 9: Correspondence between observed trends and downscaled global climate model trends for 1960–2010. Trends are averages for stations in each of the six water management regions (abscissa) for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Asterisks show observed trend and box-and-whisker plots represent the 11 downscaled model trends. Black circles indicate models lying outside 1.5 times the interquartile range. ppt [mm/year] rain_days [days/year] tmin [degC/year] tmax [degC/year] Ann SON JJA MAM DJF MAM JJA SON SON Ann Figure 9: Correspondence between observed trends and downscaled global climate model trends for 1960–2010. Trends are averages for stations in each of the six water management regions (abscissa) for summer (DJF), autumn (MAM), winter (JJA), spring (SON) and annual (Ann) means. Asterisks show observed trend and box-and-whisker plots represent the 11 downscaled model trends. Black circles indicate models lying outside 1.5 times the interquartile range. 4. Tyson PD. Climatic change and variability in southern Africa. 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Int J Climatol. 2001;21(15):1889–1922. http://dx.doi. org/10.1002/joc.680 12 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 Volume 110 \| Number 7/8 July/August 2014 Climate trends in South Africa Climate trends in South Africa 8. Kruger AC. Observed trends in daily precipitation indices in South Africa: 1910–2004. Int J Climatol. 2006;26(15):2275–2285. http://dx.doi. org/10.1002/joc.1368 26. Richard Y, Trzaska S, Roucou P, Rouault M. Modification of the southern African rainfall variability/ENSO relationship since the late 1960s. Clim Dynam. 2000;16(12):883–895. http://dx.doi.org/10.1007/s003820000086 9. Groisman PY, Knight RW, Easterling DR, Karl TR, Hegerl GC, Razuvaev VN. Trends in intense precipitation in the climate record. J Climate. 2005;18(9):1326–1350. http://dx.doi.org/10.1175/JCLI3339.1 27. Landman WA, Beraki A. Multi-model forecast skill for mid-summer rainfall over southern Africa. 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Tadross MA, Hewitson BC, Usman MT. The interannual variability of the onset of the maize growing season over South Africa and Zimbabwe. J Climate. 2005;18(16):3356–3372. http://dx.doi.org/10.1175/JCLI3423.1 30. Reason CJC. Subtropical Indian Ocean SST dipole events and southern African rainfall. Geophys Res Lett. 2001;28(11):2225–2227. http://dx.doi. org/10.1029/2000GL012735 13. Easterling DR. Maximum and minimum temperature trends for the globe. Science. 1997;277(5324):364–367. http://dx.doi.org/10.1126/ science.277.5324.364 31. Hansingo K, Reason CJC. Modelling the atmospheric response to SST dipole patterns in the South Indian Ocean with a regional climate model. Meteorol Atmos Phys. 2008;100(1–4):37–52. http://dx.doi.org/10.1007/s00703- 008-0294-7 14. Hulme M, Doherty R, Ngara T, New M, Lister D. African climate change: 1900– 2100. Clim Res. 2001;17:145–168. http://dx.doi.org/10.3354/cr017145 32. Washington R, Preston A. Extreme wet years over southern Africa: Role of Indian Ocean sea surface temperatures. J Geophys Res [serial on the Internet]. 2006 [cited 2014 Jan 10];111(D15). Available from: http://doi. wiley.com/10.1029/2005JD006724 15. Kruger AC, Shongwe S. Temperature trends in South Africa: 1960-2003. Int J Climatol. 2004;24(15):1929–1945. http://dx.doi.org/10.1002/joc.1096 16. Kruger AC, Sekele SS. Trends in extreme temperature indices in South Africa: 1962-2009. Int J Climatol. 2013;33(3):661–676. http://dx.doi.org/10.1002/ joc.3455 33. Pohl B, Richard Y, Fauchereau N. Influence of the Madden–Julian Oscillation on southern African summer rainfall. J Climate. 2007;20(16):4227–4242. http://dx.doi.org/10.1175/JCLI4231.1 17. Hewitson BC, Crane RG. Consensus between GCM climate change projections with empirical downscaling: Precipitation downscaling over South Africa. Int J Climatol. 2006;26(10):1315–1337. http://dx.doi.org/10.1002/joc.1314 34. Pohl B, Fauchereau N, Reason CJC, Rouault M. Relationships between the Antarctic Oscillation, the Madden–Julian Oscillation, and ENSO, and consequences for rainfall analysis. J Climate. 2010;23(2):238–254. http:// dx.doi.org/10.1175/2009JCLI2443.1 18. Benestad RE. Empirical-statistical downscaling in climate modeling. Eos, Trans Am Geophys Union. 2004;85(42):417. http://dx.doi.org/ 10.1029/2004EO420002 35. Reason CJC, Rouault M. Links between the Antarctic oscillation and winter rainfall over western South Africa. Geophys Res Lett. 2005;32:L007705 19. Schmidli J, Goodess CM, Frei C, Haylock MR, Hundecha Y, Ribalaygua J, et al. Statistical and dynamical downscaling of precipitation: An evaluation and comparison of scenarios for the European Alps. J Geophys Res [serial on the Internet]. 2007 Feb 20 [cited 2013 Nov 07];112(D4). Available from: http:// doi.wiley.com/10.1029/2005JD007026 36. Tyson PD, Cooper GRJ, McCarthy TS. Millennial to multi-decadal variability in the climate of southern Africa. Int J Climatol. References 2002;22(9):1105–1117. http:// dx.doi.org/10.1002/joc.787 37. Reason CJC, Rouault M. ENSO-like decadal variability and South African rainfall. Geophys Res Lett. 2002;29(13):161–164. http://dx.doi. org/10.1029/2002GL014663 20. Hoerling M, Hurrell J, Eischeid J, Phillips A. Detection and attribution of twentieth-century northern and southern African rainfall change. J Climate. 2006;19(16):3989–4008. http://dx.doi.org/10.1175/JCLI3842.1 38. Kruger AC. The influence of the decadal-scale variability of summer rainfall on the impact of El Niño and La Niña events in South Africa. Int J Climatol. 1999;19(1):59–68. http://dx.doi.org/10.1002/(SICI)1097- 0088(199901)19:1<59::AID-JOC347>3.0.CO;2-B 21. Stott PA, Gillett NP, Hegerl GC, Karoly DJ, Stone DA, Zhang X, et al. Detection and attribution of climate change: A regional perspective. Wiley Interdisciplinary Reviews: Climate Change. 2010;1(2):192–211. 22. Zhang X, Zwiers FW, Hegerl GC, Lambert FH, Gillett NP, Solomon S, et al. Detection of human influence on twentieth-century precipitation trends. Nature. 2007;448(7152):461–465. http://dx.doi.org/10.1038/nature06025 39. Department of Environmental Affairs (DEA). Long-Term Adaptation Scenarios Flagship Research Programme (LTAS) for South Africa: Climate trends and scenarios for South Africa. Pretoria: DEA; 2013. 23. Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, et al., editors. Climate change 2007: The physical science basis. Contribution of Working Group I to the fourth assessment report of the Intergovernmental Panel on Climate Change. Cambridge: Cambridge University Press; 2007. 40. Department of Environmental Affairs (DEA). Long-Term Adaptation Scenarios Flagship Research Programme (LTAS) for South Africa: Climate change implications for the water sector in South Africa. Pretoria: DEA; 2013. 41. Engelbrecht FA, McGregor JL, Engelbrecht CJ. Dynamics of the conformal- cubic atmospheric model projected climate-change signal over southern Africa. Int J Climatol. 2009;29(7):1013–1033. http://dx.doi.org/10.1002/ joc.1742 24. Lindesay JA. South African rainfall, the Southern Oscillation and a southern hemisphere semi-annual cycle. J Climatol. 1988;8(1):17–30. http://dx.doi. org/10.1002/joc.3370080103 25. Reason CJC, Allan RJ, Lindesay JA, Ansell TJ. ENSO and climatic signals across the Indian Ocean Basin in the global context. Part I: Interannual composite patterns. Int J Climatol. 2000;20(11):1285–1327. http://dx.doi. org/10.1002/1097-0088(200009)20:11<1285::AID-JOC536>3.0.CO;2-R 25. Reason CJC, Allan RJ, Lindesay JA, Ansell TJ. ENSO and climatic signals across the Indian Ocean Basin in the global context. Part I: Interannual composite patterns. Int J Climatol. 2000;20(11):1285–1327. http://dx.doi. org/10.1002/1097-0088(200009)20:11<1285::AID-JOC536>3.0.CO;2-R 42. Yao W, Zhong S. Nocturnal temperature inversions in a small, enclosed basin and their relationship to ambient atmospheric conditions. Meteorol Atmos Phys. 2009;103(1–4):195–210. http://dx.doi.org/10.1007/s00703-008-0341-4 42. Yao W, Zhong S. Nocturnal temperature inversions in a small, enclosed basin and their relationship to ambient atmospheric conditions. Meteorol Atmos Phys. 2009;103(1–4):195–210. References http://dx.doi.org/10.1007/s00703-008-0341-4 13 Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za Volume 110 \| Number 7/8 July/August 2014 South African Journal of Science http://www.sajs.co.za
W2583989724.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0171206&type=printable	en		Let them eat fruit! The effect of fruit and vegetable consumption on psychological well-being in young adults: A randomized controlled trial	PloS one	2,017	cc-by	9,733	RESEARCH ARTICLE Let them eat fruit! The effect of fruit and vegetable consumption on psychological well-being in young adults: A randomized controlled trial Tamlin S. Conner1☯, Kate L. Brookie1☯, Anitra C. Carr2‡, Louise A. Mainvil3‡, Margreet C. M. Vissers2‡ a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Psychology, University of Otago, Dunedin, Otago, New Zealand, 2 Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, New Zealand, 3 Department of Human Nutrition, University of Otago, Dunedin, Otago, New Zealand ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. tconner@psy.otago.ac.nz Abstract OPEN ACCESS Citation: Conner TS, Brookie KL, Carr AC, Mainvil LA, Vissers MCM (2017) Let them eat fruit! The effect of fruit and vegetable consumption on psychological well-being in young adults: A randomized controlled trial. PLoS ONE 12(2): e0171206. doi:10.1371/journal.pone.0171206 Editor: Jacobus P. van Wouwe, TNO, NETHERLANDS Received: November 9, 2016 Accepted: January 5, 2017 Published: February 3, 2017 Copyright: © 2017 Conner 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 Funding: This work was supported by a noncommercial University of Otago Research Grant to TS Conner (H15/010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This study tested the psychological benefits of a 14-day preregistered clinical intervention to increase fruit and vegetable (FV) consumption in 171 low-FV-consuming young adults (67% female, aged 18–25). Participants were randomly assigned into a diet-as-usual control condition, an ecological momentary intervention (EMI) condition involving text message reminders to increase their FV consumption plus a voucher to purchase FV, or a fruit and vegetable intervention (FVI) condition in which participants were given two additional daily servings of fresh FV to consume on top of their normal diet. Self-report outcome measures were depressive symptoms and anxiety measured pre- and post-intervention, and daily negative and positive mood, vitality, flourishing, and flourishing behaviors (curiosity, creativity, motivation) assessed nightly using a smartphone survey. Vitamin C and carotenoids were measured from blood samples pre- and post-intervention, and psychological expectancies about the benefits of FV were measured post-intervention to test as mediators of psychological change. Only participants in the FVI condition showed improvements to their psychological well-being with increases in vitality, flourishing, and motivation across the 14-days relative to the other groups. No changes were found for depressive symptoms, anxiety, or mood. Intervention benefits were not mediated by vitamin C, carotenoids, or psychological expectancies. We conclude that providing young adults with high-quality FV, rather than reminding them to eat more FV (with a voucher to purchase FV), resulted in significant short-term improvements to their psychological well-being. These results provide initial proof-of-concept that giving young adults fresh fruit and vegetables to eat can have psychological benefits even over a brief period of time. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12615000183583 Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 1 / 19 Effect of FV consumption on well-being: An RCT Introduction The physical health benefits of fruit and vegetables are well established. People who eat more fruit and vegetables (FV) have better cardiovascular health [1], reduced risk of some cancers [2], and greater longevity than people who eat fewer FV [3]. There is also growing evidence that people who eat more FV have better mental health. Higher consumption of FV is correlated with several psychological outcomes including a lower incidence of depression and anxiety [4, 5, 6, 7, 8, 9], greater happiness [10, 11], higher life satisfaction [10, 12, 13, 14], and greater social-emotional well-being or “flourishing” [15]. This growing body of research is intriguing because it suggests that the foods people eat have a much broader impact beyond the notable physical health benefits. However, studies examining the psychological benefits of FV have been predominantly observational and epidemiological to date, with a relative lack of intervention research that would support a causal link. Prospective research designs provide some evidence that increasing FV consumption may cause psychological benefits. At least five longitudinal studies found that dietary improvements including a diet higher in FV (i.e., a Mediterranean diet) predicted subsequent reductions in depression among adolescents, adults, and older adults [16, 17, 18, 19, 20]. However, these studies have not typically separated the effects of FV from other components of the Mediterranean diet such as olive oil or fish. More relevant are the results from Mujcic and Oswald [14] who analysed the prospective relationship between FV consumption and happiness and life satisfaction from the 2007 and 2009 waves of the Household, Income, and Labour Dynamics in Australia (HILDA) study, a nationally representative panel survey of over 12,000 people aged 15 years and older. They found that that increased FV consumption predicted greater “happiness gains” and increases in life satisfaction over the two years. Respondents with the largest increases in FV reported the greatest gains in well-being over time [14]. The opposite direction–happiness or life satisfaction predicting subsequent increases in FV consumption– was not found. Only a few intervention studies have tested the psychological benefits of increased FV consumption directly. One study of 271 low-income participants in the UK showed improvements in the composite measure of mental health from the Rand Short Form Survey (SF-36 [21]) from baseline to 8-weeks follow up in participants who underwent brief nutritional and behavioral counselling to increase their FV consumption [22]. Another study of 35 healthy young men showed that eating two kiwifruits every day for six weeks resulted in modest improvements to mood as measured by the Profile of Mood States (POMS) questionnaire [6, 23]. Mood improvements corresponded with increased vitamin C levels, were only significant for the young men with poorer mood at the start of the study, and were mainly driven by reductions in fatigue, increases in “vigor” (which included reports of feeling cheerful, energetic, lively, and full of pep), and decreases in depressive symptoms (trend only) [6]. A third study of 100 university students showed that snacking on one piece of fruit (either an apple, large clementine, or banana) each day for 10 days resulted in reductions in fatigue and anxiety compared to daily snacking on chocolate wafers or potato chips [24]. Taken together, these studies support a causal link between FV and various measures of mental health and mood. Yet each study is limited in some way–either by a small sample size [6], exclusive focus on fruit [6, 24], or use of a composite mental health index that does not separate aspects of mental health such as depression and anxiety [22]. And, to our knowledge, no intervention study has tested whether FV improves aspects of well-being aside from the impact on ill-being. It is important to fill this gap given the growing observational and prospective evidence linking FV to indicators of well-being such as happiness and life satisfaction [10, 12, 13, 14], optimism [25], and flourishing [15]. PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 2 / 19 Effect of FV consumption on well-being: An RCT Accordingly, in the present study, we tested the psychological benefits of a 14-day preregistered clinical intervention designed to increase fruit and vegetable (FV) consumption among low-FV-consuming young adults [26]. We defined low-consuming young adults as anyone aged 18 to 25 who reported eating fewer than 3 combined servings of fruit and vegetables per day. This cut-off reflects lower intake than the minimum national dietary guidelines of 5 combined servings of FV per day [27]. We implemented our intervention on a young adult population because young adults typically have the lowest FV consumption of all the age groups [28, 29, 30] and they are developing early autonomy over their health behaviors [31]. More details on the development of the intervention can be found in Brookie et al. [26]. To briefly summarize, we ran a three-arm randomized clinical trial (RCT) (n = 171) with three conditions: a diet-as-usual control condition in which participants were asked to maintain their regular food consumption for two weeks; an ecological momentary intervention condition (EMI), in which participants were sent twice daily text-messages that utilized a variety of behavioral change techniques to help them increase their fruit and vegetable consumption to at least 5 combined servings a day (plus they were given a voucher to purchase the FV); or a fruit and vegetable condition intervention (FVI), in which participants were given a bag of two weeks’ worth of fruit and vegetables (kiwifruit or oranges depending on the season, apples, and carrots) and were asked to consume at least two additional servings (1 fruit and 1 vegetable) on top of their regular daily FV consumption. Participants in both intervention conditions reported significantly higher daily FV consumption (3.7 servings / day) compared to control participants (2.8 servings / day), and compared to their own baseline (2.5 servings / day) when FV was reported nightly for two weeks using a smartphone-accessed survey [26]. Moreover, blood samples taken before and after the intervention showed that participants in the two experimental groups had small gains in vitamin C levels and plasma carotenoids, suggesting that the self-reported changes in FV consumption were legitimate. Vitamin C and carotenoids are the most consistently reliable biomarkers of FV consumption and have been used to reflect intake of nutrient-dense FV [32]. The current paper presents the psychological outcomes of that same two-week intervention reported in Brookie et al. [26]. The primary outcome measures were depressive symptoms and anxiety measured pre- and post-intervention, as well as negative and positive mood, vitality, and flourishing measured nightly through a smartphone survey. Our choice of methodology— smartphone tracking of daily psychological outcomes for two weeks—reflected our desire to minimise memory-based reporting and maximise sensitivity to detect possible group differences in mood and well-being changes over time [33]. Our choice of outcome measures reflected a desire to measure both the negative and positive aspects of mental health [34]. We predicted that participants in the two experimental conditions (EMI and FVI) would show significant improvements in the psychological outcomes relative to participants in the control condition, and that the benefits of FV consumption would be stronger for the measures of well-being (positive mood, vitality, flourishing). Although previous research has linked FV to lower levels ill-being–such as reduced depression [9, 35] and anxiety [9, 24]–there is more evidence linking FV to higher levels of well-being such as positive mood, happiness, and flourishing [6, 10, 11, 12, 13, 14, 15]. We also included three unregistered “wild-card” variables in the daily smartphone survey–self-reported curiosity, creativity, and motivation–which we grouped under the umbrella term of flourishing behaviors. Higher FV consumption was previously shown to correlate with greater curiosity and creativity in young adults [15], but research has not yet established whether that relationship is causal. And, no research has tested whether FV might improve perceptions of motivation, a key correlate of vitality [36]. Lastly, we tested whether changes in the psychological outcomes would be mediated by two key biomarkers (vitamin C and carotenoids) and/or psychological expectancies. The mechanisms linking FV PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 3 / 19 Effect of FV consumption on well-being: An RCT to well-being are unknown, but they may be due to key micronutrients like vitamin C and carotenoids, which act as cofactors for dopamine and other neurotransmitters involved in positive motivational states [37], or possibly due to positive psychological expectancies such as the perception that eating FV is virtuous or will make you feel better [38]. Materials and methods Participants and procedure This is the same sample reported in Brookie et al. [26]. The experimental design and primary outcome measures were registered before recruitment with the Australia New Zealand Clinical Trials Registry (ANZCTRN12615000183583). Recruitment began in March 2015 and follow-up data was collected by the end of November 2015. Fig 1 shows the flow of participants throughout the trial and explanations for drop outs. Participants were 171 young adults (56 men; 115 women), 18 to 25 years old (M = 19.43, SD = 1.45) who identified as European (64%), Asian (18%), Māori or Pacific Islander (8%), or another or mixed ethnicity (11%). Participants were students at the University of Otago, New Zealand who were recruited through the Psychology Department’s experimental participation program and reimbursed with partial course credit (N = 135, 79%) or recruited through a student employment agency and reimbursed with a small cash payment (N = 36, 21%). Inclusion criteria included being in the young adult age range (18–25 years), having an Internet capable smartphone, identifying as low-FV-consuming (no more than 3 combined servings of FV per day), not being on any anti-depressant medication, and having no known FV allergies. All participants provided written consent to take part by reading and signing their name on a paper consent form (later kept in secured storage). The study and consenting procedures were approved by the University of Otago Human Ethics Committee (#15/010). More detail about the intervention, including the development and complete list of intervention text-messages for the EMI condition, is described in Brookie et al. [26]. Participants attended an initial laboratory session and completed a computerized baseline survey that measured demographics and symptoms of depression and anxiety, among other measures. That evening, participants completed their first of 14 daily diary surveys delivered by hyperlink to their smartphones. The survey measured their daily mood and well-being (see Measures) and was accessible between 7:00 pm and 1:00 am. The next morning, participants attended a clinic in the Human Nutrition department where they were measured for height and weight using standardised techniques [39] and gave a fasting baseline blood sample (4 ml). Immediately after the clinic visit, participants randomly assigned to the EMI condition began to receive their twice-daily intervention messages to increase FV over the next 13 days. Participants in the FVI condition received their bag of FV to consume for the next 13 days. Participants in the control condition were asked to maintain their normal diet. After the 13 intervention days, participants returned to the Human Nutrition clinic and gave a follow up blood sample (4 ml), and completed follow-up measures of depressive symptoms and anxiety. Measures Preregistered psychological outcome measures were depressive symptoms, anxiety, negative mood, positive mood, vitality, and flourishing. Curiosity, creativity, and motivation were not registered ahead of time and were therefore considered exploratory. Demographics and covariates. Demographic measures were age, gender, and ethnicity. Height and weight measured during the first clinic visit was used to calculate body mass index (BMI) as a possible covariate. Smoking status was tested as a covariate in the vitamin C analysis because smoking increases the metabolism of vitamin C [40]. In the follow-up survey, PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 4 / 19 Effect of FV consumption on well-being: An RCT Fig 1. Participant Flow Diagram Depicting Reasons for Exclusion and Allocation to Intervention Conditions. doi:10.1371/journal.pone.0171206.g001 participants were asked “How often do you currently smoke” by choosing one of five response options: I don’t smoke now, less than once a month, at least once a month, at least once a week, or at least once a day. Participants who smoked at least once a week or day were coded as smokers (1); all others were coded as non-smokers (0). PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 5 / 19 Effect of FV consumption on well-being: An RCT Depressive symptoms. Depressive symptoms were measured before and after the intervention using the 20-item Centre for Epidemiological Studies Depression Scale (CES-D; [41]). Participants rated their depressive symptomology “in the past week” on a 4-point scale ranging from 0 (Rarely or none of the time, < 1 day) to 3 (Most or all of the time, 5–7 days). Responses were summed to give a total depressive symptom score ranging from 0 to 60 (pre α = 0.89; post α = 0.90). Anxiety. Anxiety symptoms were measured before and after the intervention using the 7-item Anxiety sub-scale of the Hospital Anxiety and Depression Scale (HADS; [42]). Participants rated each item on how they “felt recently, including today” on a 4-point scale ranging from 0 (Not at all or Only occasionally) to 3 (Very often or Most of the time). Responses were summed to give a total anxiety score ranging from 0 to 21 (baseline α = 0.77; follow up α = 0.78). Negative and positive mood. The daily survey included 3-item measures of negative mood (sad, anxious, angry) and positive mood (relaxed, happy, enthusiastic), which captured a range of activation states [43]. Participants rated each adjective on how they “felt today” on a 5-point Likert scale from 0 (Not at all) to 4 (Extremely). Responses were averaged across the three items each day for a measure of daily negative mood (α = 0.34) and daily positive mood (α = 0.42). The within-person reliabilities for the daily survey data were computed using procedures for nested data recommended by Nezlek [44]. Because of low reliability, we also analysed the items separately in supplementary analyses. Vitality. The daily survey included a modified version of the 4-item energy/fatigue scale of the Rand 36-Item Short Form Survey (SF-36 [21, 45]). Participants were asked “today”: Did you feel full of life? Did you have a lot of energy? Did you feel worn out? Did you feel tired? We changed the first item from the original “full of pep” to “full of life” to better capture feelings of vitality and to connect with a young-adult population. Each item was rated on a 6-point scale labelled None of the time, A little of the time, Some of the time, A good bit of the time, Most of the time, and All of the time. Responses were coded from 0 to 100 in 20-point intervals following SF-36 guidelines (reverse-scoring worn out and tired), and summed within each day so that highest total score was 100, indicating higher vitality that day. The modified scale showed adequate within-person reliability (α = 0.70). Flourishing. The daily survey included a shortened 3-item version of the 8-item Flourishing Scale that assesses feelings of engagement, purpose in life, and social-emotional connectedness, which previous research shows loads on a single “flourishing” factor [46]. We selected the three highest loading items from a previous dataset and adapted the items for a daily format. The three items were: Today, I was engaged and interested in my daily activities. Today, I led a purposeful and meaningful life. Today, I was a good person and lived a good life. Participants rated each item on a 7-point Likert scale from 1 (Strongly disagree) to 4 (Neither agree nor disagree) to 7 (Strongly agree). Responses were summed each day for a measure of daily flourishing ranging from 1 to 21 (α = 0.70). Flourishing behaviors. The daily survey included three items answered on a 5-point Likert scale from 0 (Not at all) to 4 (Extremely). The first item measured curiosity: How curious were you today? Did you seek out new things or experiences; look for opportunities to challenge yourself and grow as a person; or embrace the unfamiliar. This question was guided by the content of the Curiosity and Exploration Inventory II [47]. The second item measured creative activity: How creative were you today? Did you come up with novel or original ideas; express yourself in an original and useful way; or spend time doing artistic activities like, music, painting, writing, etc. This question was based on scientific definitions of creativity [48] and has been used in previous research [15]. The third item measured perceived motivation: How motivated were you today? Did you work towards your goals, or feel driven today? This question was PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 6 / 19 Effect of FV consumption on well-being: An RCT designed to capture a broad, conscious sense of motivation including working towards goals [49]. Responses to the three items were averaged each day for an index of daily flourishing behaviors (α = 0.28). Because of low reliability, these items were also analysed separately in supplementary analyses. Vitamin C and carotenoids. A 4 ml fasting blood sample was taken before and after the intervention. Samples were kept in dark conditions on ice and centrifuged within 1 hour of collection. Plasma aliquots were stored at -80C until analysis. Plasma vitamin C was assessed by high-performance liquid chromatography (HPLC) with electrochemical detection, treated with reducing agent to recover total vitamin C. Total plasma carotenoids were assessed spectrophotometrically (for full blood processing details see [26]). Four people were unable to provide a post-intervention blood sample; therefore, analyses with vitamin C and carotenoids reflected a sample size of 167. Psychological expectancies. The follow-up survey included questions about participants’ beliefs about the benefits and virtues of eating FV, as well as candy and fried foods as distractor items, and how participants thought eating these foods made them feel over the last two weeks. For analysis, we focused on the three items related to FV consumption: Do you feel that eating fruits and vegetables is virtuous (righteous, good, moral)? Do you feel better about yourself when you eat more fruits and vegetables? Did you feel that eating fruits and vegetables improved your mood during these two weeks? Each question was answered on a 5-point Likert scale from 0 (Very slightly/not at all) to 4 (Extremely). We added these expectancy items one third of the way through data collection; therefore, analyses with these items reflected a sample size of 121. Analyses and results Table 1 presents the descriptive statistics for the demographic and outcome variables. The depression and anxiety scores were within norms for this population [50]. Compliance with the smartphone surveys was high at 90% (12.6/14 surveys completed; range 9 to 14 surveys). There were no differences between the control, EMI, and FVI conditions on survey compliance (F(2,168) = 0.91, p = 0.405), gender (χ2(2, N = 171) = 0.129, p = .938), age (F(2,168) = 1.24, p = 0.291), or BMI (F(2,168) = 1.18, p = 0.309). However, the conditions varied in ethnic composition (χ2(2, N = 171) = 8.31, p = .016) with more non-Europeans in the control condition (51%) than the two intervention conditions (EMI 29%; FVI 28%). Therefore, ethnicity was included as a covariate in the analyses. The analyses consisted of two Analyses of Covariance (ANCOVA) for the measures of depressive symptoms and anxiety, and six growth curve models for the daily measures of positive mood, negative mood, vitality, flourishing, and flourishing behaviors. Based on these seven main analyses, we applied a Bonferroni correction to the alpha level (.05/7 tests) to account for multiple hypothesis testing (p = .007). Table 2 presents the raw scores for depressive symptoms and anxiety for participants in each condition. There were no differences at baseline between the three groups (depressive symptoms, F(2,168) = 0.19, p = 0.830; anxiety, F(2,168) = 0.88, p = 0.419). Results of a 3 (x 2) mixed ANCOVA with condition as the between-subjects variable, time as the within-subjects variable, and ethnicity as the covariate showed only a main effect of time in predicting depressive symptoms (F(1, 167) = 5.01, p = .027, Partial η2 = .029), no main effect of condition (F(2, 167) = 0.212, p = .809, Partial η2 = .003) and no time x condition interaction (F(2, 167) = 0.412, p = .663, Partial η2 = .005). Similar patterns were found for anxiety (time, F(1, 167) = 3.49, p = .063, Partial η2 = .020; condition, F(2, 167) = 1.33, p = .268, Partial η2 = .016; time x condition, F(2, 167) = 0.15, p = .859, Partial η2 = .002). The non-significant time x condition coefficients indicated that the interventions did not reduce depressive symptoms or anxiety relative to control. PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 7 / 19 Effect of FV consumption on well-being: An RCT Table 1. Sample Characteristics and Descriptive Statistics for the Psychological Outcome Variables. M SD Min Max 19.43 1.45 18 25 24.13 3.89 15.62 39.65 14.30 8.68 1.00 53.00 13.03 8.73 1.00 39.00 Anxiety (pre) 5.78 3.46 0.00 17.00 Anxiety (post) 5.37 3.31 0.00 17.00 Negative mood 0.77 0.46 0.00 2.18 Positive mood 2.05 0.50 0.57 3.92 Vitality 55.78 12.54 21.07 97.69 Flourishing 14.43 2.73 7.17 21.00 Flourishing behaviors b 1.48 0.54 0.19 3.42 N 171 % Female 67.3 % European 63.7 Age BMI % Regular smoker a 7.0 Depressive symptoms (pre) Depressive symptoms (post) Curiosity 1.40 0.66 0.00 3.69 Creativity 1.13 0.67 0.00 3.82 Motivation 1.91 0.58 0.50 3.62 Note. SD = standard deviation; BMI = body mass index. Descriptive statistics for the daily variables of negative mood to motivation were computed on aggregated variables. a Regular smoker was defined someone who smokes at least once a week (n = 8) or once a day (n = 4). b Daily composite of curiosity, creativity, and motivation items. doi:10.1371/journal.pone.0171206.t001 The daily survey data were analyzed using the Hierarchical Linear Modeling program to account for dependency among nested data (HLM v.6.08; [51]). We modelled linear growth curves to assess whether participants in the experimental conditions reported greater improvements in mood and well-being over time relative to participants in the control condition. Table 2. Changes in Depressive Symptoms and Anxiety for the Three Intervention Conditions, Unadjusted for Ethnicity. n Depressive Symptoms Baseline Follow-up M (SD) M (SD) Diff 95% CI diff a Control 59 14.44 (9.82) 12.78 (10.07) 1.66 (0.09, 3.24) EMI 55 13.73 (9.01) 13.07 (8.37) 0.66 (-1.57, 2.87) FVI 57 14.70 (7.06) 13.25 (7.65) 1.46 (0.06, 2.85) Control 59 5.68 (3.66) 5.44 (3.54) 0.24 (-0.35, 0.83) EMI 55 5.40 (3.17) 4.80 (3.02) 0.60 (-0.27, 1.47) FVI 57 6.25 (3.50) 5.84 (3.29) 0.40 (-0.42, 1.23) Anxiety b Note. M = mean; SD = standard deviation; EMI = ecological momentary intervention condition; FVI = fruit and vegetable intervention condition. Diff = point difference between baseline and follow-up group means. CI = Confidence Interval in the point difference between baseline and follow-up group means. * p < 0.05 a b Depressive symptoms measured with the Center for Epidemiological Studies Depression Scale (CES-D). Anxiety measured with the Hospital Anxiety and Depression Scale (HADS). doi:10.1371/journal.pone.0171206.t002 PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 8 / 19 Effect of FV consumption on well-being: An RCT Growth curves were modelled for each participant for each outcome using robust standard errors, as follows: Level 1 : Negative mood ¼ B0 þ B1 Time þ r Level 2 : B0 ¼ G00 þ G01 ConditionD1 þ G02 ConditionD2 þ G03 Eth þ u0 B1 ¼ G10 þ G11 ConditionD1 þ G12 ConditionD2 þ u1 This set of equations determined each person’s negative mood at baseline (B0), how negative mood changed over time for each participant (B1), and how these changes varied by experimental condition (G11; G12). Time was recoded so that 0 was day 1. G11 tested change in the EMI condition against change in the control condition (using Condition Dummy 1; control = 0; EMI = 1; FVI = 0). A significant G11 indicated that participants in the EMI condition exhibited a different pattern of change compared to participants in the control condition. G12 tested change in the FVI condition against the change in the control condition (using Condition Dummy 2; control = 0; EMI = 0; FVI = 1). A significant G12 indicated that participants in the FVI condition exhibited a different pattern of change compared to participants in the control condition. Thus, the null hypothesis was that participants in the EMI / FVI conditions would report similar changes over time as the control participants. For significant group differences, we plotted the growth patterns for the three groups and computed tests of the simple slopes. We also tested an additional set of dummy codes to compare the EMI and FVI groups in subsequent analyses. Ethnicity (0 = European; 1 = non-European) was entered as a covariate in the Level-2 intercept equation. Ethnicity was also entered as a covariate in the Level-2 slope equation but was not significant, did not affect results, and was removed from final models for simplicity. Table 3 presents the growth curve results. Participants in the two experimental conditions did not show any improvements in negative or positive mood relative to participants in the control condition. Supplementary analyses showed similar null effects when analysing the mood items separately (see S2 Table) with an exception: happy showed significant group differences in change over time. Analysis of simple slopes showed that happiness was stable over time for participants in the FVI condition (B(SE) = -0.001(0.006), p = .845) but happiness decreased for participants in the EMI condition (B(SE) = -0.034(0.010), p = 0.001) and control condition (B(SE) = -0.012(0.008), p = 0.018). The slopes were significantly different only between the FVI and EMI conditions (p = 0.008). However, participants in the FVI condition did show improvements to their psychological well-being compared to participants in the EMI and control conditions. As shown in Table 3, participants given fresh fruit and vegetables to consume (FVI condition) reported significant growth in vitality, flourishing, and flourishing behaviors compared to participants in the EMI and control conditions. Fig 2 presents the growth patterns for these outcomes. As shown in the top of Fig 2, participants in the FVI condition reported lower starting vitality than the other groups, but their rate of improvement was greater compared to both the EMI and control conditions. Analysis of simple slopes showed that participants in the FVI condition reported significant growth in vitality over time (B(SE) = 0.455(0.200), p = .023) whereas participants in EMI and control conditions were unchanged over time (EMI B(SE) = -0.192 (0.187), p = 0.307; control B(SE) = -0.336(0.204), p = 0.101). Group differences were even more apparent for the measure of flourishing. As shown in the middle of Fig 2, participants in the FVI condition reported significantly greater increases in flourishing compared to participants in the control and EMI conditions. Analysis of the simple slopes showed that participants in the FVI condition reported significant growth in flourishing over time (B(SE) = 0.010 (0.033), p = .004) whereas participants in the EMI and control conditions were unchanged PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 9 / 19 Effect of FV consumption on well-being: An RCT Table 3. Results from Growth Curve Modelling Testing for Differences in Change over Time between the Three Intervention Conditions. Significant intervention effects are bolded (p < .007). Outcome Negative Mood G Coef SE p Control Day 1 G00 0.744 0.068 < 0.001 EMI Day 1 diff G01 -0.068 0.095 0.471 FVI Day 1 diff G02 0.049 0.093 0.601 Ethnicity G03 -0.024 0.079 0.758 Control Change (Δ) a G10 0.011 0.006 0.085 EMI Change diff b G11 -0.001 0.010 0.897 FVI Change diff c Positive Mood G12 -0.012 0.008 0.136 G Coef SE p EMI Δ d FVI Δ diff e Coef SE p 0.009 0.007 0.204 -0.011 0.009 0.897 Control Day 1 G00 2.137 0.092 < 0.001 EMI Day 1 diff G01 0.194 0.122 0.115 FVI Day 1 diff G02 0.006 0.103 0.955 Ethnicity G03 -0.140 0.084 0.098 Control Change G10 -0.014 0.008 0.072 EMI Change diff G11 -0.013 0.012 0.261 EMI Δ -0.027 0.008 0.001 FVI Change diff G12 0.006 0.009 0.529 FVI Δ diff 0.019 0.011 0.076 G Coef SE p Vitality Control Day 1 G00 58.639 2.443 < 0.001 EMI Day 1 diff G01 -0.701 2.915 0.810 FVI Day 1 diff G02 -6.778 2.976 0.024 Ethnicity G03 -0.510 2.140 0.812 Control Change G10 -0.336 0.204 0.101 EMI Change diff G11 0.144 0.277 0.603 EMI Δ -0.192 0.187 0.307 FVI Change diff G12 0.791 0.284 0.006 FVI Δ diff 0.647 0.027 0.019 G Coef SE p Flourishing Control Day 1 G00 14.505 0.509 < 0.001 EMI Day 1 diff G01 0.440 0.588 0.455 FVI Day 1 diff G02 -0.630 0.611 0.305 Ethnicity G03 -0.200 0.470 0.671 Control Change G10 -0.031 0.027 0.242 EMI Change diff G11 -0.007 0.041 0.864 EMI Δ -0.038 0.031 0.205 FVI Change diff G12 0.128 0.042 0.003 FVI Δ diff 0.135 0.045 0.004 G Coef SE p Flourishing Behaviors Control Day 1 G00 1.548 0.100 < 0.001 EMI Day 1 diff G01 -0.079 0.111 0.476 FVI Day 1 diff G02 -0.110 0.117 0.350 Ethnicity G03 0.044 0.096 0.647 Control Change G10 -0.012 0.006 0.038 EMI Change diff G11 0.003 0.008 0.756 EMI Δ -0.009 0.006 0.093 FVI Change diff G12 0.025 0.009 0.006 FVI Δ diff 0.022 0.009 0.011 Note. Coef = coefficient from Hierarchical Linear Modeling; SE = Robust standard error; diff = difference in coefficient; EMI = ecological momentary intervention condition; FVI = fruit and vegetable intervention condition; Δ = change. Degrees of freedom were 167 for G00 –G03 and 168 for G10 –G12. a Change over time for reference group (control condition). b Difference in change between EMI condition versus control condition. c Difference in change between FVI condition versus control condition. d Change over time for reference group (EMI condition). e Difference in change between FVI condition versus EMI condition. doi:10.1371/journal.pone.0171206.t003 PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 10 / 19 Effect of FV consumption on well-being: An RCT PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 11 / 19 Effect of FV consumption on well-being: An RCT Fig 2. Changes in vitality, flourishing, and flourishing behaviors (composite of daily curiosity, creativity, and motivation) for the control, ecological momentary intervention (EMI), and fruit and vegetable intervention (FVI) conditions. Significant simple slopes are indicated. * p < .05; * p < .01. doi:10.1371/journal.pone.0171206.g002 over time (EMI B(SE) = -0.038(0.031), p = 0.215; control B(SE) = -0.031(0.027), p = 0.242). And, shown in the bottom of Fig 2, participants in the FVI condition also reported significantly greater growth in flourishing behaviors compared to participants in the control condition. Simple slope analyses revealed a trend growth in flourishing behaviors for participants in the FVI condition (B(SE) = 0.013(0.007), p = .053), a trend decrease for participants in the EMI condition (B(SE) = -0.009(0.006), p = .093), and a significant decrease for participants in the control condition (B(SE) = -0.012(0.006), p = .038). When we analyzed the three flourishing behaviors separately, all three showed similar patterns, but the strongest effects were found for motivation (see S2). Fig 3 presents the growth patterns for motivation showing that participants in the FVI condition reported significantly increasing motivation over time (B(SE) = 0.019(0.010), p = .045), whereas participants in the EMI and control conditions were unchanged over time (EMI B(SE) = -0.005(0.011), p = 0.652; control B(SE) = -0.011(0.008), p = 0.218). In terms of effect sizes, being assigned to the FVI condition (vs. control) predicted 8.7% of the variance in growth in vitality, 10.5% of the growth in flourishing, and 18.4% of the growth in flourishing behaviors using variance-explained effect size estimates of the time slopes [52]. Additional analyses testing for non-linear change over time using time and time squared as level-1 predictors found no evidence for curvilinear changes over time or moderation of curvilinear changes by intervention group. Thus, changes in well-being as a function of the intervention were strictly linear. Lastly, none of these changes in well-being were mediated by post-test vitamin C, post-test carotenoid levels, or psychological expectancies. Following procedures for testing mediation with a multicategorical independent variable [53], we added each mediator as a separate Level2 predictor (grand-mean centered) plus the two condition dummy codes (ConditionD1 and Fig 3. Changes in self-reported motivation for the control, ecological momentary intervention (EMI), and fruit and vegetable intervention (FVI) conditions. Significant simple slopes are indicated. * p < .05. doi:10.1371/journal.pone.0171206.g003 PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 12 / 19 Effect of FV consumption on well-being: An RCT ConditionD2) to predict both the intercepts (B0s) and time slopes (B1s) for vitality, flourishing, and flourishing behaviors (with ethnicity as a covariate in the intercept equation, as before). Five mediators were tested separately: post-test vitamin C levels, post-test carotenoid levels, expectancy 1 (eating FV is virtuous), expectancy 2 (I feel better about myself when eating more FV), and expectancy 3 (eating FV improved my mood). None of the condition coefficients G11 or G12 changed when entering vitamin C, carotenoids, or the three psychological expectancy questions into the model. The fact that the total effects (c) were nearly equivalent to the direct effects (c’) when partialling out each indirect mediational pathway (ab) argues against mediation by these variables. Discussion Giving young adults fruits and vegetables (FV) to eat, but not reminding them to eat their FV, improved several aspects of psychological well-being over a two-week period. Despite both intervention groups reporting modestly higher and similar consumption of FV relative to control (3.7 vs. 2.8 daily servings) [26], only young adults who were given two weeks’ worth of FV showed improvements in their feelings of vitality, flourishing, and motivation. The short duration of our study indicated that FV intake translated into improved well-being quite rapidly. The intervention effects were prominent across measures of well-being but not ill-being such as depression, anxiety, or negative mood. This is an interesting pattern that supports the observational and prospective research linking FV to greater well-being [10, 14, 15]. It is also possible that this pattern might reflect differences in the timescale of effects. The majority of research linking depression to dietary patterns has been longitudinal, meaning that possible differences in ill-being may be established over a much longer period of time rather than our brief two-week period. The accumulation of factors such as low vitality, reduced motivation, and poorer socio-emotional flourishing may precipitate the development of psychological illbeing over time [54]. In saying this, Smith and Rogers [24] did find a difference in self– reported anxiety using the same measure (HADS) over a 10-day period with snacking on one piece of fruit each day compared to snacking on chocolate. However, their participants had higher average baseline anxiety (approximately +1.5 points). Intervention studies with positive outcomes tend to have participants with higher baselines of illness [55], which may explain the discrepancy in our findings. The lack of psychological improvement among the EMI participants is challenging to interpret. Both the EMI and FVI groups reported higher and equivalent consumption of FV compared to control, but the EMI group did not show any corresponding psychological benefit. Both intervention conditions were given roughly the same amount of monetary goods at the start of the study–a $10 voucher for the EMI condition versus a little more than $10 worth of fruit and vegetables for the FVI condition. One possibility is that this difference might be due to lower control over the type, quality, and preparation of fruit and vegetables eaten by the EMI group. The EMI group were free to choose whatever fruit and vegetables they liked, and when surveyed, we found that they were more likely to eat cooked vegetables in casseroles or mixed into their main meals [26]. By contrast, for the FVI group, we chose high quality produce, which was mostly eaten raw (including the carrots, eaten as snacks). Some researchers have shown that more optimal psychological outcomes are associated with the consumption of fresh fruit and raw vegetables/ salads, but not cooked vegetables [56, 57]. It is also possible that regular text-reminders (twice daily for two weeks) might have annoyed participants in the EMI condition and wiped out any gains in well-being. However, our experience is that participant burden is relatively low with receiving only two text messages per day. Furthermore, informal feedback from the EMI participants was mostly positive; participants reported that PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 13 / 19 Effect of FV consumption on well-being: An RCT the messages were not particularly intrusive and were effective at making them more aware about what they ate [26]. Alternatively, it could be that giving FV to participants triggered more intrinsic rather than extrinsic regulation of behavior. A previous study found that households given boxes of free FV were reluctant to waste the food and therefore had to overcome internal conflicts in their personal value systems and find solutions they could live with–contributing to a largely internal source of motivation to consume the FV they had been given [58]. Given that intrinsic goals tend to be more rewarding and lead to greater well-being [59], this could potentially explain the psychological improvement in the FVI condition, but not in the EMI condition. The significant changes in well-being occurred despite relatively small changes in FV consumption (+ 1 serving per day more than control, and +1.2 servings from baseline for the FVI condition). In fact, participants in the FVI condition still fell short of the “5+ a day” FV servings target [27], let alone the optimal 7–9 daily servings that has been associated with longterm health [1]. Psychological improvements could have been larger had participants in the experimental conditions eaten even more FV (or higher quality FV) or had they achieved saturation levels of vitamin C and carotenoids. As we reported in Brookie et al. [26], the micronutrient increases in vitamin C and carotenoids were modest for our intervention conditions and well-below saturation levels, suggesting that there was still room for improvement. In future research, it would be important to increase consumption more than we did here. Neither vitamin C and carotenoids nor psychological expectancies about FV mediated the psychological benefits of FV consumption in this study. This was despite us choosing the two most responsive micronutrient biomarkers of FV consumption [32] and measuring a range of psychological expectancies. The literature on micronutrient status and mental health suggests that single nutrients are unlikely to play a large role in the protection against mental illness [60]. Instead, better mental health may arise from the cumulative effects of a broad spectrum of vitamins, minerals, and antioxidants [61]. Future research should consider how FV consumption affects well-being through multiple pathways, including through improved blood flow to the brain [62] or changes in gut microbiota [63, 64]. Strengths of the study included the three-arm randomized design that compared two experimental conditions to a diet-as-usual control condition; use of a smartphone survey to track mood and well-being in near-to-real time which provided a more accurate assessment than retrospective self-report; high compliance with the smartphone survey; and, inclusion of blood samples as an objective marker of FV consumption. Limitations included issues with measurement reliability for some of the diary measures, which was a consequence of efforts to keep the survey short. This could explain why we did not see intervention effects on mood. Future research should include a more extensive measure of mood, such as the 9-item measure of positive mood previously found associated with FV [11, 15]. Another limitation is the short timeframe of our intervention (two weeks) and lack of longer-term follow-up. In this regard, our approach was more of a ‘light touch’ intervention to evoke near-term behavioral change. For this reason, we consider these results preliminary, offering proof of concept that small changes to FV consumption over a short time period may confer changes in well-being. Our findings have implications for campaigns designed to increase FV consumption. FV campaigns reflect an ‘information is power’ ethos and largely consist of educative programmes operating through child health and wellness services, schools, communities, and public service television ads. However, our research suggests that simply educating people about FV and reminding them to eat their recommended daily intake may not be sufficient in ensuring the wider population reaps the psychological benefits of FV consumption. It is already established that successful interventions tend to be more personal and more intensive [55], but perhaps greater emphasis needs to be placed on actually providing people with fresh FV (stocking PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 14 / 19 Effect of FV consumption on well-being: An RCT more FV in dorms, cafeterias, workplaces, substituting fruit for dessert, and offering free fruit for people when they shop). Additionally, conveying the immediate psychological benefits of FV consumption may have more impact on behavior. Behavioral change messages are more effective when they are immediately relevant to the target population [65] suggesting that near-term benefits–such as increases in vitality and motivation–may be more salient than longer-term health benefits–such as the prevention of heart disease. This may be particularly pertinent to young people, who are endowed with feelings of immortality and quite focused on the ‘here and now’. Conclusions Providing young adults with high-quality FV, not texting them reminders to eat more FV and giving them a voucher, resulted in improvements to their psychological well-being over a twoweek period. This is the first study to show that providing high-quality FV to young adults can result in short-term improvements in vitality, flourishing, and motivation. Findings provide initial validation of a causal relationship between FV and well-being, suggesting that largescale intervention studies are warranted. Supporting information S1 File. Pre- and Post-intervention Variables. (SAV) S2 File. Daily diary Variables. (SAV) S1 Table. CONSORT Checklist for Reporting a Randomized Trial. (PDF) S2 Table. Separate Analyses of Mood Items and Flourishing Behaviours. (PDF) S1 Text. Research Protocol. (PDF) Acknowledgments We are grateful to Hadyn Youens for web programming and members of the Daily Experiences Lab for their assistance with data collection and running of the blood clinic. We thank Angel Temple and Ashley Duncan for their work taking and preparing bloods and Simone Bayer for technical assistance with the analysis of biomarkers. Author contributions Conceptualization: TC KB. Data curation: TC KB. Formal analysis: TC KB MV AC. Funding acquisition: TC. Investigation: KB. Methodology: TC KB. PLOS ONE \| DOI:10.1371/journal.pone.0171206 February 3, 2017 15 / 19 Effect of FV consumption on well-being: An RCT Project administration: KB. Resources: KB. Supervision: TC. Visualization: TC KB. Writing – original draft: TC KB. 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https://openalex.org/W3125212064	http://journal.asu.ru/wv/article/download/8966/7349	Russian	null	Archeology of the early medieval turks of Central Asia: main stages of research and interpretations of the complexes	Narody i religii Evrazii	2,020	cc-by	9,652	НАРОДЫ И РЕЛИГИИ ЕВРАЗИИ Барнаул Издательство Алтайского государственного университета 2020 ISSN 2542-2332 (Print) ISSN 2686-8040 (Online) 2020 № 4 (25) 2020 № 4 (25) ISSN 2542-2332 (Print) ISSN 2686-8040 (Online) ISSN 2542-2332 (Print) ISSN 2686-8040 (Online) НАРОДЫ И РЕЛИГИИ ЕВРАЗИИ Издание основано в 2007 г. Учредитель: ФГБОУ ВО «Алтайский государственный университет» Учредитель: ФГБОУ ВО «Алтайский государственный университет» Учредитель: ФГБОУ ВО «Алтайский государственный университет» Главный редактор: П. К. Дашковский, доктор исторических наук (Россия, Барнаул) Редакционная коллегия: С. А. Васютин, доктор исторических наук (Россия, Кемерово) Н. Л. Жуковская, доктор исторических наук (Россия, Москва) А. П. Забияко, доктор философских наук (Россия, Благовещенск) А. А. Тишкин, доктор исторических наук (Россия, Барнаул) Н. А. Томилов, доктор исторических наук (Россия, Омск) Т. Д. Скрынникова, доктор исторических наук (Россия, Санкт-Петербург) О. М. Хомушку, доктор философских наук (Россия, Кызыл) М. М. Шахнович, доктор философских наук (Россия, Санкт-Петербург) Л. И. Шерстова, доктор исторических наук (Россия, Томск) А. Г. Ситдиков, доктор исторических наук (Россия, Казань) Е. А. Шершнева (отв. секретарь), кандидат исторических наук (Россия, Барнаул) Редакционный совет журнала: Л. Н. Ермоленко, доктор исторических наук (Россия, Кемерово) Ю. А. Лысенко, доктор исторических наук (Россия, Барнаул) Л. С. Марсадолов, доктор культурологии (Россия, Санкт-Петербург) Г. Г. Пиков, доктор исторических наук, доктор культурологии (Россия, Новосибирск) А. К. Погасий, доктор философских наук (Россия, Казань) К. А. Руденко, доктор исторических наук (Россия, Казань) С. А. Яценко, доктор исторических наук (Россия, Москва) А. С. Жанбасинова, доктор исторических наук (Казахстан, Усть-Каменогорск) Н. И. Осмонова, доктор философских наук (Кыргыстан, Бишкек) Н. Цэдэв, кандидат педагогических наук (Монголия, Улан-Батор) Ц. Степанов, доктор исторических наук (Болгария, София) З. С. Самашев, доктор исторических наук (Казахстан, Астаны). Главный редактор: П. К. Дашковский, доктор исторических наук (Россия, Барнаул) П. К. Дашковский, доктор исторических наук (Россия, Барнаул) Редакционная коллегия: С. А. Васютин, доктор исторических наук (Россия, Кемерово) Н. Л. Жуковская, доктор исторических наук (Россия, Москва) А. П. Забияко, доктор философских наук (Россия, Благовещенск) А. А. Тишкин, доктор исторических наук (Россия, Барнаул) Н. А. Томилов, доктор исторических наук (Россия, Омск) Т. Д. Скрынникова, доктор исторических наук (Россия, Санкт-Петербург) О. М. Хомушку, доктор философских наук (Россия, Кызыл) М. М. Шахнович, доктор философских наук (Россия, Санкт-Петербург) Л. И. Шерстова, доктор исторических наук (Россия, Томск) А. Г. Ситдиков, доктор исторических наук (Россия, Казань) Е. А. Шершнева (отв. секретарь), кандидат исторических наук (Россия, Барнаул) ISSN 2542-2332 (Print) ISSN 2686-8040 (Online) 2020 № 4 (25) NATIONS AND RELIGIONS OF THE EURASIA Barnaul Publishing house of Altai State University 2020 ISSN 2542-2332 (Print) ISSN 2686-8040 (Online) 2020 № 4 (25) Редакционный совет журнала: Редакционный совет журнала: Л. Н. Ермоленко, доктор исторических наук (Россия, Кемерово) Ю. А. Лысенко, доктор исторических наук (Россия, Барнаул) Л. С. Марсадолов, доктор культурологии (Россия, Санкт-Петербург) Г. Г. Пиков, доктор исторических наук, доктор культурологии (Россия, Новосибирск) А. К. Погасий, доктор философских наук (Россия, Казань) К. А. Руденко, доктор исторических наук (Россия, Казань) С. А. Яценко, доктор исторических наук (Россия, Москва) А. С. Жанбасинова, доктор исторических наук (Казахстан, Усть-Каменогорск) Н. И. Осмонова, доктор философских наук (Кыргыстан, Бишкек) Н. Цэдэв, кандидат педагогических наук (Монголия, Улан-Батор) Ц. Степанов, доктор исторических наук (Болгария, София) З. С. Самашев, доктор исторических наук (Казахстан, Астаны). Журнал утвержден научно-техническим советом Алтайского государственного университета и зарегистрирован Комитетом РФ по печати. у р р р р Свидетельство о регистрации ПИ № ФС 77–78911 от 07.08.2020 г. Все права защищены. Ни одна из частей журнала либо издание в целом не могут быть перепечатаны без письменного разрешения авторов или издателя. у Свидетельство о регистрации ПИ № ФС 77–78911 от 07.08.2020 г. Все права защищены. Ни одна из частей журнала либо издание в целом не могут быть перепечатаны без письменного разрешения авторов или издателя. Журнал подготовлен при поддержке РНФ «Религия и власть: исторический опыт государственного регулирования деятельности религиозных общин в Западной Сибири и сопредельных районах Казахстана в XIX–XX вв.» (проект № 19‑18‑00023). Журнал подготовлен при поддержке РНФ «Религия и власть: исторический опыт государственного регулирования деятельности религиозных общин в Западной Сибири и сопредельных районах Казахстана в XIX–XX вв.» (проект № 19‑18‑00023). Адрес редакции: 656049, Барнаул, ул. Димитрова, 66, Адрес редакции: 656049, Барнаул, ул. Димитрова, 66, Алтайский государственный университет, кафедра религиоведения России, национальных и государственно-конфессиональных отношений. Адрес редакции: 656049, Барнаул, ул. Димитрова, 66, Алтайский государственный университет, кафедра религиоведения России, национальных и государственно-конфессиональных отношений. Алтайский государственный университет, кафедра религиоведения России, национальных и государственно-конфессиональных отношений. The editorial Board: The editorial Board: S. A. Vasutin, doctor of historical sciences (Russia, Kemerovo) N. L. Zhukovskay, doctor of historical sciences (Russia, Moskow) A. P. Zabiyako, doctor of philosophical sciences (Russia, Blagoveshchensk) A. A. Tishkin, doctor of historical sciences (Russia, Barnaul) N. А. Tomilov, doctor of historical sciences (Russia, Omsk) T. D. Skrynnikova, doctor of historical sciences (Russia, Saint-Petersburg) O. M. Homushku, doctor of philosophical sciences (Russia, Kyzyl) M. M. Shakhnovich, doctor of philosophical sciences (Russia, Saint-Petersburg) L. I. Sherstova, doctor of historical sciences (Russia, Tomsk) A. G. Sitdikov, doctor of historical sciences (Russia, Kazan) E. A. Shershneva (resp. secretary), candidate of historical sciences (Russia, Barnaul) NATIONS AND RELIGIONS OF THE EURASIA The journal was founded in 2007 The journal editorial Board: h j L. N. Yarmolenko, doctor of historical sciences (Russia, Kemerovo) U. A. Lusenko, doctor of historical sciences Russia, Barnaul) L. S. Marsadolov, doctor of Culturology (Russia, St. Petersburg) G. G. Pikov, doctor of historical sciences, doctor of cultural studies (Russia, Novosibirsk) A. K. Pogassiy, doctor of philosophical sciences (Russia, Kazan) K. A. Rudenko, doctor of historical sciences (Russia, Kazan) S. A. Yatsenko, doctor of historical sciences (Russia, Moscow) A. S. Zhanbosynov, doctor of historical sciences (Kazakhstan, Ust-Kamenogorsk) N. I. Osmonovа, candidate of philosophical sciences (Kyrgyzstan, Bishkek) N. Cedev, candidate of pedagogical sciences (Mongolia, Ulaanbaatar) Ts. Stepanov, doctor of historical sciences (Bolgariy, Sofiy) Z. S. Samashev, doctor of historical sciences (Kazakhstan, Astana) Approved for publication by the Joint Scientific and Technical Council of Altai State University. All rights reserved. No publication in whole or in part may be reproduced without the written permission of the authors or the publisher. Registered with the RF Committee on Printing. Registration certificate PI № ФС 77–78911. Registration date 07.08.2020 г. Approved for publication by the Joint Scientific and Technical Council of Altai State University. All rights reserved. No publication in whole or in part may be reproduced without the written permission of the authors or the publisher. Registered with the RF Committee on Printing. Registration certificate PI № ФС 77–78911. Registration date 07.08.2020 г. Approved for publication by the Joint Scientific and Technical Council of Altai State University. All rights reserved. No publication in whole or in part may be reproduced without the written permission of the authors or the publisher. Registered with the RF Committee on Printing. Registration certificate PI № ФС 77–78911. Registration date 07.08.2020 г. The journal was prepared with the support of the RSF grant on “Religion and power: the historical experience of state regulation of religious communities in Western Siberia and neighboring regions of Kazakhstan in the XIX–XX centuries” (project № 19‑18‑00023). The journal was prepared with the support of the RSF grant on “Religion and power: the historical experience of state regulation of religious communities in Western Siberia and neighboring regions of Kazakhstan in the XIX–XX centuries” (project № 19‑18‑00023). Editorial office address: 656049, Barnaul, ul. Dimitrova, 66, Altai State University, Department of regional studies of Russia, national and state-confessional relations. © Altai State University Publisher, 2020 СОДЕРЖАНИЕ Раздел I АРХЕОЛОГИЯ И ЭТНОКУЛЬТУРНАЯ ИСТОРИЯ Руденко К. А. Клады эпохи Волжской Булгарии на Старо-Куйбышевском археологическом комплексе в Татарстане: состав, датировка, интерпретация...............7 Дашковский П. К. Кочевые народы Центральной Азии эпохи Средневековья в исторических исследованиях российских ученых второй половины XIX — начала XX в.........................................................................................................................34 Нарудцева Е. А., Радовский С. С., Серегин Н. Н. К вопросу о значении полевых исследований Э. М. Медниковой в развитии археологии Алтая........................................50 Серегин Н. Н. Археология раннесредневековых тюрок Центральной Азии: основные этапы исследований и интерпретации комплексов...........................................65 Папин Д. В. Проблемы этнокультурного взаимодействия населения степного и лесостепного Алтая в период поздней бронзы...................................................................80 Раздел II ЭТНОЛОГИЯ И НАЦИОНАЛЬНАЯ ПОЛИТИКА Кожа М. Б., Балтабаева А. Ю. Туркестанская легенда о Ноевом ковчеге на вершине горы Казыкурт.........................................................................................................87 Гундова О. Е. Российская интеллектуальная элита XVIII — середины XIX в. об экономике казахов....................................................................................................................98 Лысенко Ю. А. Процесс возвращения беженцев — участников Туркестанского восстания 1916 г. из Китая органами советской власти в 20‑е гг. ХХ в..........................112 Раздел III РЕЛИГИОВЕДЕНИЕ И ГОСУДАРСТВЕННО-КОНФЕССИОНАЛЬНАЯ ПОЛИТИКА Дробышев Ю. И. Монгольский хаган как сын Бога..............................................................123 Шерстюков С. А. «Раскрепощение» мусульманских женщин в Центральной Азии: стратегии сопротивления и способы адаптации (1920–1930‑е гг.)..................... 148 ДЛЯ АВТОРОВ...........................................................................................................................161 ISSN 2542-2332 (Print) • ISSN 2686-8040 (Online) CONTENT Section I ARCHAEOLOGY AND ETNO-CULTURAL HISTORY Rudenko K. A. Treasures of the era of the Volga Bulgaria at the old kuibyshev’s archaeological complex in Tatarstan: composition, dating, interpretation................................7 Dashkovskiy P. K. The nomadic nations of Central Asia of the Middle ages in historical research of russian scientists of the second half of XIX — early XX century archeology and etno-kultural history.............................................................34 Narudtseva E. A., Radovsky S. S., Seregin N. N. To the question of the importance of field research of E. M. Mednikova in the development of archeology in Altay...................50 Seregin N. N. Archeology of the early medieval turks of Central Asia: main stages of research and interpretations of the complexes........................................................................65 Papin D. V. Problems of ethnic and cultural interaction of the population steppe and forest-steppe Altai in the late bronze age...............................................................................80 Section II ETHNOLOGY AND NATIONAL POLICY Kozha M. B., Baltabayeva A. Yu. Turkestan legend of Noah’s ark on top of mount Kazygurt...........................................................................................................................87 Gundova O. E. Russian intellectual elite of the XVIII — middle of XIX centuries about the economy of the kazakhs.................................................................................................98 Lysenko Y. A. The process of returning refugees –participants of the Turkestan uprising of 1916 from China by the organs of Soviet power in the 1920s of the 20th century...........................................................................................................................112 Section III RELIGIOUS STUDIES AND STATE-CONFESSIONAL RELATIONS Drobyshev Yu. I. Mongolian khagan as a son of the God..........................................................123 Sherstyukov S. A. “Emancipation” of muslim women in Central Asia: strategies of resistance and ways of adaptation (1920s–1930s)................................................................ 148 FOR AUTHORS............................................................................................................................161 Алтайский государственный университет, Барнаул (Россия лтайский государственный университет, Барнаул (Россия) АРХЕОЛОГИЯ РАННЕСРЕДНЕВЕКОВЫХ ТЮРОК ЦЕНТРАЛЬНОЙ АЗИИ: ОСНОВНЫЕ ЭТАПЫ ИССЛЕДОВАНИЙ И ИНТЕРПРЕТАЦИИ КОМПЛЕКСОВ1 Статья посвящена характеристике истории исследования и интерпретации археоло- гических памятников раннесредневековых тюрок Центральной Азии. Учтен значитель- ный объем сведений о результатах изучения погребальных и «поминальных» комплек- сов второй половины I тыс. н. э., расположенных в разных частях обширного региона. Обоснована периодизация, предполагающая выделение четырех основных этапов в ис- тории исследования памятников тюрок: 1) XVIII в. — начало XX в.; 2) 1920‑е — 1940‑е гг.; 3) 1950‑е — начало 1990‑х гг.; 4) вторая половина 1990‑х гг. — начало XXI в. Для обозначен- ных периодов характерны особенности методологии и методики работ, различная интен- сивность практических и теоретических изысканий, уровень интерпретации имеющих- ся сведений. Установлено, что наиболее разработанными являются вопросы исследова- ния предметного комплекса номадов. Кроме того, значительное количество публикаций посвящено решению проблем хронологии конкретных объектов и периодизации куль- туры кочевников. Наименее изученными остаются проблемы реконструкции сложных процессов формирования и эволюции общности тюрок по археологическим материалам. Ключевые слова: тюрки, Центральная Азия, история исследований, археологиче- ские памятники, периодизация, интерпретация. N. N. Seregin Altai State University, Barnaul (Russia) Section I ARCHAEOLOGY AND ETNO-CULTURAL HISTORY Rudenko K. A. Treasures of the era of the Volga Bulgaria at the old kuibyshev’s archaeological complex in Tatarstan: composition, dating, interpretation................................7 Dashkovskiy P. K. The nomadic nations of Central Asia of the Middle ages in historical research of russian scientists of the second half of XIX — early XX century archeology and etno-kultural history.............................................................34 Narudtseva E. A., Radovsky S. S., Seregin N. N. To the question of the importance of field research of E. M. Mednikova in the development of archeology in Altay...................50 Seregin N. N. Archeology of the early medieval turks of Central Asia: main stages of research and interpretations of the complexes........................................................................65 Papin D. V. Problems of ethnic and cultural interaction of the population steppe and forest-steppe Altai in the late bronze age...............................................................................80 Section II ETHNOLOGY AND NATIONAL POLICY Kozha M. B., Baltabayeva A. Yu. Turkestan legend of Noah’s ark on top of mount Kazygurt...........................................................................................................................87 Gundova O. E. Russian intellectual elite of the XVIII — middle of XIX centuries about the economy of the kazakhs.................................................................................................98 Lysenko Y. A. The process of returning refugees –participants of the Turkestan uprising of 1916 from China by the organs of Soviet power in the 1920s of the 20th century...........................................................................................................................112 Section III RELIGIOUS STUDIES AND STATE-CONFESSIONAL RELATIONS Drobyshev Yu. I. Mongolian khagan as a son of the God..........................................................123 Sherstyukov S. A. “Emancipation” of muslim women in Central Asia: strategies of resistance and ways of adaptation (1920s–1930s)................................................................ 148 FOR AUTHORS............................................................................................................................161 ISSN 2542-2332 (Print) • ISSN 2686-8040 (Online) 65 Nations and religions of Eurasia • 2020 № 4 (25). P. 65–79. УДК 902/904 DOI: 10.14258/nreur(2020)4–04 УДК 902/904 DOI: 10.14258/nreur(2020)4–04 1 Работа подготовлена в рамках государственного задания Алтайского государственного универси‑ тета, проект № 748715Ф.99.1. ББ97АА00002 «Тюрко-монгольский мир «Большого Алтая»: един‑ ство и многообразие в истории и современности». Cайт журнала: http://journal.asu.ru/wv • Journal homepage: http://journal.asu.ru/wv Н. Н. Серегин Н. Н. Серегин Алтайский государственный университет, Барнаул (Россия) ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES The article presents the characteristics of the history of research and interpretation of archaeological sites of the early medieval Turks in Central Asia. A significant amount 1 Работа подготовлена в рамках государственного задания Алтайского государственного универси‑ тета, проект № 748715Ф.99.1. ББ97АА00002 «Тюрко-монгольский мир «Большого Алтая»: един‑ ство и многообразие в истории и современности». 66 Народы и религии Евразии • 2020 № 4 (25). C. 65–79. of information on the results of the study of burial and «memorial» complexes of the second half of the I millennium AD, located in different parts of the vast region, was taken into account. The author created the periodization, which implies the allocation of four main stages in the history of the study of the Turkic sites: 1) the XIX century — the beginning of the XX century; 2) 1920s — 1940s; 3) 1950s — early 1990s; 4) the second half of the 1990s — the beginning of the XXI century. The designated periods are characterized by the peculiarities of the methodology and methodology of work, different intensity of practical and theoretical research, the level of interpretation of the available information. It has been established that the most developed are the issues of research of the subject complex of nomads. In addition, a significant number of publications are devoted to solving the problems of the chronology of specific objects and the periodization of the culture of nomads. The problems of reconstruction of the complex processes of formation and evolution of the Turks’ community based on archaeological materials remain the least studied. Keywords: Turks, Central Asia, history of research, archaeological sites, periodization, interpretation Серегин Николай Николаевич, доктор исторических наук, доцент кафедры археоло- гии, этнографии и музеологии Алтайского государственного университета, Барнаул (Россия). Адрес для контактов: nikolay-seregin@mail.ru Seregin Nikolay Nikolaevich, Doctor of Historical Sciences, Associate Professor of the Department of Archeology, Ethnography and Museology of Altai State University, Barnaul (Russia). Contact address: nikolay-seregin@mail.ru И зучение этногенетических и социокультурных процессов, происходивших на территории Центральной Азии в раннем Средневековье, основывается глав- ным образом на исследовании материалов раскопок археологических памятни- ков. Это обусловлено фрагментарностью письменных источников, в которых представ- лены лишь отдельные сюжеты истории кочевников. Одной из ярких общностей нома- дов рассматриваемого региона второй половины I тыс. н. э. является культура ранне- средневековых тюрок. ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES Основным источником для реконструкции истории ее носителей выступают археологические комплексы — погребения, «поминальные» объекты (ме- мориалы элиты номадов, «рядовые» оградки, изваяния, балбалы и другие сопроводи- тельные сооружения), наскальные изображения. Важным аспектом их изучения пред- ставляется анализ процесса накопления материалов, полученных в ходе полевых иссле- дований, а также рассмотрение выводов, ставших результатом интерпретации памят- ников. Проведение такой работы позволит не только охарактеризовать сформирован- ную источниковую базу и суммировать ключевые выводы ученых, но также будет спо- собствовать определению приоритетных направлений дальнейших изысканий и обо- значению путей решения вопросов, остающихся дискуссионными. И Специального исследования, посвященного анализу истории изучения археологи- ческих комплексов раннесредневековых тюрок Центральной Азии, до сих пор не пред- принималось. В ряде работ археологов представлен опыт рассмотрения основных эта- ISSN 2542-2332 (Print) • ISSN 2686-8040 (Online) 67 Nations and religions of Eurasia • 2020 № 4 (25). P. 65–79. пов исследований памятников на отдельных территориях [Гаврилова, 1965: 5–6; Кыз- ласов, 1969: 5–14; Кубарев, 1984: 4–11; Митько, 1995: 5–6; Войтов, 1996: 11–22; Худя- ков, 2004: 6–14; Кубарев, 2005: 6–9; Серегин, Матренин, 2014: 61–116; Идэрхангай, 2017: 8–10]. Кроме того, результаты раскопок погребальных и «поминальных» комплексов тюрок, а также раннесредневековых наскальных изображений учтены в специальных монографиях по истории археологического изучения Сибири [Белокобыльский, 1986; Демин, 1989; Борисенко, Худяков, 2005; Китова, 2007; Матющенко, 2009; Артюх, 2010]. у В целом, несмотря на то, что различные аспекты истории изучения археологиче- ских памятников раннесредневековых тюрок затрагивались в целом ряде работ, мно- гие вопросы в этом направлении по‑преднему рассмотрены лишь частично. В боль- шинстве исследований представлена лишь общая история археологических раскопок без выявления основных тенденций в интерпретации материалов, а также в развитии методики исследований. Фрагментарным является опыт осмысления развития зна- ний о комплексах рассматриваемой общности на обширных территориях Централь- ной Азии, так как ученые в большинстве случаев ограничивались рассмотрением про- цесса исследования памятников в отдельных регионах. Наконец, отсутствует разрабо- танная схема периодизации истории изучения археологических комплексов раннесред- невековых тюрок, что также не способствует эффективной систематизации обширно- го накопленного опыта. Учитывая огромный объем имеющихся данных, необходимо признать, что резуль- таты анализа обозначенных историографических аспектов требуют развернутого изло- жения в работе монографического характера. Поэтому в настоящей статье представле- ны только основные вехи в изучении и интерпретации комплексов раннесредневеко- вых тюрок Центральной Азии с краткой характеристикой выделенных этапов и опре- делением ключевых тенденций исследовательского процесса. Анализ работ исследователей позволяет выделить четыре этапа в процессе накоп- ления и интерпретации археологических материалов по культуре раннесредневековых тюрок Центральной Азии. Cайт журнала: http://journal.asu.ru/wv • Journal homepage: http://journal.asu.ru/wv ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES Для каждого из периодов характерны особенности методо- логии и методики работ, различная интенсивность практических и теоретических изы- сканий, уровень интерпретации имеющихся сведений и др. Характеристики выделен- ных этапов в значительной степени определялись спецификой развития исторической и археологической науки, а также общей социально-экономической ситуацией в раз- личных частях Центрально-Азиатского региона. Первый этап (XVIII в. — начало XX в.) — время первоначального ознакомления ученых с раннесредневековыми материалами Центральной Азии. Основной объем по- лученных материалов связан с выявлением и фиксацией каменных изваяний, а также немногочисленными раскопками «поминальных» комплексов второй половины I тыс. н. э. Кроме того, в рамках рассматриваемого периода были впервые раскопаны погре- бения раннесредневековых тюрок. Важными событиями, определившими на многие годы направления развития отечественной и зарубежной тюркологии, стали открытия «элитных» мемориалов тюрок Монголии и дешифровка рунической письменности. За- вершение первого этапа связано с началом систематических исследований археологи- ческих комплексов раннесредневековых номадов Центральной Азии в 1920‑е гг. и по- 68 Народы и религии Евразии • 2020 № 4 (25). C. 65–79. лучением значительного объема материалов, анализ которых дал возможность для ре- конструкции различных аспектов истории кочевников данного обширного региона. лучением значительного объема материалов, анализ которых дал возможность для ре- конструкции различных аспектов истории кочевников данного обширного региона. Отдельно отметим большое значение раскопок, осуществленных В. В. Радловым на комплексе Катанда в юго-западной части Алтая. В 1865 г. известным ученым иссле- дованы несколько небольших курганов, четыре из которых не были потревожены и от- носились к культуре тюрок, а также четыре каменные оградки и впускные захоронения второй половины I тыс. н. э. [Захаров, 1926: 76, 81; Радлов, 1989: 448–449]. Анализ мате- риалов, полученных в ходе экспедиции, позволил В. В. Радлову выделить несколько эта- пов в древней и средневековой истории Сибири. Памятники культуры тюрок не обо- значены в каком‑либо из них, однако общая логика построений ученого позволяет утверждать, что данные объекты, наряду с «киргизскими могилами», отнесены к «но- вейшей эпохе железного века». Отметим, что в настоящее время актуальным остается осуществление полной публикации результатов исследований В. В. Радлова на Алтае. Хорошо известна также роль ученого в процессе исследования «элитных» тюркских комплексов на территории Монголии. Результатом его изысканий, а также работ дру- гих специалистов, осуществленных в данном регионе в конце XIX в., стало обоснова- ние назначения таких объектов как мемориальных памятников верхушки каганатов и их идентификация с конкретными фигурами политической истории тюрок, деши- фровка рунической письменности и прочтение обнаруженных текстов, а также под- тверждение того, что подобные сооружения не являются единичными. ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES Полученные све- дения показали перспективы дальнейших исследований истории раннесредневековых тюрок по археологическим материалам. Второй этап (1920–1940‑е гг.) — осуществление первых крупных исследований по- гребальных и «поминальных» комплексов раннесредневековых тюрок на Алтае, в Туве, Минусинской котловине. Реализация подобных изысканий в Монголии была связана, главным образом, с изучением мемориальных памятников элиты номадов. Результа- ты интерпретации материалов раскопок комплексов стали основой для создания ряда обобщающих работ. Начало нового этапа в исследовании археологических памятников Алтае-Саянского региона, в том числе комплексов раннего Средневековья, связано с работами экспеди- ций С. А. Теплоухова. Наиболее масштабные изыскания были им реализованы на тер- ритории Минусинской котловины. Отряд Минусинской экспедиции под руководством С. А. Теплоухова проводил раскопки в 1920–1921 гг. и 1923–1925 гг. [Белокобыльский, 1986: 117]. Важно отметить, что археолог изначально предполагал осуществить рабо- ты на небольшой по площади территории, однако насыщенной различными по хро- нологии и культурной принадлежности памятниками. Именно в результате таких ис- следований могла быть прослежена преемственность в развитии культур и специфи- ка исторической ситуации в регионе [Белокобыльский, 1986: 133]. Наряду с комплек- сами различных хронологических периодов, раскопаны и памятники раннесредневе- ковых тюрок. В 1924 г. исследованы пять погребений у с. Батени на Енисее, а в 1925 г. — одиночный курган на р. Таштык. ур р В 1926–1927 гг. и 1929 гг. в соседнем регионе археологические работы проводи- лись Тувинской археологической экспедицией, руководителем которой также являл- ISSN 2542-2332 (Print) • ISSN 2686-8040 (Online) 69 Nations and religions of Eurasia • 2020 № 4 (25). P. 65–79. ся С. А. Теплоухов [Китова, 2007: 79]. Сведения о погребениях тюрок, исследованных С. А. Теплоуховым на некрополях Бай-Даг и Улуг-Хову, а также раннесредневековых оградках, раскопанных на комплексах Саадак-Терек и Элегест, частично введены в на- учный оборот Л. Р. Кызласовым [1979: 132–135, 188–191]. ся С. А. Теплоухов [Китова, 2007: 79]. Сведения о погребениях тюрок, исследованных С. А. Теплоуховым на некрополях Бай-Даг и Улуг-Хову, а также раннесредневековых оградках, раскопанных на комплексах Саадак-Терек и Элегест, частично введены в на- учный оборот Л. Р. Кызласовым [1979: 132–135, 188–191]. Раскопки в Минусинской котловине в районе с. Батени, а также в Туве позволили С. А. Теплоухову осуществить систематизацию памятников от энеолита до эпохи Сред- невековья и представить их культурную, хронологическую и этническую характери- стику. Памятники раннего Средневековья не были включены в какую‑либо археоло- гическую культуру, однако распределены по этапам с обозначением датировки и этни- ческой принадлежности [Теплоухов, 1929: 55, 58]. Погребения тюрок отнесены ученым к VII–X вв. По мнению С. А. Cайт журнала: http://journal.asu.ru/wv • Journal homepage: http://journal.asu.ru/wv ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES Теплоухова [1929: 55], коренным населением Минусинской котловины были кыргызы, которых в VII в. покорили «турки». В это же время после долгого перерыва осуществлялись раскопки памятников ран- несредневековых тюрок на Алтае. Работы на ряде памятников, в том числе и относя- щихся ко второй половине I тыс. н. э., проведены в 1924–1925 гг. Алтайской экспедици- ей этнографического отдела Русского музея под руководством С. И. Руденко и при уча- стии А. Н. Глухова. В ходе исследований участников экспедиции обнаружена и раско- пана серия «поминальных» объектов, расположенных в долинах рек Бугузун, Чулыш- ман, Улаган и Балыктуюль (Восточный Алтай). Наиболее значимыми для развития ран- несредневековой археологии не только Алтая, но и всей Центральной Азии стали ис- следования погребений и оградок на комплексе Кудыргэ, расположенном в низовьях р. Чулышман, в восточной части региона [Руденко, Глухов, 1927]. По точному замечанию А. А. Гавриловой [1965: 3], после выхода статьи С. И. Руден- ко и А. Н. Глухова материалы раскопок памятника Кудыргэ так или иначе упоминались практически во всех работах, посвященных различным аспектам раннесредневековой археологии. До сих пор данный комплекс остается одним из крупнейших комплексов тюрок. Несмотря на неизбежные на тот момент неточности в интерпретации результа- тов раскопок могильника, выводы и наблюдения, сделанные С. И. Руденко и А. Н. Глу- ховым, представляли большой интерес для современников. Отметим, что в последую- щие годы хронология известного памятника, а также этнокультурная атрибуция по- гребальных и «поминальных» объектов комплекса неоднократно становились предме- том специального рассмотрения целого ряда специалистов. К 1920‑м гг. относится начало систематических исследований на территории Мон- голии экспедиций, организованных Академией наук СССР совместно с местными ис- следователями. В 1925 гг. И. Боровкой [1927] впервые раскопано тюркское погребение на обозначенной территории, а также изучены мемориальные комплексы. Исследова- ния «поминальных» памятников элиты каганатов в это время осуществлены Б. Я. Вла- димирцовым [1927], который представил первый опыт классификации таких объектов. р р р р ф Серия тюркских погребений и оградок в конце 1920‑х — 1930‑е гг. раскопана П. М. Грязновым на Алтае. Кроме того, им совместно с Е. Р. Шнейдером [1929] обобщены сведения о каменных изваяниях Минусинской котловины, в том числе о нескольких ран- несредневековых скульптурах. Объекты второй половины I тыс. н. э. учтены М. П. Гряз- новым в разработанной периодизации древних и средневековых культур Алтая. 70 Народы и религии Евразии • 2020 № 4 (25). C. 65–79. Масштабные исследования памятников различных хронологических периодов, в том числе объектов раннего Средневековья, осуществлены в 1928–1940 гг. Саяно-Ал- тайской экспедицией под руководством С. В. Киселева. ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES Ру- ISSN 2542-2332 (Print) • ISSN 2686-8040 (Online) Nations and religions of Eurasia • 2020 № 4 (25). P. 65–79. 71 денко. В последующие годы А. А. Гавриловой осуществлены работы и на других памят- никах Алтая. Анализ полученных материалов с использованием результатов раскопок на сопредельных территориях отражен в обобщающей монографии [Гаврилова, 1965]. В послевоенные годы наиболее интенсивные раскопки погребальных и «поминаль- ных» комплексов тюрок осуществлялись на территории Тувы. В 1950–1960‑е гг. в дан- ном регионе проводились исследования под руководством С. И. Вайнштейна, А. Д. Гра- ча, Л. Р. Кызласова, М. X. Маннай-оола. В работах археологов представлен опыт опреде- ления хронологии изученных памятников, интерпретации каменных изваяний и огра- док, реконструкции этнокультурной истории региона [Грач, 1961; Вайнштейн, 1966; Кызласов, 1969]. К середине XX столетия относится начало следующего этапа в изучении раннесред- невековой археологии Монголии. Он связан главным образом с работами местных спе- циалистов, к этому времени накопивших необходимый опыт полевых работ и продол- живших исследования памятников региона самостоятельно, а также совместно с уче- ными из социалистических стран. Новые сведения о погребальных и «поминальных» комплексах тюрок Монголии получены в 1950–1960‑е гг. в ходе исследований Ц. До- ржсурэна, Д. Навана, Х. Пэрлээ, Н. Сэр-Оджава, И. Эрдели. Значительный объем материалов по культуре раннесредневековых тюрок получен в результате работ «новостроечных» экспедиций. Серия тюркских погребений исследо- вана участниками Красноярской экспедиции Института археологии АН СССР в Мину- синской котловине. Большое количество курганов, «поминальных» объектов и наскаль- ных изображений раннего Средневековья изучено в ходе изысканий Саяно-Тувинской экспедиции Ленинградского отделения Института археологии АН СССР в Туве. Резуль- таты анализа массива новых материалов представлены в серии тематических публика- ций [Трифонов, 1971; Длужневская, 1976; Овчинникова, 1982]. Интенсивные исследования памятников тюрок в 1970‑е — начале 1990‑х гг. проводи- лись на Алтае. Изучение раннесредневековых погребальных и «поминальных» комплек- сов, а также наскальных изображений в данном регионе осуществлялось А. П. Бородов- ским, А. С. Васютиным, А. М. Илюшиным, С. М. Киреевым, Ю. Ф. Кирюшиным, В. Д. Ку- баревым, В. А. Кочеевым, Ю. Т. Мамадаковым, А. И. Мартыновым, Е. А. Миклашевич, О. А. Митько, В. А. Могильниковым, В. И. Молодиным, С. В. Неверовым, Е. А. Окладни- ковой, Д. Г. Савиновым, В. И. Соеновым, А. И. Соловьевым, С. С. Сорокиным, А. С. Су- разаковым, Ю. В. Тетериным, Ю. С. Худяковым, А. В. Эбелем. Определенный объем ма- териалов по культуре тюрок получен в ходе работ на территории Минусинской кот- ловины и Тувы, реализованных Г. П. Данченок, Э. Н. Киргинековым, Л. Р. Кызласовым, О. А. Митько, Ю. А. ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES В ходе этих работ изучена пред- ставительная серия погребальных и «поминальных» комплексов тюрок Алтая и Ми- нусинской котловины [Киселев, 1929; Евтюхова, Киселев, 1941]. Тюркские захороне- ния в последнем из обозначенных регионов исследованы под руководством В. П. Ле- вашовой [1952]. В целом, к середине XX в. была сформирована определенная источниковая база для изучения и интерпретации археологических комплексов раннесредневековых тю- рок, включавшая главным образом памятники Алтая, Минусинской котловины и Мон- голии. Материалы исследований, частично опубликованные археологами, стали извест- ны широкому кругу специалистов. Памятники раннего Средневековья были отдель- но рассмотрены в ряде обобщающих работ, в которых получил отражение опыт хро- нологической и этнокультурной атрибуции объектов, а также наблюдения о специфи- ке социальной организации раннесредневековых кочевников [Евтюхова, 1948; Кисе- лев, 1949]. Вместе с тем практически неизвестными археологам оставались погребаль- ные и «поминальные» комплексы тюрок на территории Тувы. Материалы раскопок С. А. Теплоухова в указанном регионе были опубликованы значительно позже. Имен- но на этой территории в последующие годы проводились наиболее интенсивные иссле- дования археологических памятников раннего Средневековья. Весьма перспективны- ми оставались также раскопки тюркских погребений в Монголии, остающейся в этом плане «белым пятном» на археологической карте Центральной Азии. Третий этап (1950‑е — начало 1990‑х гг.) — время наиболее интенсивных иссле- дований археологических комплексов раннесредневековых тюрок Центральной Азии. Развитие науки в начале данного периода в значительной степени связано с общим по- слевоенным экономическим подъемом в России, а также сложением группы специали- стов-археологов в Монголии. Главной тенденцией третьего периода стало преимуще- ственное развитие региональной тематики, заключавшееся в рассмотрении различных аспектов истории раннесредневековых кочевников Центральной Азии на основе изуче- ния памятников отдельных территорий. Характерной чертой научной деятельности ар- хеологов в рамках данного этапа было стремление к историчности выводов, сделанных на основе анализа материалов раскопок. Этому способствовало активное привлечение сведений письменных источников, главным образом, китайских династийных хроник и тюркских рунических текстов. К рассматриваемому периоду относится публикация ряда крупных работ, в которых были обобщены обширные накопленные материалы. Конец 1940‑х — 1950‑е гг. в истории археологических исследований в различных ча- стях Центральной Азии связаны в значительной степени с продолжением намеченных ранее работ. Это объяснялось как вынужденным перерывом в уже организованных изысканиях на ряде комплексов, так и очевидными перспективами в доисследовании некоторых известных объектов. Одним из таких памятников стал могильник Кудыргэ, материалы раскопок которого имели огромное значение для развития представлений по раннесредневековой археологии региона. В 1948 г. работы на данном погребально- поминальном комплексе были продолжены А. А. Гавриловой при содействии С. И. Cайт журнала: http://journal.asu.ru/wv • Journal homepage: http://journal.asu.ru/wv ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES Плотниковым, А. И. Поселяниным, Д. Г. Савиновым, Ю. В. Тетери- ным, Ю. С. Худяковым. у Увеличение объема сведений о памятниках тюрок Монголии в 1969–1989 гг. связа- но с работами совместной Советско-Монгольской историко-культурной экспедиции. В ходе исследований В. Е. Войтова, С. Г. Кляшторного, В. А. Лившиц, Н. Сэр-Оджава получены значительные результаты в области выявления новых, а также детального изучения уже известных мемориалов знати раннесредневековых кочевников. В 1979– 1982 гг. серия «рядовых» оградок раннего Средневековья обследована Ю. С. Худяко- 72 Народы и религии Евразии • 2020 № 4 (25). C. 65–79. вым. Раскопки нескольких тюркских погребений в разных частях страны проведены Х. Лхагвасурэном, Д. Наваном, Ю. С. Худяковым, Д. Цэвээндоржем. вым. Раскопки нескольких тюркских погребений в разных частях страны проведены Х. Лхагвасурэном, Д. Наваном, Ю. С. Худяковым, Д. Цэвээндоржем. Разноплановая интерпретация накопленных обширных материалов осуществлена рядом специалистов. В большей части работ представлен опыт систематизации па- мятников тюрок, изученных в отдельных регионах [Сэр-Оджав, 1970; Худяков, 1979; Васютин, 1983; Кубарев, 1984]. Кроме того, подготовлена серия обобщающих иссле- дований, посвященных анализу результатов раскопок археологических комплексов тюрок Центральной Азии [Могильников, 1981; Савинов, 1984; Худяков, 1986; Овчи- никова, 1990]. Таким образом, 1950‑е — начало 1990‑х гг. стали весьма важным периодом в из- учении археологических памятников раннесредневековых тюрок Центральной Азии, имеющим определяющее значение для формирования и развития представлений по различным аспектам истории номадов. В результате полевых исследований, осуще- ствленных в это время, был накоплен обширный материал, демонстрирующий вариа- бельность культуры кочевников на различных территориях ее распространения во вто- рой половине I — начале II тыс. н. э. В работах целого ряда ученых состоялась систе- матизация этих сведений, что заложило основы для дальнейших исследований в обо- значенном направлении. Не менее важным стало определение дискуссионных вопро- сов, требующих детальной проработки. Многие из них были раскрыты в статьях и мо- нографиях археологов конца XX — начала XXI вв. Четвертый этап (вторая половина 1990‑х гг. — начало XXI в.). Во второй поло- вине 1990‑х гг. объем археологических исследований во многих частях Центральной Азии существенно сократился. В определенной степени это было связано с недостат- ком финансирования экспедиций. Однако одной из основных причин, затруднивших проведение раскопок в ряде регионов, прежде всего в Республике Алтай, стала слож- ная этносоциальная обстановка, проявившаяся, в том числе, в противодействии осу- ществлению полевых исследований. Работы археологических экспедиций осложнялись негативным отношением к ним местного населения, в значительной степени сформи- рованным искусственно. ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES К сожалению, определенная степень напряженности между археологами и жителями Республики Алтай, несмотря на отдельные позитивные тен- денции в этом плане, сохраняется [Плетц и др., 2014]. Очевидно, проводившиеся в рам- ках рассматриваемого периода сравнительно немногочисленные исследования были сосредоточены преимущественно в северной части обозначенного региона, где ситуа- ция несколько более спокойная. Кроме того, в большей степени в это время изучались «поминальные» комплексы, а раскопки погребений стали возможными, главным об- разом, только в ходе охранных археологических работ в зонах строительства хозяй- ственных объектов. Напротив, в Монголии в обозначенный период наблюдается активизация архео- логических изысканий. Изменение политической ситуации в стране в начале 1990‑х гг. серьезным образом отразилось на проводимых полевых исследованиях. С этого времени в стране работают многочисленные экспедиции ученых из стран ближне- го и дальнего зарубежья — Бельгии, Великобритании, Германии, Италии, Казахста- на, Кореи, США, Турции, Франции, Японии и др. Эти исследования, осуществляе- ISSN 2542-2332 (Print) • ISSN 2686-8040 (Online) 73 Nations and religions of Eurasia • 2020 № 4 (25). P. 65–79. мые на высоком уровне и с применением современных методик, не только позволи- ли получить и ввести в научный оборот значительный объем материалов, но также послужили важным стимулом для развития археологической науки в Монголии в це- лом. Результатом работ совместных экспедиций монгольских исследователей и зару- бежных специалистов стали исследованные памятники различных хронологических периодов, в том числе серия погребений и «поминальных» комплексов раннесредне- вековых тюрок. Наряду с исследованиями, осуществляемыми совместно с зарубеж- ными учеными, монгольские археологи проводят и самостоятельные работы в раз- ных частях страны. Ключевой тенденцией современного этапа в изучении археологических комплек- сов раннесредневековых тюрок Центральной Азии является не интенсивность рас- копок, а в большей степени повышение уровня интерпретации уже накопленных обширных материалов. Важным моментом стало появление серии обобщающих ра- бот. В большом количестве статей, ряде монографий и диссертационных сочинени- ях представлены результаты исследований, посвященных анализу специфики фор- мирования и развития культуры номадов во второй половине I тыс. н. э. [Митько, 1995; Худяков, 2004; Кубарев, 2005; Тишкин, Горбунов, 2005; Горбунов, 2006; Шелепо- ва, 2009; Идэрхангай, 2017]. В результате полевых работ, проведенных в разных частях Центрально-Азиатско- го региона, сформирована представительная источниковая база археологических па- мятников, отражающая различные этапы истории тюрок. К настоящему времени рас- копаны более 400 погребений, а также свыше 450 «поминальных» комплексов, вклю- чающих различные сооружения. В музейных центрах, локализованных главным обра- зом в России и Монголии, хранится значительный объем коллекций, демонстрирую- щих материальную культуру раннесредневековых номадов. Накоплена огромная ин- формация о результатах разноплановых обследований тюркских комплексов. Cайт журнала: http://journal.asu.ru/wv • Journal homepage: http://journal.asu.ru/wv БИБЛИОГРАФИЧЕСКИЙ СПИСОК Артюх Е. А. Алтайский период в научной деятельности В. В. Радлова. Барнаул : Изд-во Алт. ун-та, 2010. 295 с. Белокобыльский Ю. Г. Бронзовый и ранний железный век Южной Сибири. История идей и исследований (XVIII — первая треть XX в.). Новосибирск : Наука, 1986. 168 с. Борисенко А. Ю., Худяков Ю. С. Изучение древностей Южной Сибири немецкими учеными XVIII–XIX вв. Новосибирск : изд. НГУ, 2005. 270 с. Боровка Г. И. Археологическое обследование среднего течения р. Толы // Северная Монголия. Предварительные отчеты лингвистической и археологической экспедиций о работах, произведенных в 1925 году. 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Отчет о работах Саяно-Алтайской археологической экспедиции в 1935 г. // Труды ГИМ. 1941. ARCHEOLOGY OF THE EARLY MEDIEVAL TURKS OF CENTRAL ASIA: MAIN STAGES OF RESEARCH AND INTERPRETATIONS OF THE COMPLEXES Нет со- мнений в том, что информационный потенциал этих обширных данных реализован далеко не полностью. Проведенный историографический анализ позволил определить степень изучен- ности различных аспектов исследования археологических памятников тюрок Цен- тральной Азии. Установлено, что наиболее разработанными являются вопросы иссле- дования предметного комплекса номадов. Кроме того, значительное количество пуб- ликаций посвящено решению проблем хронологии конкретных объектов и периоди- зации культуры кочевников. Наименее изученными остаются проблемы реконструк- ции сложных процессов формирования и эволюции различных элементов материаль- ной и духовной культуры тюрок. Перспективы расширения и детализации представле- ний об истории тюрок связаны с проведением точечных полевых исследований в сла- бо изученных частях Центральной Азии. Наиболее важными являются раскопки по- гребальных и «поминальных» комплексов на территории Монголии, а также в Китае (Синьцзян). Не меньшее значение имеет выявление и исследование памятников тю- рок в Казахстане и Средней Азии, что позволит более подробно рассматривать исто- рию недолгого пребывания номадов в данных регионах, а также процессы последую- щей трансформации их традиций. Народы и религии Евразии • 2020 № 4 (25). C. 65–79. 74 БИБЛИОГРАФИЧЕСКИЙ СПИСОК Вып 16. С. 75–117. Захаров А. А. Материалы по археологии Сибири (раскопки В. В. Радлова в 1965 г.) // Труды ГИМ. 1926. Вып. 1. С. 71–106. Идэрхангай Т.-О. Тюркские оградки Монгольского Алтая: систематизация, хроно- логия и интерпретация : автореф. дис. … канд. ист. наук. Барнаул, 2017. 26 с. Киселев С. В. 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ISSN 2542-2332 (Print) • ISSN 2686-8040 (Online) 79 Nations and religions of Eurasia • 2020 № 4 (25). P. 65–79. Цитирование статьи: Серегин Н. Н. Археология раннесредневековых тюрок Центральной Азии: основ- ные этапы исследований и интерпретации комплексов // Народы и религии Евразии. 2020. № 4 (25). С. 65–79. Citation: Seregin N. N. Archeology of the early medieval turks of Central Asia: main stages of research and interpretations of the complexes. Nations and religions of Eurasia. 2020. № 4 (25). P. 65–79. Цитирование статьи: Цитирование статьи: Ц р Серегин Н. Н. Археология раннесредневековых тюрок Центральной Азии: основ- ные этапы исследований и интерпретации комплексов // Народы и религии Евразии. 2020. № 4 (25). С. 65–79. Ц р Серегин Н. Н. Археология раннесредневековых тюрок Центральной Азии: основ- ные этапы исследований и интерпретации комплексов // Народы и религии Евразии. 2020. № 4 (25). С. 65–79. Seregin N. N. Archeology of the early medieval turks of Central Asia: main stages of research and interpretations of the complexes. Nations and religions of Eurasia. 2020. № 4 (25). P. 65–79.
https://openalex.org/W2299507679	https://europepmc.org/articles/pmc2980245?pdf=render	Latin	null	6-(1-Methylethyl)-12-phenyl-5,6,7,12-tetrahydrodibenz[<i>c</i>,<i>f</i>][1,5]azasilocine	Acta crystallographica. Section E	2,009	cc-by	3,906	organic compounds b = 10.269 (7) A˚ c = 18.912 (12) A˚ = 92.745 (3) V = 1893 (2) A˚ 3 Z = 4 Mo K radiation = 0.13 mm1 T = 120 K 0.20 0.20 0.10 mm Data collection Rigaku Mercury CCD diffractometer Absorption correction: multi-scan (REQAB; Jacobson, 1998) Tmin = 0.975, Tmax = 0.987 11962 measured reﬂections 3278 independent reﬂections 2798 reﬂections with I > 2(I) Rint = 0.029 Reﬁnement R[F 2 > 2(F 2)] = 0.041 wR(F 2) = 0.100 S = 1.08 3278 reﬂections 232 parameters H atoms treated by a mixture of independent and constrained reﬁnement max = 0.30 e A˚ 3 min = 0.30 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Mo K radiation = 0.13 mm1 T = 120 K 0.20 0.20 0.10 mm Kei Goto,a* Akihiro Fukushimab and Takayuki Kawashimab Reﬁnement R[F 2 > 2(F 2)] = 0.041 wR(F 2) = 0.100 S = 1.08 3278 reﬂections 232 parameters H atoms treated by a mixture of independent and constrained reﬁnement max = 0.30 e A˚ 3 min = 0.30 e A˚ 3 aInteractive Research Center of Science, Graduate School of Science and Engineering, Tokyo Instiute of Technology, Ookayama, Meguro-ku, Tokyo, 152-8551, Japan, and bDepartment of Chemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Correspondence e-mail: goto@chem.titech.ac.jp Received 20 November 2009; accepted 26 November 2009 Data collection: CrystalClear (Rigaku, 2004); cell reﬁnement: CrystalClear data reduction: CrystalClear; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEPIII (Burnett & Johnson, 1996); software used to prepare material for publication: yadokari-XG (Wakita, 2005). Key indicators: single-crystal X-ray study; T = 120 K; mean (C–C) = 0.003 A˚; R factor = 0.041; wR factor = 0.100; data-to-parameter ratio = 14.1. The title compound, C23H25NSi, has an eight-membered silicon-containing heterocyclic ring with an intramolecular N Si close contact, the transannular distance of which is 2.6294 (18) A˚ . The resulting geometry about the Si atom is distorted trigonal-bypyramidal, with the N and H atoms occupying apical sites. The dihedral angle between the aromatic rings fused to the eight-membered ring is 63.27 (7). This work was partly supported by the Global COE Program (Chemistry) and Grants-in-Aid for Scientiﬁc Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. KG is grateful to Tokuyama Science Foundation for ﬁnancial support. We also thank Tosoh Finechem Corporation for the generous gifts of alkyllithiums. Related literature Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: IS2498). For highly coordinated organosilanes, see: Brelle`re et al. (1986); Carre´ et al. (1997); Paton et al. (1977); Woning & Verkade (1991); Yoshida et al. (2006). For a related structure, see: Saruhashi et al. (2001). 6-(1-Methylethyl)-12-phenyl-5,6,7,12- tetrahydrodibenz[c,f][1,5]azasilocine Kei Goto,a* Akihiro Fukushimab and Takayuki Kawashimab Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: IS2498). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: IS2498). References Brelle`re, C., Carre´, F., Corriu, R. J. P., Poirier, M. & Royo, G. (1986). Organometallics, 5, 388–390. Experimental Crystal data C23H25NSi Mr = 343.53 Monoclinic, P21=c a = 9.756 (7) A˚ Burnett, M. N. & Johnson, C. K. (1996). ORTEPIII. Report ORNL-6895. Oak Ridge National Laboratory, Tennessee, USA. Carre´, F. H., Corriu, R. J. P., Lanneau, G. F., Merle, P., Soulairol, F. & Yao, J. (1997). Organometallics, 16, 3878–3888. Jacobson, R. (1998). REQAB. Molecular Structure Corporation, The Wood- lands, Texas, USA. Paton, W. F., Corey, E. R., Corey, J. Y. & Glick, M. D. (1977). Acta Cryst. B33, 3322–3325. Rigaku (2004). CrystalClear. Rigaku Corporation, Tokyo, Japan. Saruhashi, K., Goto, K. & Kawashima, T. (2001). Chem. Heterocycl. Compd, 37, 1394–1395. Experimental Crystal data C23H25NSi Mr = 343.53 Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Experimental Crystal data C23H25NSi Mr = 343.53 Experimental Wakita, K. (2005). yadokari-XG. http://www.hat.hi-ho.ne.jp/k-wakita/yado- kari/index.html. Woning, J. & Verkade, J. G. (1991). Organometallics, 10, 2259–2266. Monoclinic, P21=c a = 9.756 (7) A˚ Monoclinic, P21=c a = 9.756 (7) A˚ Yoshida, A., Goto, K. & Kawashima, T. (2006). Bull. Chem. Soc. Jpn, 79, 793– 795. o20 Acta Cryst. (2010). E66, o20 o20 Goto et al. Goto et al. doi:10.1107/S1600536809050909 Experimental A solution of n-butyllithium in hexane (1.6 M; 4.2 ml, 6.7 mmol) was added dropwise to a solution of N,N-bis(2- bromobenzyl)isopropylamine (1.25 g, 3.16 mmol) in ether (3 ml) at 233 K. The solution was stirred at the same temperature for 30 min and then allowed to warm to room temperature. After stirring for additional 2 h, the solution was cooled to 233 K, and a solution of phenylsilane (345 mg, 3.19 mmol) in ether (2 ml) was added dropwise. The mixture was allowed to warm to room temperature, and stirred overnight. After addition of water, the mixture was extracted with ether, and the organic layer was dried over anhydrous magnesium sulfate. After filtration and removal of the solvent, the residue was purified by gel permeation liquid chromatography (eluting with chloroform) and then recrystallization from hexane to give the title compound (101 mg, 0.295 mmol, 9.3%) as colorless crystals. Physical data: m.p. 354.1–355.8 K (decomposition); 1NMR (400 MHz, CDCl3, 300 K): δ 0.76 (br, 6H), 2.53 (br, 1H), 3.78 (s, 4H), 5.53 (brs, 1H), 7.12–7.32 (m, 9H), 7.51 (br, 2H), 7.72 (br, 2H). Anal. Calcd for C23H25NSi: C 80.41, H 7.34, N 4.08%. Found: C 80.19, H 7.48, N, 3.94%. Comment Highly coordinated hydrosilanes have been of great interest for their unique structures and reactivities. It has been known that, in highly coordinated monohydrosilanes, the Si—H bond has high affinity for equatorial position (Brellère et al., 1986) and there are only a few examples with the Si—H bond at the apical position (Woning & Verkade, 1991). A dibenz[c,f][1,5]azasilocine framework has been utilized for the synthesis of various highly coordinated silicon compounds (Paton et al., 1977; Carré et al., 1997; Yoshida et al., 2006). Recently, we reported the synthesis and structural characteriz- ation of a pentacoordinated monohydrosilane bearing this molecular framework with the apical Si—H bond (Saruhashi et al., 2001). As a further investigation of this work, the crystal structure of the title new hydrosilane is reported. The title compound was synthesized by the reaction of N,N-bis(2-bromobenzyl)isopropylamine (Carré et al., 1997) with n-butyllithium followed by treatment with phenylsilane. The molecular structure of the title compound is shown in Fig. 1. It was found that the geometry around the silicon atom is that of a distorted trigonal bypyramid with the sum of the equatorial C—Si—C bond angles of 346.3°. The SiH hydrogen atom occupies the apical site in spite of its lower apicophilicity than that of a phenyl group, which is similar to the related N-butyl compound we previously reported (Saruhashi et al., 2001). The Si···N transannular distance is 2.6294 (18) Å, which is slightly longer than that of the N-butyl derivative [2.516 (2) Å] probably because of the steric repulsion between the isopropyl group and the phenyl ring. 6-(1-Methylethyl)-12-phenyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azasilocine K. Goto, A. Fukushima and T. Kawashima K. Goto, A. Fukushima and T. Kawashima K. Goto, A. Fukushima and T. Kawashima supplementary materials Acta Cryst. (2010). E66, o20 [ doi:10.1107/S1600536809050909 ] Acta Cryst. (2010). E66, o20 [ doi:10.1107/S1600536809050909 ] supplementary materials supplementary materials Refinement The H atom of the SiH group was found in a difference Fourier map and refined isotropically, while the C-bound H atoms were treated as riding, with C—H = 0.95–0.99 Å, and with Uiso(H) = 1.2 (1.5 for methyl groups) times Ueq(C). The methyl groups were allowed to rotate freely about the C-C bond. sup-1 supplementary materials Figures Fig. 1. The molecular structure of (I) with 50% probability displacement ellipsoids (arbitrary spheres for H atoms). Fig. 2. Packing diagram. 6-(1-Methylethyl)-12-phenyl-5,6,7,12- tetrahydrodibenz[c,f][1,5]azasilocine Figures Fig. 1. The molecular structure of (I) with 50% probabil spheres for H atoms). Fig. 2. Packing diagram. 6-(1-Methylethyl)-12-phenyl-5,6,7,12- tetrahydrodibenz[c,f][1,5]azasilocine Fig. 1. The molecular structure of (I) with 50% probability displacement ellipsoids (arbitrary spheres for H atoms). 6-(1-Methylethyl)-12-phenyl-5,6,7,12- tetrahydrodibenz[c,f][1,5]azasilocine 6-(1-Methylethyl)-12-phenyl-5,6,7,12- tetrahydrodibenz[c,f][1,5]azasilocine Crystal data C23H25NSi Z = 4 Mr = 343.53 F(000) = 736 Monoclinic, P21/c Dx = 1.206 Mg m−3 Hall symbol: -P 2ybc Mo Kα radiation, λ = 0.71070 Å a = 9.756 (7) Å θ = 3.0–25.0° b = 10.269 (7) Å µ = 0.13 mm−1 c = 18.912 (12) Å T = 120 K β = 92.745 (3)° Block, colourless V = 1893 (2) Å3 0.20 × 0.20 × 0.10 mm Data collection Rigaku Mercury CCD diffractometer 3278 independent reflections Radiation source: fine-focus sealed tube 2798 reflections with I > 2σ(I) graphite Rint = 0.029 ω scans θmax = 25.0°, θmin = 3.0° Absorption correction: multi-scan (REQAB; Jacobson, 1998) h = −11→11 Tmin = 0.975, Tmax = 0.987 k = −12→9 11962 measured reflections l = −21→21 Refinement Refinement on F2 Primary atom site location: structure-invariant direct methods Crystal data C23H25NSi Z = 4 Mr = 343.53 F(000) = 736 Monoclinic, P21/c Dx = 1.206 Mg m−3 Hall symbol: -P 2ybc Mo Kα radiation, λ = 0.71070 Å a = 9.756 (7) Å θ = 3.0–25.0° b = 10.269 (7) Å µ = 0.13 mm−1 c = 18.912 (12) Å T = 120 K β = 92.745 (3)° Block, colourless V = 1893 (2) Å3 0.20 × 0.20 × 0.10 mm 3278 independent reflections 2798 reflections with I > 2σ(I) Rint = 0.029 θmax = 25.0°, θmin = 3.0° h = −11→11 k = −12→9 l = −21→21 supplementary materials Least-squares matrix: full Secondary atom site location: difference Fourier map R[F2 > 2σ(F2)] = 0.041 Hydrogen site location: inferred from neighbouring sites wR(F2) = 0.100 H atoms treated by a mixture of independent and constrained refinement S = 1.08 w = 1/[σ2(Fo 2) + (0.0402P)2 + 1.0221P] where P = (Fo 2 + 2Fc 2)/3 3278 reflections (Δ/σ)max < 0.001 232 parameters Δρmax = 0.30 e Å−3 0 restraints Δρmin = −0.30 e Å−3 Least-squares matrix: full Secondary atom site location: difference Fourier map R[F2 > 2σ(F2)] = 0.041 Hydrogen site location: inferred from neighbouring sites wR(F2) = 0.100 H atoms treated by a mixture of independent and constrained refinement S = 1.08 w = 1/[σ2(Fo 2) + (0.0402P)2 + 1.0221P] where P = (Fo 2 + 2Fc 2)/3 3278 reflections (Δ/σ)max < 0.001 232 parameters Δρmax = 0.30 e Å−3 0 restraints Δρmin = −0.30 e Å−3 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.041 wR(F2) = 0.100 S = 1.08 3278 reflections 232 parameters 0 restraints Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo 2) + (0.0402P)2 + 1.0221P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.30 e Å−3 Δρmin = −0.30 e Å−3 w = 1/[σ2(Fo 2) + (0.0402P)2 + 1.0221P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.30 e Å−3 Δρmin = −0.30 e Å−3 Refinement Primary atom site location: structure-invariant direct methods Primary atom site location: structure-invariant direct methods sup-2 supplementary materials supplementary materials supplementary materials C13 0.17620 (17) 0.17561 (17) 0.02855 (9) 0.0223 (4) H10 0.2334 0.1872 −0.0128 0.027* H11 0.0823 0.2062 0.0153 0.027* C14 0.16617 (18) 0.37943 (17) 0.09530 (10) 0.0230 (4) H12 0.1655 0.4257 0.0494 0.028* H13 0.2192 0.4327 0.1306 0.028* N1 0.23330 (14) 0.25313 (14) 0.08823 (7) 0.0196 (3) C15 0.38542 (17) 0.26790 (18) 0.08917 (10) 0.0231 (4) H14 0.4150 0.3025 0.1369 0.028* C16 0.45915 (18) 0.13896 (18) 0.08041 (10) 0.0267 (4) H15 0.4229 0.0745 0.1128 0.040* H16 0.5576 0.1508 0.0912 0.040* H17 0.4445 0.1084 0.0315 0.040* C17 0.4350 (2) 0.3648 (2) 0.03466 (11) 0.0331 (5) H18 0.4060 0.3354 −0.0130 0.050* H19 0.5353 0.3705 0.0387 0.050* H20 0.3954 0.4507 0.0434 0.050* C18 0.25657 (18) 0.18885 (17) 0.24348 (9) 0.0210 (4) C19 0.37831 (18) 0.11897 (18) 0.25521 (9) 0.0253 (4) H21 0.3880 0.0367 0.2330 0.030* C20 0.48570 (19) 0.1670 (2) 0.29869 (10) 0.0317 (5) H22 0.5678 0.1178 0.3056 0.038* C21 0.4733 (2) 0.2858 (2) 0.33186 (11) 0.0361 (5) H23 0.5466 0.3186 0.3616 0.043* C22 0.3540 (2) 0.3568 (2) 0.32163 (11) 0.0356 (5) H24 0.3448 0.4386 0.3444 0.043* C23 0.24691 (19) 0.30841 (19) 0.27785 (10) 0.0281 (4) H25 0.1652 0.3581 0.2712 0.034* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Si1 0.0192 (3) 0.0210 (3) 0.0206 (3) −0.0018 (2) −0.00047 (19) −0.0006 (2) C1 0.0149 (8) 0.0215 (9) 0.0243 (10) −0.0014 (7) −0.0028 (7) −0.0001 (8) C2 0.0130 (8) 0.0251 (10) 0.0246 (10) −0.0005 (7) −0.0015 (7) −0.0029 (8) C3 0.0195 (9) 0.0328 (11) 0.0256 (10) −0.0031 (8) −0.0001 (7) −0.0060 (8) C4 0.0201 (9) 0.0293 (10) 0.0361 (12) −0.0003 (8) −0.0025 (8) −0.0141 (9) C5 0.0238 (10) 0.0184 (9) 0.0460 (13) −0.0011 (8) −0.0079 (9) −0.0037 (9) C6 0.0212 (9) 0.0233 (10) 0.0268 (10) −0.0031 (7) −0.0044 (7) 0.0023 (8) C7 0.0201 (9) 0.0248 (9) 0.0189 (9) −0.0010 (7) −0.0019 (7) −0.0079 (8) C8 0.0215 (9) 0.0220 (9) 0.0207 (9) 0.0015 (7) −0.0046 (7) −0.0063 (8) C9 0.0290 (10) 0.0235 (10) 0.0256 (10) 0.0036 (8) −0.0055 (8) −0.0070 (8) C10 0.0255 (10) 0.0353 (11) 0.0297 (11) 0.0100 (9) −0.0081 (8) −0.0127 (9) C11 0.0179 (9) 0.0450 (12) 0.0300 (11) 0.0035 (9) −0.0020 (8) −0.0149 (10) C12 0.0238 (10) 0.0327 (11) 0.0203 (10) −0.0022 (8) 0.0007 (7) −0.0094 (8) C13 0.0188 (9) 0.0274 (10) 0.0206 (10) −0.0002 (7) 0.0007 (7) 0.0020 (8) C14 0.0234 (9) 0.0183 (9) 0.0272 (10) −0.0002 (7) −0.0011 (7) 0.0025 (8) N1 0.0165 (7) 0.0190 (8) 0.0232 (8) −0.0006 (6) −0.0006 (6) 0.0012 (6) Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Si1 0.10808 (5) 0.12541 (5) 0.18691 (3) 0.02030 (15) H1 0.0293 (18) 0.0512 (18) 0.2351 (9) 0.023 (5) C1 0.14424 (17) −0.00037 (17) 0.11833 (9) 0.0204 (4) C2 0.17207 (17) 0.03310 (17) 0.04842 (9) 0.0210 (4) C3 0.19436 (18) −0.06347 (19) −0.00083 (10) 0.0260 (4) H2 0.2108 −0.0400 −0.0483 0.031* C4 0.19306 (18) −0.19363 (19) 0.01801 (11) 0.0286 (4) H3 0.2105 −0.2589 −0.0160 0.034* C5 0.16617 (19) −0.22838 (18) 0.08669 (11) 0.0297 (5) H4 0.1659 −0.3175 0.1001 0.036* C6 0.13964 (18) −0.13231 (17) 0.13580 (10) 0.0239 (4) H5 0.1179 −0.1569 0.1824 0.029* C7 −0.01585 (18) 0.25743 (17) 0.15815 (9) 0.0213 (4) C8 0.02098 (18) 0.36529 (17) 0.11797 (9) 0.0216 (4) C9 −0.07488 (19) 0.46221 (18) 0.10020 (10) 0.0263 (4) H6 −0.0488 0.5345 0.0725 0.032* C10 −0.20760 (19) 0.4539 (2) 0.12252 (10) 0.0305 (5) H7 −0.2716 0.5213 0.1113 0.037* C11 −0.24688 (19) 0.3469 (2) 0.16127 (10) 0.0311 (5) H8 −0.3385 0.3398 0.1758 0.037* C12 −0.15189 (18) 0.25012 (19) 0.17883 (9) 0.0256 (4) H9 −0.1797 0.1771 0.2055 0.031* sup-3 supplementary materials Atomic displacement parameters (Å2) sup-4 supplementary materials supplementary mater C15 0.0162 (9) 0.0270 (10) 0.0261 (10) −0.0027 (7) −0.0007 (7) 0.0053 (8) C16 0.0172 (9) 0.0296 (10) 0.0334 (11) 0.0003 (8) 0.0029 (8) 0.0043 (9) C17 0.0226 (10) 0.0357 (11) 0.0410 (12) −0.0047 (9) 0.0020 (8) 0.0129 (10) C18 0.0220 (9) 0.0243 (9) 0.0168 (9) −0.0031 (7) 0.0018 (7) 0.0027 (8) C19 0.0262 (10) 0.0259 (10) 0.0238 (10) −0.0007 (8) 0.0015 (8) 0.0040 (8) C20 0.0211 (10) 0.0394 (12) 0.0341 (12) −0.0002 (8) −0.0043 (8) 0.0106 (10) C21 0.0276 (11) 0.0470 (13) 0.0328 (12) −0.0103 (10) −0.0081 (9) 0.0000 (10) C22 0.0334 (11) 0.0378 (12) 0.0352 (12) −0.0057 (9) −0.0035 (9) −0.0114 (10) C23 0.0233 (10) 0.0324 (11) 0.0285 (11) 0.0012 (8) −0.0007 (8) −0.0047 (9) Geometric parameters (Å, °) Si1—C1 1.876 (2) C13—H10 0.9900 Si1—C18 1.876 (2) C13—H11 0.9900 Si1—C7 1.880 (2) C14—N1 1.462 (2) Si1—H1 1.438 (18) C14—H12 0.9900 C1—C6 1.396 (3) C14—H13 0.9900 C1—C2 1.405 (3) N1—C15 1.491 (2) C2—C3 1.385 (3) C15—C16 1.520 (3) C2—C13 1.512 (3) C15—C17 1.528 (3) C3—C4 1.384 (3) C15—H14 1.0000 C3—H2 0.9500 C16—H15 0.9800 C4—C5 1.384 (3) C16—H16 0.9800 C4—H3 0.9500 C16—H17 0.9800 C5—C6 1.388 (3) C17—H18 0.9800 C5—H4 0.9500 C17—H19 0.9800 C6—H5 0.9500 C17—H20 0.9800 C7—C8 1.400 (3) C18—C23 1.394 (3) C7—C12 1.403 (3) C18—C19 1.396 (3) C8—C9 1.396 (3) C19—C20 1.391 (3) C8—C14 1.506 (3) C19—H21 0.9500 C9—C10 1.384 (3) C20—C21 1.380 (3) C9—H6 0.9500 C20—H22 0.9500 C10—C11 1.385 (3) C21—C22 1.380 (3) C10—H7 0.9500 C21—H23 0.9500 C11—C12 1.388 (3) C22—C23 1.393 (3) C11—H8 0.9500 C22—H24 0.9500 C12—H9 0.9500 C23—H25 0.9500 C13—N1 1.469 (2) Si1···N1 2.6294 (18) C1—Si1—C18 117.89 (8) C2—C13—H11 109.6 C1—Si1—C7 115.86 (8) H10—C13—H11 108.1 C18—Si1—C7 112.52 (9) N1—C14—C8 111.83 (14) C1—Si1—N1 75.14 (8) N1—C14—H12 109.2 C18—Si1—N1 81.91 (8) C8—C14—H12 109.2 C7—Si1—N1 75.47 (8) N1—C14—H13 109.2 C1—Si1—H1 101.4 (7) C8—C14—H13 109.2 C18—Si1—H1 104.2 (7) H12—C14—H13 107.9 C7—Si1—H1 102.1 (7) C14—N1—C13 113.33 (14) sup-5 supplementary materials supplementary materials 173.9 (7) C14—N1—C15 118.03 (16) C13—N1—C15 119.71 (14) C14—N1—Si1 122.24 (14) C13—N1—Si1 120.09 (17) C15—N1—Si1 121.27 (17) N1—C15—C16 118.63 (15) N1—C15—C17 120.99 (18) C16—C15—C17 119.5 N1—C15—H14 119.5 C16—C15—H14 119.69 (18) C17—C15—H14 120.2 C15—C16—H15 120.2 C15—C16—H16 119.64 (18) H15—C16—H16 120.2 C15—C16—H17 120.2 H15—C16—H17 121.51 (18) H16—C16—H17 119.2 C15—C17—H18 119.2 C15—C17—H19 117.67 (17) H18—C17—H19 123.33 (14) C15—C17—H20 118.98 (14) H18—C17—H20 120.56 (17) H19—C17—H20 119.41 (17) C23—C18—C19 120.01 (15) C23—C18—Si1 120.55 (19) C19—C18—Si1 119.7 C20—C19—C18 119.7 C20—C19—H21 119.83 (18) C18—C19—H21 120.1 C21—C20—C19 120.1 C21—C20—H22 119.74 (18) C19—C20—H22 120.1 C22—C21—C20 120.1 C22—C21—H23 121.62 (19) C20—C21—H23 119.2 C21—C22—C23 119.2 C21—C22—H24 110.32 (14) C23—C22—H24 109.6 C22—C23—C18 109.6 C22—C23—H25 109.6 C18—C23—H25 90.22 (15) C7—C8—C14—N1 −132.20 (14) C8—C14—N1—C13 162.45 (15) C8—C14—N1—C15 −91.80 (15) C8—C14—N1—Si1 45.78 (17) C2—C13—N1—C14 −19.57 (13) C2—C13—N1—C15 −0.2 (2) C2—C13—N1—Si1 −178.19 (13) C1—Si1—N1—C14 sup-6 N1—Si1—H1 173.9 (7) C14—N1—C15 C6—C1—C2 118.03 (16) C13—N1—C15 C6—C1—Si1 119.71 (14) C14—N1—Si1 C2—C1—Si1 122.24 (14) C13—N1—Si1 C3—C2—C1 120.09 (17) C15—N1—Si1 C3—C2—C13 121.27 (17) N1—C15—C16 C1—C2—C13 118.63 (15) N1—C15—C17 C4—C3—C2 120.99 (18) C16—C15—C17 C4—C3—H2 119.5 N1—C15—H14 C2—C3—H2 119.5 C16—C15—H14 C3—C4—C5 119.69 (18) C17—C15—H14 C3—C4—H3 120.2 C15—C16—H15 C5—C4—H3 120.2 C15—C16—H16 C4—C5—C6 119.64 (18) H15—C16—H16 C4—C5—H4 120.2 C15—C16—H17 C6—C5—H4 120.2 H15—C16—H17 C5—C6—C1 121.51 (18) H16—C16—H17 C5—C6—H5 119.2 C15—C17—H18 C1—C6—H5 119.2 C15—C17—H19 C8—C7—C12 117.67 (17) H18—C17—H19 C8—C7—Si1 123.33 (14) C15—C17—H20 C12—C7—Si1 118.98 (14) H18—C17—H20 C9—C8—C7 120.56 (17) H19—C17—H20 C9—C8—C14 119.41 (17) C23—C18—C19 C7—C8—C14 120.01 (15) C23—C18—Si1 C10—C9—C8 120.55 (19) C19—C18—Si1 C10—C9—H6 119.7 C20—C19—C18 C8—C9—H6 119.7 C20—C19—H21 C9—C10—C11 119.83 (18) C18—C19—H21 C9—C10—H7 120.1 C21—C20—C19 C11—C10—H7 120.1 C21—C20—H22 C10—C11—C12 119.74 (18) C19—C20—H22 C10—C11—H8 120.1 C22—C21—C20 C12—C11—H8 120.1 C22—C21—H23 C11—C12—C7 121.62 (19) C20—C21—H23 C11—C12—H9 119.2 C21—C22—C23 C7—C12—H9 119.2 C21—C22—H24 N1—C13—C2 110.32 (14) C23—C22—H24 N1—C13—H10 109.6 C22—C23—C18 C2—C13—H10 109.6 C22—C23—H25 N1—C13—H11 109.6 C18—C23—H25 C18—Si1—C1—C6 90.22 (15) C7—C8—C14—N1 C7—Si1—C1—C6 −132.20 (14) C8—C14—N1—C13 N1—Si1—C1—C6 162.45 (15) C8—C14—N1—C15 C18—Si1—C1—C2 −91.80 (15) C8—C14—N1—Si1 C7—Si1—C1—C2 45.78 (17) C2—C13—N1—C14 N1—Si1—C1—C2 −19.57 (13) C2—C13—N1—C15 C6—C1—C2—C3 −0.2 (2) C2—C13—N1—Si1 Si1—C1—C2—C3 −178.19 (13) C1—Si1—N1—C14 173.9 (7) C14—N1—C15 111.02 (14) 118.03 (16) C13—N1—C15 113.88 (14) 119.71 (14) C14—N1—Si1 98.76 (11) 122.24 (14) C13—N1—Si1 95.92 (11) 120.09 (17) C15—N1—Si1 122.57 (10) 121.27 (17) N1—C15—C16 112.71 (15) 118.63 (15) N1—C15—C17 113.89 (14) 120.99 (18) C16—C15—C17 109.11 (16) 119.5 N1—C15—H14 106.9 119.5 C16—C15—H14 106.9 119.69 (18) C17—C15—H14 106.9 120.2 C15—C16—H15 109.5 120.2 C15—C16—H16 109.5 119.64 (18) H15—C16—H16 109.5 120.2 C15—C16—H17 109.5 120.2 H15—C16—H17 109.5 121.51 (18) H16—C16—H17 109.5 119.2 C15—C17—H18 109.5 119.2 C15—C17—H19 109.5 117.67 (17) H18—C17—H19 109.5 123.33 (14) C15—C17—H20 109.5 118.98 (14) H18—C17—H20 109.5 120.56 (17) H19—C17—H20 109.5 119.41 (17) C23—C18—C19 117.03 (16) 120.01 (15) C23—C18—Si1 120.26 (14) 120.55 (19) C19—C18—Si1 122.68 (14) 119.7 C20—C19—C18 121.52 (18) 119.7 C20—C19—H21 119.2 119.83 (18) C18—C19—H21 119.2 120.1 C21—C20—C19 120.14 (18) 120.1 C21—C20—H22 119.9 119.74 (18) C19—C20—H22 119.9 120.1 C22—C21—C20 119.69 (18) 120.1 C22—C21—H23 120.2 121.62 (19) C20—C21—H23 120.2 119.2 C21—C22—C23 119.9 (2) 119.2 C21—C22—H24 120.1 110.32 (14) C23—C22—H24 120.1 109.6 C22—C23—C18 121.72 (18) 109.6 C22—C23—H25 119.1 109.6 C18—C23—H25 119.1 90.22 (15) C7—C8—C14—N1 27.9 (2) −132.20 (14) C8—C14—N1—C13 68.82 (19) 162.45 (15) C8—C14—N1—C15 −161.54 (14) −91.80 (15) C8—C14—N1—Si1 −31.55 (15) 45.78 (17) C2—C13—N1—C14 −142.27 (15) −19.57 (13) C2—C13—N1—C15 89.55 (17) −0.2 (2) C2—C13—N1—Si1 −40.07 (14) −178.19 (13) C1—Si1—N1—C14 148.32 (11) —H1 173.9 (7) C14—N1—C15 111.02 (14) —C2 118.03 (16) C13—N1—C15 113.88 (14) —Si1 119.71 (14) C14—N1—Si1 98.76 (11) —Si1 122.24 (14) C13—N1—Si1 95.92 (11) —C1 120.09 (17) C15—N1—Si1 122.57 (10) —C13 121.27 (17) N1—C15—C16 112.71 (15) —C13 118.63 (15) N1—C15—C17 113.89 (14) —C2 120.99 (18) C16—C15—C17 109.11 (16) —H2 119.5 N1—C15—H14 106.9 —H2 119.5 C16—C15—H14 106.9 —C5 119.69 (18) C17—C15—H14 106.9 —H3 120.2 C15—C16—H15 109.5 —H3 120.2 C15—C16—H16 109.5 —C6 119.64 (18) H15—C16—H16 109.5 —H4 120.2 C15—C16—H17 109.5 —H4 120.2 H15—C16—H17 109.5 —C1 121.51 (18) H16—C16—H17 109.5 —H5 119.2 C15—C17—H18 109.5 —H5 119.2 C15—C17—H19 109.5 —C12 117.67 (17) H18—C17—H19 109.5 —Si1 123.33 (14) C15—C17—H20 109.5 7—Si1 118.98 (14) H18—C17—H20 109.5 —C7 120.56 (17) H19—C17—H20 109.5 —C14 119.41 (17) C23—C18—C19 117.03 (16) —C14 120.01 (15) C23—C18—Si1 120.26 (14) 9—C8 120.55 (19) C19—C18—Si1 122.68 (14) 9—H6 119.7 C20—C19—C18 121.52 (18) —H6 119.7 C20—C19—H21 119.2 0—C11 119.83 (18) C18—C19—H21 119.2 0—H7 120.1 C21—C20—C19 120.14 (18) 10—H7 120.1 C21—C20—H22 119.9 11—C12 119.74 (18) C19—C20—H22 119.9 11—H8 120.1 C22—C21—C20 119.69 (18) 11—H8 120.1 C22—C21—H23 120.2 12—C7 121.62 (19) C20—C21—H23 120.2 12—H9 119.2 C21—C22—C23 119.9 (2) 2—H9 119.2 C21—C22—H24 120.1 3—C2 110.32 (14) C23—C22—H24 120.1 3—H10 109.6 C22—C23—C18 121.72 (18) 3—H10 109.6 C22—C23—H25 119.1 3—H11 109.6 C18—C23—H25 119.1 1—C1—C6 90.22 (15) C7—C8—C14—N1 27.9 (2) —C1—C6 −132.20 (14) C8—C14—N1—C13 68.82 (19) —C1—C6 162.45 (15) C8—C14—N1—C15 −161.54 (14) 1—C1—C2 −91.80 (15) C8—C14—N1—Si1 −31.55 (15) —C1—C2 45.78 (17) C2—C13—N1—C14 −142.27 (15) —C1—C2 −19.57 (13) C2—C13—N1—C15 89.55 (17) —C2—C3 −0.2 (2) C2—C13—N1—Si1 −40.07 (14) —C2—C3 −178.19 (13) C1—Si1—N1—C14 148.32 (11) sup-6 supplementary materials supplementary materials C6—C1—C2—C13 179.50 (15) C18—Si1—N1—C14 −89.90 (12) Si1—C1—C2—C13 1.5 (2) C7—Si1—N1—C14 25.98 (11) C1—C2—C3—C4 −1.6 (3) C1—Si1—N1—C13 33.57 (10) C13—C2—C3—C4 178.74 (16) C18—Si1—N1—C13 155.34 (11) C2—C3—C4—C5 1.4 (3) C7—Si1—N1—C13 −88.77 (11) C3—C4—C5—C6 0.5 (3) C1—Si1—N1—C15 −89.73 (13) C4—C5—C6—C1 −2.4 (3) C18—Si1—N1—C15 32.04 (13) C2—C1—C6—C5 2.2 (3) C7—Si1—N1—C15 147.92 (14) Si1—C1—C6—C5 −179.79 (13) C14—N1—C15—C16 −178.81 (15) C1—Si1—C7—C8 −79.90 (16) C13—N1—C15—C16 −49.5 (2) C18—Si1—C7—C8 59.91 (17) Si1—N1—C15—C16 65.15 (18) N1—Si1—C7—C8 −14.73 (13) C14—N1—C15—C17 −53.8 (2) C1—Si1—C7—C12 101.44 (15) C13—N1—C15—C17 75.5 (2) C18—Si1—C7—C12 −118.76 (14) Si1—N1—C15—C17 −169.85 (12) N1—Si1—C7—C12 166.60 (15) C1—Si1—C18—C23 158.83 (14) C12—C7—C8—C9 0.7 (2) C7—Si1—C18—C23 19.91 (17) Si1—C7—C8—C9 −177.93 (13) N1—Si1—C18—C23 90.44 (15) C12—C7—C8—C14 178.98 (16) C1—Si1—C18—C19 −23.30 (18) Si1—C7—C8—C14 0.3 (2) C7—Si1—C18—C19 −162.22 (14) C7—C8—C9—C10 0.6 (3) N1—Si1—C18—C19 −91.69 (15) C14—C8—C9—C10 −177.69 (17) C23—C18—C19—C20 −0.6 (3) C8—C9—C10—C11 −1.7 (3) Si1—C18—C19—C20 −178.53 (14) C9—C10—C11—C12 1.4 (3) C18—C19—C20—C21 0.4 (3) C10—C11—C12—C7 −0.1 (3) C19—C20—C21—C22 −0.1 (3) C8—C7—C12—C11 −1.0 (3) C20—C21—C22—C23 −0.1 (3) Si1—C7—C12—C11 177.76 (14) C21—C22—C23—C18 0.0 (3) C3—C2—C13—N1 −145.26 (16) C19—C18—C23—C22 0.4 (3) C1—C2—C13—N1 35.1 (2) Si1—C18—C23—C22 178.38 (15) C9—C8—C14—N1 −153.86 (16) sup-7 supplementary materials supplementary materials Fig. 1 sup-8 supplementary materials Fig. 2 sup-9
https://openalex.org/W2771989903	https://ueaeprints.uea.ac.uk/id/eprint/65736/1/Nanoscale_c7nr06020c.pdf	English	null	Unravelling the mechanisms that determine the uptake and metabolism of magnetic single and multicore nanoparticles in a<i>Xenopus laevis</i>model	Nanoscale	2,018	public-domain	14,017	View Article Online View Journal View Article Online View Journal ough the has been a, es Nanoscale Volume 8 Number 1 7 January 2016 Pages 1–660 Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the author guidelines. You can find more information about Accepted Manuscripts in the author guidelines. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the ethical guidelines, outlined in our author and reviewer resource centre, still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. Nanoscale Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: M. Marín-Barba, H. Gavilán, L. Gutierrez, E. Lozano-Velasco, I. Rodriguez-Ramiro, G. Wheeler, C. J. Morris, M. P. Morales and A. Ruiz, Nanoscale, 2017, DOI: 10.1039/C7NR06020C. This article can be cited before page numbers have been issued, to do this please use: M. Marín-Barba, H. Gavilán, L. Gutierrez, E. Lozano-Velasco, I. Rodriguez-Ramiro, G. Wheeler, C. J. Morris, M. P. Morales and A. Ruiz, Nanoscale, 2017, DOI: 10.1039/C7NR06020C. This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Nanoscale www.rsc.org/nanoscale ISSN 2040-3364 PAPER Qian Wang et al. TiC2: a new two-dimensional sheet beyond MXenes Volume 8 Number 1 7 January 2016 Pages 1–660 a. School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR4 7TJ, UK. b. Instituto de Ciencia de Materiales de Madrid, Sor Juana Inés de la Cruz 3, 28049 Madrid, Spain. c. Dept. Química Analítica, Instituto de Nanociencia de Aragón, Universidad de Zaragoza, 50018 Zaragoza, Spain. d. School of Medicine, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR4 7TJ, UK. e. School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR4 7TJ, UK. ⊥ These authors contributed equally to this work. * corresponding author: G.Ruiz-Estrada@uea.ac.uk DOI: 10.1039/x0xx00000x Nanoscale Unravelling the mechanisms that determine the uptake and metabolism of magnetic single and multicore nanoparticles in a Xenopus laevis model M. Marín-Barbaa⊥, H. Gavilánb⊥, L. Gutiérrezc, E. Lozano-Velascoa, I. Rodríguez-Ramirod, G. N. Wheelera, C. J. Morrise, M. P. Moralesb and A. Ruize* Unravelling the mechanisms that determine the uptake and metabolism of magnetic single and multicore nanoparticles in a Xenopus laevis model M. Marín-Barbaa⊥, H. Gavilánb⊥, L. Gutiérrezc, E. Lozano-Velascoa, I. Rodríguez-Ramirod, G. N. Wheelera, C. J. Morrise, M. P. Moralesb and A. Ruize* scale Accepted Manuscript Nanoscale Accepted Manuscript Nanoscale Accepted Manuscri Multicore superparamagnetic nanoparticles have been proposed as ideal tools for some biomedical applications because of their high magnetic moment per particle, high specific surface area and long term colloidal stability. Through controlled aggregation and packing of magnetic cores it is possible to obtain not only single-core but also multicore and hollow spheres with internal voids. In this work, we compare toxicological properties of single and multicore nanoparticles. Both types of particles showed moderate in vitro toxicity (MTT assay) tested in Hep G2 (human hepatocellular carcinoma) and Caco-2 (human colorectal adenocarcinoma) cells. The influence of surface chemistry in their biological behavior was also studied after functionalization with O,O′-bis(2-aminoethyl) PEG (2000 Da). For the first time, these nanoparticles were evaluated in a Xenopus laevis model studying their whole organism toxicity and their impact upon iron metabolism. The degree of activation of the metabolic pathway depends on the size and surface charge of the nanoparticles which determine their uptake. The results also highlight the potential of Xenopus laevis model bridging the gap between in vitro cell-based assays and rodent models for toxicity assessment to develop effective nanoparticles for biomedical applications. Nanoscale Accepted Manu or therapeutic objective needs to be studied. Thus, the metabolism of the particles and degradation by-products must be assessed and assured. Since iron is involved in diverse cellular processes,8 organisms display mechanisms to transport and store iron in non-toxic forms.9 Therefore, IOMNPs are predicted to be safely incorporated into biological systems. Increasing evidence demonstrates that IOMNPs trigger iron- coping mechanisms in cells and that the degradation products of these materials are incorporated into normal iron metabolic routes.10–16 However, nanoparticle coating has been shown to be a determinant of the IOMNPs uptake and degradation pathways.17,18 In order to establish solid conclusions about IOMNPs toxicity and biodistribution and their cellular effects, it would be ideal to have biologically pertinent models. rsc.li/nanoscale rsc.li/nanoscale rsc.li/nanoscale Nanoscale ARTICLE Please do not adjust margins ge 1 of 15 Nanoscale View Article Online DOI: 10.1039/C7NR06020C Please do not adjust margins Nanoscale Page 1 of 15 Page 1 of 15 View Article Online DOI: 10.1039/C7NR06020C Introduction determining how best to characterize nanoparticles and follow their transformation/degradation.33 The study of in vivo fate of IOMNPs is imperative to develop successful biomedical applications. In this paper, we present for the first time, a study, by means of AC magnetic susceptibility measurements, of the intake of iron-containing particles in X. laevis embryos. Introduction particles from 60 to 150 nm in size are taken up by the reticuloendothelial system leading to rapid uptake in the liver and spleen in humans. Intravenously injected nanoparticles with diameters of 10-40 nm allow longer blood circulation and can cross capillary walls, and they are often phagocytosed by macrophages which traffic to lymph nodes and bone marrow.35 However, how these parameters affect the greater picture of toxicity and biodegradability mechanisms is still poorly understood. 142 nm (± 23 nm) nanoparticles with a well-defined size and shape (Figure 1 D, F). These MC nanoparticles consist of smaller cores of approximately 10 nm. HRTEM and X-ray diffraction patterns have already been reported.34 Particle structure and size were selected intentionally, since they are two key parameters that directly influence in vivo biological behavior. The size of intravenously injected nanoparticles greatly affects their in vivo biodistribution, e.g. particles from 60 to 150 nm in size are taken up by the reticuloendothelial system leading to rapid uptake in the liver and spleen in humans. Intravenously injected nanoparticles with diameters of 10-40 nm allow longer blood circulation and can cross capillary walls, and they are often phagocytosed by macrophages which traffic to lymph nodes and bone marrow.35 However, how these parameters affect the greater picture of toxicity and biodegradability mechanisms is still poorly understood. In this work, we report the effect of γ-Fe2O3 magnetic single and multicore nanoparticles suitable for bioapplications in X. laevis embryos. The particles have uniform size in the nanoscale and are coated with biocompatible shells. IOMNPs colloids used in this work can be classified as single-core (with only one magnetic core per particle) and multicore (with several magnetic cores per particle).24 Single-core superparamagnetic nanoparticles have been proposed as ideal tools in biomedicine, since they display longer circulation times post-injection.25 Indeed, this may favour their uptake in leaky vasculature regions such as tumors.26 In contrast, for some medical applications, such as bioseparation or magnetic fluid hyperthermia, it can be advantageous to use larger multicore particles that have a large magnetic moment per particle.27 Few in vitro and in vivo studies have been dedicated to the comparison of single-core and multicore nanoparticles28 and there is a lack of knowledge still on how the aggregation of magnetic cores forming multicore nanoparticles affects the nanoparticle uptake and degradation. Published on 08 December 2017. Downloaded by University of East Anglia Library on 13/12/2017 10:14:31. Introduction scale Accepted Manuscript Nanoscale Accepted Manuscri Iron oxide nanoparticles obtained by thermal decomposition are hydrophobic. To stabilize them in aqueous media and make them suitable for biological applications, oleic acid on the surface of the nanoparticle was removed with DMSA via ligand exchange reaction (SC-DMSA). Polyol mediated process render hydrophilic nanoparticles along with polyvinylpyrrolidone (PVP40), present in the reaction, however an extra capping agent like citric acid enhances the electrostatic repulsion and facilitates the final dispersion of the MC nanoparticles in aqueous media (MC-Cit). Hydrodynamic sizes are always higher than sizes measured by TEM, indicating the presence of the coating or some degree of agglomeration after surface modification, but both types of particles remain below 200 nm (34 and 181 nm were obtained for SC-DMSA and MC-Cit, respectively), an important requirement for biomedical applications. Both particles have high negative surface charge at pH 7 (Z-potential equal to -38 and -25 mV for SC-DMSA and MC-Cit, respectively). In order to evaluate the influence of the surface charge in particle absorption and biodegradation, we conjugated covalently a diamine PEG derivative to the carboxylic groups (from both DMSA and citric acid) to the surface of the nanoparticles. After PEG conjugation, average hydrodynamic size at pH 7 increased from 34 to 65 nm and from 181 to 183 nm for SC-DMSA and MC-Cit, respectively (SC-DMSA-PEG and MC-Cit-PEG) and net surface charge decreased from approximately -38 to -24 mV for SC-DMSA-PEG samples and from -25 to -18 mV for MC-Cit- PEG. Colloidal properties of aqueous suspensions of the nanoparticles at pH 7 are summarized in Table 1. Also, hydrodynamic sizes of the particles have been studied in different biological media (Figure 1S). Nanoscale Accepted Manu Here we report for the first-time toxicity and metabolization of single-core and multicore nanoparticles using a combination of in vitro cell culture models and an in vivo X. laevis embryo model. The application of AC magnetic susceptibility measurements has proven to be an excellent tool to study magnetic nanoparticles in complex matrices, since the diamagnetic signal coming from tissues does not interfere with the superparamagnetic signal of the NPs.29 This way it was possible to follow the signal of the magnetic nanoparticles in animal models30–32 and to quantify the iron content. This is one of the greatest challenges in the nanomaterials area nowadays, i.e. Introduction Intravenously injected nanoparticles with diameters of 10-40 nm allow longer blood circulation and can cross capillary walls, and they are often phagocytosed by macrophages which traffic to lymph nodes and bone marrow.35 However, how these parameters affect the greater picture of toxicity and biodegradability mechanisms is still poorly understood. Iron oxide nanoparticles obtained by thermal decomposition are hydrophobic. To stabilize them in aqueous media and make them suitable for biological applications, oleic acid on the surface of the nanoparticle was removed with DMSA via ligand exchange reaction (SC-DMSA). Polyol mediated process render hydrophilic nanoparticles along with polyvinylpyrrolidone (PVP40), present in the reaction, however an extra capping agent like citric acid enhances the electrostatic repulsion and facilitates the final dispersion of the MC nanoparticles in aqueous media (MC-Cit). Hydrodynamic sizes are always higher than sizes measured by TEM, indicating the presence of the coating or some degree of agglomeration after surface modification, but both types of particles remain below 200 nm (34 and 181 nm were obtained for SC-DMSA and MC-Cit, respectively), an important requirement for biomedical applications. Both particles have high negative surface charge at pH 7 (Z-potential equal to -38 and -25 mV for SC-DMSA and MC-Cit, respectively). In order to evaluate the influence of the surface charge in particle absorption and biodegradation, we conjugated covalently a diamine PEG derivative to the carboxylic groups (from both DMSA and citric acid) to the surface of the nanoparticles. After PEG conjugation, average hydrodynamic size at pH 7 increased from 34 to 65 nm and from 181 to 183 nm for SC-DMSA and MC-Cit, respectively (SC-DMSA-PEG and MC-Cit-PEG) and net surface charge decreased from approximately -38 to -24 mV for SC-DMSA-PEG samples and from -25 to -18 mV for MC-Cit- PEG. Colloidal properties of aqueous suspensions of the nanoparticles at pH 7 are summarized in Table 1. Also, hydrodynamic sizes of the particles have been studied in different biological media (Figure 1S). 142 nm (± 23 nm) nanoparticles with a well-defined size and shape (Figure 1 D, F). These MC nanoparticles consist of smaller cores of approximately 10 nm. HRTEM and X-ray diffraction patterns have already been reported.34 Particle structure and size were selected intentionally, since they are two key parameters that directly influence in vivo biological behavior. The size of intravenously injected nanoparticles greatly affects their in vivo biodistribution, e.g. This journal is © The Royal Society of Chemistry 20xx This journal is © The Royal Society of Chemistry 20xx Introduction Iron oxide nanoparticles obtained by thermal decompositio are hydrophobic. To stabilize them in aqueous media an make them suitable for biological applications, oleic acid o the surface of the nanoparticle was removed with DMSA vi ligand exchange reaction (SC-DMSA). Polyol mediated proces render hydrophilic nanoparticles along wit polyvinylpyrrolidone (PVP40), present in the reaction, howeve an extra capping agent like citric acid enhances th electrostatic repulsion and facilitates the final dispersion of th MC nanoparticles in aqueous media (MC-Cit). Hydrodynami sizes are always higher than sizes measured by TEM, indicatin the presence of the coating or some degree of agglomeratio after surface modification, but both types of particles remai below 200 nm (34 and 181 nm were obtained for SC-DMSA and MC-Cit, respectively), an important requirement fo biomedical applications. Both particles have high negativ surface charge at pH 7 (Z-potential equal to -38 and -25 mV fo SC-DMSA and MC-Cit, respectively). In order to evaluate th influence of the surface charge in particle absorption an biodegradation, we conjugated covalently a diamine PEG derivative to the carboxylic groups (from both DMSA and citri acid) to the surface of the nanoparticles. After PEG conjugation, average hydrodynamic size at pH 7 increase from 34 to 65 nm and from 181 to 183 nm for SC-DMSA an MC-Cit, respectively (SC-DMSA-PEG and MC-Cit-PEG) and ne surface charge decreased from approximately -38 to -24 mV for SC-DMSA-PEG samples and from -25 to -18 mV for MC-Cit PEG. Colloidal properties of aqueous suspensions of th nanoparticles at pH 7 are summarized in Table 1 Also 142 nm (± 23 nm) nanoparticles with a well-defined size and shape (Figure 1 D, F). These MC nanoparticles consist of smaller cores of approximately 10 nm. HRTEM and X-ray diffraction patterns have already been reported.34 Particle structure and size were selected intentionally, since they are two key parameters that directly influence in vivo biological behavior. The size of intravenously injected nanoparticles greatly affects their in vivo biodistribution, e.g. particles from 60 to 150 nm in size are taken up by the reticuloendothelial system leading to rapid uptake in the liver and spleen in humans. Introduction Iron oxide magnetic nanoparticles (IOMNPs) are extensively studied nowadays for their potential in biomedical applications.1,2 Their surface chemistry can be modified, adding functionality to the material and enabling their use for gene therapy, tissue regeneration and drug delivery, to specifically target tumours using external magnetic fields3. Their magnetic properties can be exploited for magnetic resonance imaging and magnetic-fluid hyperthermia, which has raised hope for improved imaging techniques4 and cancer treatment.5,6 However, in spite of their potential, few of these biomaterials have reached clinical practice.7 A crucial issue for magnetic nanoparticle utilization in biomedicine and their approval by regulatory agencies depends on their biotransformation and toxicity. The course and fate of the nanoparticles once they deliver their imaging Xenopus laevis is an amphibian model ideal to study the course and fate of the nanoparticles as it is an easy and flexible system in which to evaluate vertebrate embryology, basic cell and molecular biology, genomics, neurobiology and toxicology.19 Recently, this model was used to identify the risk of exposure to contaminated water.20 The mortality, malformations and growth inhibition of Xenopus laevis were studied, after challenge with metal oxide-based nanomaterial contaminants (γ-Fe2O3, TiO2, ZnO and CuO). Notably, γ-Fe2O3 did not pose risks to amphibian populations up to 1 mg/mL where it causes developmental abnormalities. In contrast, the other nanoparticles caused gastrointestinal, spinal, and other abnormalities at concentrations of ~3 mg/L. Furthermore, it was shown that nanoparticle sizes above 200 nm had toxic effects.21 Despite available literature on the effects of many J. Name., 2013, 00, 1-3 \| 1 This journal is © The Royal Society of Chemistry 20xx Please do not adjust m Nanoscale Please do not adjust margins Nanoscale do not adjust margins Nanoscale Page 2 of 15 Page 2 of 15 Nanoscale View Article Online DOI: 10.1039/C7NR06020C ARTICLE Nanoscale on the larval development of X. laevis in ntal studies, there are only few reports evaluating es toxicity and biodistribution designed for applications,22,23 and there is still a lack of bridging biotransformation studies in cell-based data generated from rodent in vivo systems. k, we report the effect of γ-Fe2O3 magnetic single ore nanoparticles suitable for bioapplications in X. ryos. The particles have uniform size in the nd are coated with biocompatible shells. Introduction IOMNPs d in this work can be classified as single-core (with magnetic core per particle) and multicore (with magnetic cores per particle).24 Single-core agnetic nanoparticles have been proposed as ideal medicine, since they display longer circulation times on.25 Indeed, this may favour their uptake in leaky regions such as tumors.26 In contrast, for some plications, such as bioseparation or magnetic fluid ia, it can be advantageous to use larger multicore at have a large magnetic moment per particle.27 o and in vivo studies have been dedicated to the of single-core and multicore nanoparticles28 and ack of knowledge still on how the aggregation of ores forming multicore nanoparticles affects the e uptake and degradation. port for the first-time toxicity and metabolization of and multicore nanoparticles using a combination of culture models and an in vivo X. laevis embryo e application of AC magnetic susceptibility nts has proven to be an excellent tool to study nanoparticles in complex matrices, since the c signal coming from tissues does not interfere with aramagnetic signal of the NPs.29 This way it was follow the signal of the magnetic nanoparticles in els30–32 and to quantify the iron content. This is one eatest challenges in the nanomaterials area i.e. determining how best to characterize es and follow their transformation/degradation.33 of in vivo fate of IOMNPs is imperative to develop biomedical applications. In this paper, we present st time, a study, by means of AC magnetic y measurements, of the intake of iron-containing X. laevis embryos. nd discussion 142 nm (± 23 nm) nanoparticles with a well-defined size an shape (Figure 1 D, F). These MC nanoparticles consist o smaller cores of approximately 10 nm. HRTEM and X-ra diffraction patterns have already been reported.34 Particl structure and size were selected intentionally, since they ar two key parameters that directly influence in vivo biologica behavior. The size of intravenously injected nanoparticle greatly affects their in vivo biodistribution, e.g. particles from 60 to 150 nm in size are taken up by the reticuloendothelia system leading to rapid uptake in the liver and spleen i humans. Intravenously injected nanoparticles with diameter of 10-40 nm allow longer blood circulation and can cros capillary walls, and they are often phagocytosed b macrophages which traffic to lymph nodes and bon marrow.35 However, how these parameters affect the greate picture of toxicity and biodegradability mechanisms is sti poorly understood. Nanoparticles synthesis and characterization Two different types of iron oxide nanoparticles were synthesized in this work. Single core nanoparticles (SC) were obtained by thermal decomposition of the iron(III) oleate precursor in 1-octadecene (Figure 1 A, B). Particles were 13 nm (± 1 nm) in diameter, uniform in size (Figure 1 C), relatively round and well dispersed, owing to the presence of oleic acid around the particles. In a different approach, multicore nanoparticles (MC) were obtained by polyol mediated reduction of iron(III) chloride. MC are composed of spherical 2 \| J. Name., 2012, 00, 1-3 Nanoscale Page 3 of 15 ARTICLE View Article Online DOI: 10.1039/C7NR06020C ARTICLE View Article Online DOI: 10.1039/C7NR06020C Journal Name Table 1. Colloidal properties of aqueous suspensions of the nanoparticles at pH 7. Hydrodynamic sizes, PDI (= standard deviation/mean size) and surface charge of single and multicore nanoparticles after PEG conjugation Sample Hydrodynamic size (nm) PDI ζ-Potential (mV) SC-DMSA 34 0.054 -38 ± 12 SC-DMSA-PEG 65 0.084 -24 ± 7 MC-CIT 181 0.201 -25 ± 9 MC-CIT-PEG 183 0.225 -18 ± 9 Published on 08 December 2017. Downloaded by University of East Anglia Library on 13/12/2017 10:14:31. oscale Accepted Manuscript Nanoscale Accepted Manuscript Nanoscale Accepted Manuscri Nanoscale Accepted Manu This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 \| 3 Please do not adjust margins Nanoscale Accepted Man Nanoscale Accepted TGA of the unconjugated nanoparticles reveals a weight loss of ∼15 % and 8 % for SC-DMSA and MC-CIT respectively, due to the removal of physical and chemical water and capping molecules (DMSA or citric acid) (Figure 1 I). Particles modified with diamine PEG nonetheless showed a larger amount of conjugated polymer (∼20 % in the case of SC-DMSA-PEG and 13 % for MC-Cit-PEG) which indicates greater reaction efficiency. In the case of SC- DMSA-PEG, the presence of ~2 molecules of PEG per nm2 was calculated. In the case of MC-Cit-PEG is not possible to make an accurate calculation of the number of PEG molecules bounded to the surface because is not a homogeneous spherical surface due to their multicore structure. A notable decrease in the surface charge of the particles is observed after PEG conjugation, however not all carboxyl groups are modified during the reaction. The surface is not saturated probably due to the steric hindrance caused by the PEG chains, and therefore we have a limited balance of the negative surface charge coming from the DMSA or citric acid. oscale Accepted Manuscript Nanoscale Accepted Manusc Nanoscale Accepted M This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 \| 3 ry 20xx J. Name., 2013, 00, 1-3 \| 3 This journal is © The Royal Society of Chemist ARTICLE Nanoscale Please do not adjust margins Figure 1. Transmission electron microscopy (TEM) images of single and multicore nanoparticles. (A, B) SC-DMSA. (D, E) MC-Cit. (C, F) Size distribution histograms. Red lines indicate the log-normal fitting function of TEM particle size data. Physical chemical characterization of the nanoparticles after conjugation with PEG. FTIR spectra of (G) Single-core nanoparticles. (H) Multicore nanoparticles. (I) Thermogravimetric analysis of four formulations Nanoscale View Article Online DOI: 10.1039/C7NR06020C Please do not adjust margins Nanoscale Page 4 of 15 ne Nanoscale View Article Online DOI: 10.1039/C7NR06020C ARTICLE Figure 1. Transmission electron microscopy (TEM) images of single and multicore nanoparticles. (A, B) SC-DMSA. (D, E) MC-Cit. (C, F) Size distribution histograms. Red lines indicate the log-normal fitting function of TEM particle size data. Physical chemical characterization of the nanoparticles after conjugation with PEG. FTIR spectra of (G) Single-core nanoparticles. (H) Multicore nanoparticles. (I) Thermogravimetric analysis of four formulations Figure 1. Transmission electron microscopy (TEM) images of single and multicore nanoparticles. (A, B) SC-DMSA. (D, E) MC-Cit. (C, F) Size distribution histograms. Red lines indicate the log-normal fitting function of TEM particle size data. Physical chemical characterization of the nanoparticles after conjugation with PEG. FTIR spectra of (G) Single-core nanoparticles. (H) Multicore nanoparticles. (I) Thermogravimetric analysis of four formulations Figure 2. Evaluation of cell viability by MTT assay. (A) Hep G cells. (B) Caco-2 cells. Cells were treated with the nanoparticle for 24 h. (C) Ferritin formation measured by ELISA in Caco- cells exposed to 40 µg/mL of single or multicore nanoparticles Data represent means ± SD (n = 3). * shows statistically significance compared with the control (t test, p<0.05, p<0.01, *p<0.001, *p<0.0001). Nanoparticle surface modification was also confirmed by FTIR (Figure 1 G, H). For all the samples, the typical bands of metal skeleton vibration (Fe-O) in the region of 550-600 cm-1 and a broad peak between 3000 and 3500 cm-1 due to surface -OH groups are observed. After PEG conjugation, some peaks appeared at 1354 and 1102 cm-1, indicating asymmetric and symmetric stretching of C-O-C, and out-of-plane bending of the -CH of the PEG chains at 956 cm-1. 4 \| J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx This journal is © The Royal Society of Chemistry 20xx Toxicity in vitro In vitro toxicological characterization of the nanoparticles was evaluated through the degree of cell survival by means of the standard MTT assay. The cell lines used, Hep G2 and Caco-2, represent the liver and the intestine, which are two important target organs to encounter the NPs after oral exposure or intravenous injection in a biomedical application. The analysis of cytotoxicity after incubation with the nanoparticles showed that viability of cell culture is not significantly reduced by the presence of the nanoparticles up to 160 µg/mL Fe concentration after 24 h of treatment (80-100% viability compared with the control) (Figure 2 A, B). At iron concentrations of 320 µg/mL, SC decreased the viability of both cell lines down to values of 20 %. In the case of MC, viability percentage of both cell lines is in the range of 60-80 %, indicating lower toxicity even at high iron concentrations. The effect of PEG coating is not very clear at high iron concentration. In the case of Hep G2 cells, PEG functionalization improved the cytotoxicity for SC while in Caco-2 it had no effect. For MC, PEG functionalization reduces the viability to 60 % in Caco-2 cells (Figure 2 A, B). Even though Figure 2. Evaluation of cell viability by MTT assay. (A) Hep G2 cells. (B) Caco-2 cells. Cells were treated with the nanoparticles for 24 h. (C) Ferritin formation measured by ELISA in Caco-2 cells exposed to 40 µg/mL of single or multicore nanoparticles. Data represent means ± SD (n = 3). shows statistically- significance compared with the control (t test, p<0.05, * p<0.01, *p<0.001, *p<0.0001). 4 \| J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Page 5 of 15 Nanoscale View Article Online DOI: 10.1039/C7NR06020C View Article Online DOI: 10.1039/C7NR06020C Journal Name ARTICLE 0.5, 0.75 and 1 mg/mL. In these experiments, nanoparticles were added at stage 38 and survival was evaluated every 24 h until embryos reach stage 45, after approximately 72 h of exposure. None of the conditions was found lethal for the embryos, therefore we decided to perform the rest of the experiments comparing a low and a high dose of nanoparticles (0.5 and 1 mg/mL). The use of the Xenopus model has advantages with respect to other animal models. First, embryos develop externally, allowing experiments to be performed prior to, or directly following fertilization. Toxicity in vitro Also, they have a rapid embryo growth and development. A larval tadpole has a fully functional set of organs, and it can be examined to determine if any experimental intervention (in this case a solution containing nanoparticles) has had an effect. this method needs further refinement and standardization for toxicity evaluation of nanomaterials, it offers a quick, inexpensive and high-throughput methodology to perform in vitro cytotoxicity screens.36 Published on 08 December 2017. Downloaded by University of East Anglia Library on 13/12/2017 10:14:31. In order to characterize the absorption and biotransformation of the nanoparticles, we determined by ELISA the ferritin formation in Caco-2 cells as a measure of iron uptake (Figure 2 C). Single core nanoparticles seem to be internalized by the cells more efficiently than multicore nanoparticles as evidenced by a 5.4-fold increase in intracellular ferritin (cf. control) compared with a 3.7-fold increase for MC-Cit probably due to the smaller size of the SC. PEGylated MC particles failed to alter (p>0.05) the ferritin levels in Caco-2 cells. The surface modification with PEG in both types of nanoparticles reduces the internalization by the cells. The coating with short-chain PEG molecules of iron oxide nanoparticles was found to shield their surface charge and decreases their internalization.37 scale Accepted Manuscript Nanoscale Accepted Manuscript Tadpoles treated with SC-DMSA and MC-Cit at 1 mg/mL displayed some general body toxicity compared with the control, characterized by defects in embryo body shape like bent spine or enlarged ventral fin (Figure 3 B). In the case of SC-DMSA, 20 % of the animals showed enlarged ventral fin and MC-Cit provokes a significant increase (27 %) of embryos with bent spine. Nanoscale Accepted Manusc In vivo evaluation of nanoparticles The intestine coiled structure is preserved although it is visibly enlarged. Embryos treated with MC-Cit at the same dose showed more drastic effects (20 % mild and 36 % severe inflammation). In the case of severe inflammation, the gut is not maintained or not well formed, as we are unable to see the intestinal loops. Regarding the effects on inflammation we didn’t observed significant differences in PEGylated particles compared with the uncoated ones. In addition, 30 % of the animals treated with MC showed a significant decrease of touch response after 72 hours of exposure (Figure 3 A). Figure 4. Scanning electron microscopy (SEM) images of pharynx sections from different embryos. (A) Schematic representation of the transversal section analyzed. (B) Control. (C) Embryos exposed to SC-DMSA nanoparticles (1 mg/mL, 72 h). (D) Embryos exposed to MC-Cit nanoparticles (1 mg/mL, 72 h). Right panel shows elemental analysis of red selected areas in the images obtained by Energy Dispersive X-ray Spectrometry (SEM-EDS). oscale Accepted Manuscript Nanoscale Accepted Manuscri Nanoscale Accepted Manu p ( g ) Electron microscopy analysis of pharynx sections (Figure 4), taken from different embryos, affords the localization of the ingested particles. The presence of the NPs in the tissue sample is evidenced by the Energy Dispersive X-ray (EDS) elemental mapping which delivers spots with brighter contrast in areas of NP accumulation, due to the higher Z atomic number of iron. NP intake is visibly detected in the images of pharynx sections of the embryos because the EDS spectra clearly showed K electron shell (K-α and K-β lines) of iron, whereas in the case of control these peaks are absent (Figure 4). EDS analysis confirmed that only at the dose of 1 mg/mL, SC-DMSA and MC-Cit, could be detected in the upper body tissue sections, in contrast with the control. PEGylated nanoparticles (SC or MC) were undetectable in all organisms. We also observed SC-DMSA in the gills of the embryos (Figure 2S). These results agree with the size-dependent deposition in the intestine and/or the gills in Zebrafish observed for silver nanoparticles. Particles from 10-20 nm showed increased bioavailability compared with 140 nm particles.38 In our case, the deposition of SC-DMSA in the gills is directly related to the smaller size and larger surface area compared with MC-Cit, providing increased adherence, penetration, and deposition in the organism. Figure 4. Scanning electron microscopy (SEM) images of pharynx sections from different embryos. In vivo evaluation of nanoparticles (A) Schematic representation of the transversal section analyzed. (B) Control. (C) Embryos exposed to SC-DMSA nanoparticles (1 mg/mL, 72 h). (D) Embryos exposed to MC-Cit nanoparticles (1 mg/mL, 72 h). Right panel shows elemental analysis of red selected areas in the images obtained by Energy Dispersive X-ray Spectrometry (SEM-EDS). In vivo evaluation of nanoparticles The presence of the NPs in the tissue sample is evidenced by the Energy Dispersive X-ray (EDS) elemental mapping which delivers spots with brighter contrast in areas of NP accumulation, due to the higher Z atomic number of iron. NP intake is visibly detected in the images of pharynx sections of the embryos because the EDS spectra clearly showed K electron shell (K-α and K-β lines) of iron, whereas in the case of control these peaks are absent (Figure 4). EDS analysis confirmed that only at the dose of 1 mg/mL, SC-DMSA and MC-Cit, could be detected in the upper body tissue sections, in contrast with the control. PEGylated nanoparticles (SC or MC) were undetectable in all organisms. We also observed SC-DMSA in the gills of the embryos (Figure 2S). These results agree with the size-dependent deposition in the intestine and/or the gills in Zebrafish observed for silver nanoparticles. Particles from 10-20 nm showed increased bioavailability compared with 140 nm particles.38 In our case, the deposition of SC-DMSA in the gills is directly related to the smaller size and larger surface area compared with MC-Cit, providing increased adherence, penetration, and deposition in Figure 3. Xenopus laevis phenotypic nanotoxicity assay after 72 h of exposure from stage 38-45. (A) Quantification of embryonic abnormalities. Histograms shown are of 30 embryos per group (Chi-Square test, (p<0.05, p<0.01, p<0.001, *p<0.0001). (B) Representative images from the X. laevis phenotypic nanotoxicity assay. Figure 3. Xenopus laevis phenotypic nanotoxicity assay after 72 abnormalities. Histograms shown are of 30 embryos per p<0.0001). (B) Representative images from the X. laevis phe Interestingly, the effect of the PEG coating for each type of NPs reduced the toxicological effect as evidenced in a 2.6-fold decrease of embryos with bent spine and a 2-fold decrease of mild gut inflammation for MC; and a significant reduction in the enlarged ventral fin phenotype for SC treated tadpoles (Figure 3 A). In the case of embryos treated with 0.5 mg/mL, defects were subtler. These results suggested a dose- dependent uptake of the NPs by the embryos. We next evaluated gut morphology as it is the main organ involved in NPs’ absorption after oral ingestion. Different degrees of inflammation were observed according to the dose of the nanoparticles, but also according to their chemical structure. Mild inflammation is observed in 43 % of animals treated with SC-DMSA at 1 mg/mL. In vivo evaluation of nanoparticles To characterize the general toxicity of the nanoparticles, embryos were treated with four different concentrations, 0.25, Nanoscale Accepted M This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 \| 5 Please do not adjust margins Nanoscale Accepted N l A t d This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 \| 5 Please do not adjust margins Please do not adjust margins Nanoscale Page 6 of 15 ne Page 6 of 15 e 15 Nanoscale View Article Online DOI: 10.1039/C7NR06020C ARTICLE ARTICLE Figure 3. Xenopus laevis phenotypic nanotoxicity assay after 72 h abnormalities. Histograms shown are of 30 embryos per *p<0.0001). (B) Representative images from the X. laevis phen Interestingly, the effect of the PEG coating for each type of NPs reduced the toxicological effect as evidenced in a 2.6-fold decrease of embryos with bent spine and a 2-fold decrease of mild gut inflammation for MC; and a significant reduction in the enlarged ventral fin phenotype for SC treated tadpoles (Figure 3 A). In the case of embryos treated with 0.5 mg/mL, defects were subtler. These results suggested a dose- dependent uptake of the NPs by the embryos. We next evaluated gut morphology as it is the main organ involved in NPs’ absorption after oral ingestion. Different degrees of inflammation were observed according to the dose of the nanoparticles, but also according to their chemical structure. Mild inflammation is observed in 43 % of animals treated with SC-DMSA at 1 mg/mL. The intestine coiled structure is preserved although it is visibly enlarged. Embryos treated with MC-Cit at the same dose showed more drastic effects (20 % mild and 36 % severe inflammation). In the case of severe inflammation, the gut is not maintained or not well formed, as we are unable to see the intestinal loops. Regarding the effects on inflammation we didn’t observed significant differences in PEGylated particles compared with the uncoated ones. In addition, 30 % of the animals treated with MC showed a significant decrease of touch response after 72 hours of exposure (Figure 3 A). Electron microscopy analysis of pharynx sections (Figure 4), taken from different embryos, affords the localization of the ingested particles. Iron quantification. AC magnetic susceptibility and ICP-OES In order to quantify the iron present in the samples through SEM-EDS analysis, the application of quantitative correction procedures is needed, which are sometimes referred to as matrix corrections.39 As SEM only provides local information, the quantitative analysis of NP intake, was performed through magnetic characterization of freeze-dried tissue samples and iron elemental analysis of acid digested embryos. Moreover, these techniques allow the comparison of the accumulation depending on the coating and the surface charge (DMSA, citric acid, and PEG functionalization). In order to evaluate the accumulation of the nanoparticles in the animals, we performed AC susceptibility of pools of seven animals. Magnetic measurements, especially AC magnetic susceptibility, are extremely sensitive being able to distinguish the contribution from the magnetic nanoparticles from that of 6 \| J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx e ARTICLE Figure5-01.tif C magnetic susceptibility measurement of embryo tissue samples. In-phase (χ’(T)) and out-of-phase (χ’’(T)) of the magnetic susceptibility, per mass of sample, corresponding to freeze-dried tissues from different embryos e (72 h): (A, C) Embryos exposed to SC. (B, D) Embryos exposed to MC. (n = 7 per group). Nanoscale View Article Online DOI: 10.1039/C7NR06020C ame Figure5-01.tif Nanoscale al Name Figure5-01.tif Nanoscale Name., 2013, 00, 1-3 \| 7 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Nanoscale do not adjust margins Nanoscale Page 8 of 15 Page 8 of 1 Nanoscale View Article Online DOI: 10.1039/C7NR06020C ARTICLE Nanoscale Nanoscale facilitates the reduction of ferric to ferrous ions enabling the absorption of iron. were detected. In addition, a quantitative analysis of NP intake was performed by ICP-OES (Figure 6). Elemental analysis results evidenced a dose-dependent iron absorption, which is in agreement with the iron content detected by AC magnetic susceptibility. The absorption of SC-DMSA detected in the embryos has been confirmed by ICP-OES, a three or five-fold increase in comparison with MC-Cit for iron concentrations of 0.5 and 1 mg/mL, respectively. Interestingly, the iron content in the animals treated with PEGylated NPs is comparable to the control indicating a very low absorption of these NPs. were detected. In addition, a quantitative analysis of NP intake was performed by ICP-OES (Figure 6). Elemental analysis results evidenced a dose-dependent iron absorption, which is in agreement with the iron content detected by AC magnetic susceptibility. The absorption of SC-DMSA detected in the embryos has been confirmed by ICP-OES, a three or five-fold increase in comparison with MC-Cit for iron concentrations of 0.5 and 1 mg/mL, respectively. Interestingly, the iron content in the animals treated with PEGylated NPs is comparable to the control indicating a very low absorption of these NPs. In the basolateral membrane of the enterocytes the ferroportin transporter is located together with hephaestin which oxidize ferrous ions and allows transferrin to bind iron and carry it in the bloodstream through the different organs. Hepcidin negatively regulates the entry of iron into circulation through inhibiting ferroportin in case of iron overload (Figure 8 A). Ferritin is the most important protein involved in iron storage within cells, and the levels of Fe2+ present in the organism regulates its expression.43 g y p One possible explanation for the visual disturbance of the intestine structure of the embryos treated with MC-Cit-PEG, could be that the particles are rapidly excreted by the organism and therefore the iron content in these animals is seen as low. As we previously observed in Caco-2 cells, PEG functionalization reduces net surface charge of the particles and this effect could reduce the absorption of the particles in the gut favouring their excretion. al Name Figure5-01.tif Nanoscale Surface functionalization with PEG has been reported to enable particles to diffuse through mucus in the gastrointestinal tract at a rate similar to diffusion through water. It has been hypothesized that this occurs by reduction of particle-mucus interactions.40 There is also evidence that PEG-coating can decrease macrophage or HeLa uptake of iron particles where the polymer increases uptake hindrance compared with uncoated NPs.41 Another possible explanation for the morphological alterations observed at higher doses of nanoparticles could be the presence of polyol used during the MC synthesis. Other studies have shown that polyol-made nanoparticles produce in vitro (10 µg/mL) and in vivo (0.8 mg/kg) toxicity.42 From magnetic measurements and elemental analysis, we detected a higher absorption of SC-DMSA compared with MC- Cit. We propose that the rate of particle internalization into the embryos will dictate the time at which iron metabolic pathway activation is measurable. In the case of SC-DMSA treated embryos, dmt-1, transferrin and ferritin have the highest levels of transcription. The increase in ferritin in embryos treated with SC-DMSA compared with MC-Cit, follows the same pattern observed in Caco-2 cells where the protein was detected by ELISA suggesting a higher iron bioavailability from single-core nanoparticles. Hepcidin is downregulated for SC-DMSA, which implies a saturation of the metabolic pathway of iron. In the case of MC-Cit only ferritin and hepcidin are upregulated corroborating a slower activation of the iron metabolic pathway depending on the amount of internalized particles after 72 h of treatment. scale Accepted Manuscript Nanoscale Accepted Manuscript Nanoscale Accepted Manuscri Nanoscale Accepted Manu Nanoscale Accepted Figure 6. Iron quantification though ICP-OES of acid digested tissues from embryos treated at different concentrations of IOMNPs. Striped pattern: 0.5 mg/mL. Solid pattern: 1 mg/mL. (n = 7 per group). Iron metabolism Since iron resulting from NP degradation is predicted to be processed by iron metabolic pathways, we next studied the expression of different genes involved in iron metabolism and oxidative stress. The expression of genes linked to both processes was assessed by real-time quantitative PCR (RT- qPCR) in embryos treated with SC-DMSA and MC-Cit after 72 h of treatment with the nanoparticles at 1 mg/mL, taking into account that only at highest doses could we detect a significant amount of absorbed nanoparticles (Figure 7 A). In the case of SC-DMSA treated embryos, transferrin and dmt1 increased 2.6 and 2-fold respectively (cf. control) coupled with a 70 % downregulation of hepcidin. In contrast, the MC-Cit treated embryos displayed marginal increases (p<0.05) in hepcidin, and transferrin and dmt1 levels remained unchanged. In both cases the transcription of ferritin is increased after nanoparticle treatment, 2.9-fold change for SC- DMSA and 1.9-fold change for MC-Cit treated embryos Figure 7 A). From these data, we can describe differences in the metabolic activation pathway of single-core or multicore nanoparticles. After iron ingestion, ferrous ions (Fe2+) are absorbed in the enterocytes through the divalent metal transporter-1 (DMT1). In the apical membrane of the enterocytes, the cytochrome B Since iron resulting from NP degradation is predicted to be processed by iron metabolic pathways, we next studied the expression of different genes involved in iron metabolism and oxidative stress. The expression of genes linked to both processes was assessed by real-time quantitative PCR (RT- qPCR) in embryos treated with SC-DMSA and MC-Cit after 72 h of treatment with the nanoparticles at 1 mg/mL, taking into account that only at highest doses could we detect a significant amount of absorbed nanoparticles (Figure 7 A) significant amount of absorbed nanoparticles (Figure 7 A). In the case of SC-DMSA treated embryos, transferrin and dmt1 increased 2.6 and 2-fold respectively (cf. control) coupled with a 70 % downregulation of hepcidin. In contrast, the MC-Cit treated embryos displayed marginal increases (p<0.05) in hepcidin, and transferrin and dmt1 levels remained unchanged. In both cases the transcription of ferritin is increased after nanoparticle treatment, 2.9-fold change for SC- DMSA and 1.9-fold change for MC-Cit treated embryos Figure 7 A). From these data, we can describe differences in the metabolic activation pathway of single-core or multicore nanoparticles. After iron ingestion, ferrous ions (Fe2+) are absorbed in the enterocytes through the divalent metal transporter-1 (DMT1). 8 \| J. Name., 2012, 00, 1-3 al Name Figure5-01.tif Nanoscale Page 7 of 15 oscale Accepted Manuscript Nanoscale Accepted Manuscript Nanoscale Accepted Manuscri Nanoscale Accepted Manu Nanoscale Accepted Figure 5. AC magnetic susceptibility measurement of embryo tissue samples. In-phase (χ’(T)) and out-of-phase (χ’’(T)) components of the magnetic susceptibility, per mass of sample, corresponding to freeze-dried tissues from different embryos after exposure (72 h): (A, C) Embryos exposed to SC. (B, D) Embryos exposed to MC. (n = 7 per group). coating (Figure 5), independently if they are SC or MC. Interestingly PEG coated nanoparticles, both SC and MC, were below the detection limits of the technique. These results are in agreement with what we observed by SEM-EDS. In this work, the presence of ferritin, the iron storage protein with an out-of-phase susceptibility maxima located at around 10 K,18 has not been observed. The absence of a substantial paramagnetic contribution, usually observed at the lowest temperatures, indicates that the concentration of free iron atoms is very low, indicating that NP degradation process is limited if not absent in the timescale of these experiments. Through AC magnetic susceptibility measurements, dose- dependent amounts of nanoparticles (SC-DMSA and MC-Cit) other endogenous iron-containing species, usually present in a bigger concentration but with weaker magnetic signal than the nanoparticles. The presence of magnetic nanoparticles can be identified by a maximum in the in-phase magnetic susceptibility (χ’) accompanied by a maximum at slightly lower temperatures in the out-of-phase susceptibility (χ’’). The temperature location of the maxima depends on the nanoparticle (material, size, aggregation degree, etc.). The height of the maxima is a surrogate measurement of the concentration of nanoparticles. other endogenous iron-containing species, usually present in a bigger concentration but with weaker magnetic signal than the nanoparticles. The presence of magnetic nanoparticles can be identified by a maximum in the in-phase magnetic susceptibility (χ’) accompanied by a maximum at slightly lower temperatures in the out-of-phase susceptibility (χ’’). The temperature location of the maxima depends on the nanoparticle (material, size, aggregation degree, etc.). The height of the maxima is a surrogate measurement of the concentration of nanoparticles. In our case, a dose-dependent amount of nanoparticles is observed in the animals treated with particles without PEG J. Oxidative stress MC-Cit (sod 0.5- fold, catalase 0.67-fold and gsr 1.14-fold) (Figure 7 B). These results suggest the activation of protective mechanisms depending on nanoparticle uptake in order to reduce reactive oxygen species (ROS) generated during the biodegradation process. This is the early response of the organism when nanoparticle clearance induces the formation of ROS. However, we are not in the presence of an acute state of oxidative stress when a general decrease of antioxidant enzymes takes place.46 In embryos treated with SC-DMSA or MC-Cit at 1 mg/mL we observed a 2.2 and 2.96-fold increase in hmox1 (cf. control) respectively after 72 h of exposure (Figure 7 B). Increases in transcript levels of hmox1 have been described in macrophages treated with iron oxide hybrids nanomaterials.44 Hmox1 is induced after oxidative stress, reactive oxygen species or heavy metals, thus degrading heme group to form biliverdin and Fe2+. The production of Fe2+ leads to the activation of iron regulatory protein (IRP) which is able to control the translation of iron sensitive proteins such as ferritin. In the absence of iron, IRP binds to ferritin mRNA and inhibits its translation. However, when iron ions are available, they bind to IRP and release it from ferritin mRNA, thus allowing its translation (Figure 8 B).45 The increase in Fe2+ in the embryos 72 h after the treatment, activates iron response proteins, which activates the translation of proteins involved in iron metabolism such as ferritin. We show in vivo the activation by Hmox-1 of iron regulatory proteins and their effect in the transcription of ferritin mRNA associated to the treatment with iron oxide nanoparticles (Figure 7 A, B). Other oxidative stress response genes showed an increase in expression in the case of embryos treated with SC-DMSA (sod 1 35- fold catalase 3 27-fold and gsr 2 85-fold) compared with With these results, we provide evidence that an early developmental vertebrate model such as Xenopus laevis is a rapid and inexpensive system for NP toxicity assessment, compared with adult mammalian models. Because most theranostic applications need nanoparticles to be taken up by the cells, intracellular nano-biodegradation in an in vitro model needs to correlate with in vivo observations. In the case of IONPs degradation in vitro and their bioavailability by the cells translated into an increase of ferritin levels (Figure 2 C), should correlate with the corresponding activation of the iron metabolic pathway as shown in Figure 7 A). Iron metabolism In the apical membrane of the enterocytes, the cytochrome B Figure 6. Iron quantification though ICP-OES of acid digested tissues from embryos treated at different concentrations of IOMNPs. Striped pattern: 0.5 mg/mL. Solid pattern: 1 mg/mL. (n = 7 per group). 8 \| J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Page 9 of 15 Nanoscale View Article Online DOI: 10.1039/C7NR06020C View Article Online DOI: 10.1039/C7NR06020C Journal Name Figure 7. Effect on the expression of different genes in X. laevis embryos exposed to SC-DMSA or MC-Cit after 72 h at 1 mg/mL. (A) Genes involved in iron metabolism. (B) Genes involved in oxidative stress response. (n = 7 per group). shows statistically- significance compared with the control (t test, ns >0.05, p<0.05, * p<0.01, *p<0.001, *p<0.0001). Published on 08 December 2017. Downloaded by University of East Anglia Library on 13/12/ oscale Accepted Manuscript Nanoscale Accepted Manuscri Nanoscale Accepted Manu Figure 7. Effect on the expression of different genes in X. laevis embryos exposed to SC-DMSA or MC-Cit after 72 h at 1 mg/mL. (A) Genes involved in iron metabolism. (B) Genes involved in oxidative stress response. (n = 7 per group). shows statistically- significance compared with the control (t test, ns >0.05, p<0.05, * p<0.01, *p<0.001, **p<0.0001). J. Name., 2013, 00, 1-3 \| 9 Oxidative stress dictate the time at which iron metabolic pathway activation is measurable. Also, the NP deposition and uptake likely depends on the physico-chemical characteristics of the nanoparticles in terms of structure, size, and chemical modification of the surface. We can consider Xenopus as the bridge between cell- based assays and mammalian models taking into account that the activation of the iron metabolic pathway, especially with SC-DMSA, correlates with previous results in different murine models tracking IONPs biodegradation.18,32,47 The significant increase of transcript levels of ferritin or dmt1 correlates with previous observations in a Wistar rat animal model where these proteins were quantified by ELISA in tissue samples from the liver, spleen and kidneys, 24 hours after SC-DMSA intravenous injection.18 Also, multicore nanoparticles degradation has been described in vitro recently, starting with the dissociation of the multigrain structure and following with the massive dissolution of the iron oxide cores48. These results highlight the necessity to study at longer times the nanoparticle biotransformation in X. laevis. on 08 December 2017. Downloaded by University of East Anglia Library on 13/12/2017 10:14:31. Published on 08 December 2017. Downloaded by University of East Anglia Library on 13/12/2017 10:14:31. Published on 08 December 2017. Downloaded by University of East Anglia Library on 13/12 scale Accepted Manuscript Nanoscale Accepted Manuscript Materials Commercial products: iron(III) chloride hexahydrate, oleylamine, oleic acid, meso-2,3-dimercaptosuccinic acid (DMSA), 1-octadecene, toluene, dimethyl sulfoxide, O,O′-bis(2- aminoethyl)PEG, 2000 Da, polyvinylpyrrolidone PVP40, sodium acetate trihydrated, and citric acid (Sigma-Aldrich), sodium oleate (Riedel-de Haen), n-hexane (Scharlau), ethyl-3-(3- dimethylaminopropyl)-carbodiimide, ethylene glycol (Fluka), and ethanol (Panreac). Dialysis tubing cellulose membranes were purchased from Sigma-Aldrich and washed prior to use. Figure 8. Schematic representation of iron metabolic pathway. (A) Enterocyte uptake and transfer of iron: Ferrous ions (Fe2+) are absorbed in the enterocytes through the divalent metal transporter-1 (DMT1). In the apical membrane of the enterocytes, the cytochrome B (DcytB) facilitates the reduction of ferric to ferrous ions enabling the absorption of iron. In the basolateral membrane of the enterocytes the ferroportin transporter (Fpn) is located together with hephaestin (Hp), which oxidize ferrous ions and allows transferrin to bind iron and carry it in the bloodstream through the different organs. In the liver iron will be stored in complex with ferritin (Ftn). In the This journal is © The Royal Society of Chemistry 20xx Conclusions In this work, the toxicity and metabolization of DMSA, citric acid and PEG coated single and multicore iron oxide magnetic nanoparticles have been studied in Hep G2 and Caco-2 cell lines, and in an amphibian animal (Xenopus laevis) model during its embryo development. The viability of both cell lines is preserved in all cases when treated with the nanoparticles up to an iron concentration of 160 µg/mL. Above that concentration, only PEGylated nanoparticles didn’t cause toxicity on Hep G2 cells but Caco-2 cells were more sensitive. In the case of the in vivo viability, although none of the single- core nor multicore coated nanoparticles were found lethal for the embryos, more dramatic effects were observed in the treatment with multicore nanoparticles. Single-core nanoparticles are absorbed (three or five-fold increase for doses of 0.5 and 1 mg/mL, respectively) in comparison with multicore ones suggesting size-dependent differences in the deposition an uptake of the nanoparticles. Monitoring nanoparticle biodegradation is critical for therapeutic efficiency and safety issues. Depending on their structural organization (single or multicore) and surface chemistry, the embryos will trigger the iron metabolic pathway to varying degrees. These findings advance the understanding of iron oxide nanoparticle metabolization in an early developmental vertebrate model like Xenopus laevis, which offers a quick, inexpensive and high-throughput alternative prior toxicity assessment of nanotherapeutics in rodent models. Nanoscale Accepted Manuscri Nanoscale Accepted Manu Figure 8. Schematic representation of iron metabolic pathway. (A) Enterocyte uptake and transfer of iron: Ferrous ions (Fe2+) are absorbed in the enterocytes through the divalent metal transporter-1 (DMT1). In the apical membrane of the enterocytes, the cytochrome B (DcytB) facilitates the reduction of ferric to ferrous ions enabling the absorption of iron. In the basolateral membrane of the enterocytes the ferroportin transporter (Fpn) is located together with hephaestin (Hp), which oxidize ferrous ions and allows transferrin to bind iron and carry it in the bloodstream through the different organs. In the liver iron will be stored in complex with ferritin (Ftn). In the Oxidative stress Here we demonstrated for the first time the study, by means of AC magnetic susceptibility measurements, of the intake of iron-containing particles in X. laevis embryos. We postulate that the rate of particle internalization into the embryos will Other oxidative stress response genes showed an increase in expression in the case of embryos treated with SC-DMSA (sod 1.35- fold, catalase 3.27-fold and gsr 2.85-fold) compared with J. Name., 2013, 00, 1-3 \| 9 J. Name., 2013, 00, 1-3 \| 9 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Nanoscale do not adjust margins Nanoscale Page 10 of 15 i Page 10 of 15 i Nanoscale View Article Online DOI: 10.1039/C7NR06020C ARTICLE Nanoscale dictate the time at which iron metabolic pathway activation is measurable. Also, the NP deposition and uptake likely depends on the physico-chemical characteristics of the nanoparticles in terms of structure, size, and chemical modification of the surface. We can consider Xenopus as the bridge between cell- based assays and mammalian models taking into account that the activation of the iron metabolic pathway, especially with SC-DMSA, correlates with previous results in different murine models tracking IONPs biodegradation.18,32,47 The significant increase of transcript levels of ferritin or dmt1 correlates with previous observations in a Wistar rat animal model where these proteins were quantified by ELISA in tissue samples from the liver, spleen and kidneys, 24 hours after SC-DMSA intravenous injection.18 Also, multicore nanoparticles degradation has been described in vitro recently, starting with the dissociation of the multigrain structure and following with the massive dissolution of the iron oxide cores48. These results highlight the necessity to study at longer times the nanoparticle biotransformation in X. laevis. case of iron overload Hepcidin transcription is activated and it will regulate the entry of iron into circulation through inhibiting ferroportin. (B) Regulation of ferritin mRNA translation by Hmox1 and IRP: Iron overload induces ROS generation. Hmox1 is induced after oxidative stress, thus degrading heme group to form biliverdin and ferrous ions. The production of Fe2+ leads to the activation of iron regulatory protein (IRP) which is able to control the translation of iron sensitive proteins such as ferritin. In the absence of iron, IRP binds to ferritin mRNA and inhibits its translation. However, when iron ions are available, they bind to IRP and release it from ferritin mRNA, thus allowing its translation. Journal Name Journal Name Journal Name ARTICLE Synthesis of iron oxide single-core nanoparticles (SC). The synthesis of iron oxide nanoparticles has been based on a previous work described in the literature.49 The reaction was carried out under nitrogen. In a round-bottomed flask (500 mL), equipped with a mechanical stirrer (glass stirrer shaft), thermometer, entry for nitrogen flow and reflux condenser, iron(III) oleate (4.54 g) was mixed with oleic acid (0.724 g) in 1- octadecene (50 mL). The mixture was stirred (100 rpm) and slowly heated (5 ºC·min-1 for T< 100ºC, and 2ºC·min-1 for T> 100ºC) until reflux (325ºC) with a heating mantle. The heating mantle was withdrawn, and the system was cooled down to room temperature. The resulting black mixture was washed with ethanol several times by centrifugation (RCF = 7500) and magnetic decantation. The resulting dried black solid was redispersed in hexane. Synthesis of iron oxide single-core nanoparticles (SC). The synthesis of iron oxide nanoparticles has been based on a previous work described in the literature.49 The reaction was carried out under nitrogen. In a round-bottomed flask (500 mL), equipped with a mechanical stirrer (glass stirrer shaft), thermometer, entry for nitrogen flow and reflux condenser, iron(III) oleate (4.54 g) was mixed with oleic acid (0.724 g) in 1- octadecene (50 mL). The mixture was stirred (100 rpm) and slowly heated (5 ºC·min-1 for T< 100ºC, and 2ºC·min-1 for T> 100ºC) until reflux (325ºC) with a heating mantle. The heating mantle was withdrawn, and the system was cooled down to room temperature. The resulting black mixture was washed with ethanol several times by centrifugation (RCF = 7500) and magnetic decantation. The resulting dried black solid was redispersed in hexane. -Surface coating with citric acid on multicore NPs (MC-Cit). For citric acid coating a standard procedure was used.24,52 First, sample volume equivalent to 20 mg of Fe was adjusted to pH 2 and then dispersed in 13 mL of a solution of citric acid 0.1 M. Afterwards, the mixture was heated at 80 °C for 30 min. The solution was centrifuged and washed with distilled water. Finally, the pH was adjusted first to 11 with KOH 1M and then to 7 with HNO3 0.01 M. -Surface coating with polyethylene glycol (SC-DMSA-PEG and MC-Cit-PEG). Nanoparticle structural characterization The particle sizes and morphologies were determined by transmission electron microscopy (TEM) with a JEM1010 microscope (JEOL, Peabody, USA) operating at 100 kV. Samples were prepared by placing a drop of the uncoated particles suspended in water onto a carbon coated copper grid and allowing it to dry at room temperature. The size distributions were determined by manual measurement of more than 100 particles using the public domain software ImageJ. The presence of the coating and the washing process was also confirmed and studied by Fourier transform infrared spectroscopy (FTIR) in the range of 4000-250 cm-1 by use of a Bruker (USA) IFS 66VS. The samples for FTIR were prepared diluting the dried powder in KBr at 2 % by weight and pressing it into a pellet. The presence of the coating was also studied by thermogravimetric analyses (TGA). They were performed in a Seiko TG/DTA 320U thermobalance, whose temperature scanning range is from room temperature up to 900 °C. For this work, samples were heated from room temperature to 700 °C at 10 °C/min under an air flow of 100 mL·min-1. Platinum pans were used and α-Al2O3 was used as reference. Colloidal properties were studied in a Zetasizer Nano S, from Malvern Instruments (UK). The hydrodynamic size was determined by Dynamic Light Scattering (DLS) and the zeta potential was measured as a function of pH at 25 °C, using HNO3 and KOH to change the pH of the suspensions. Hydrodynamic size is given as the intensity-weighted mean. Surface modification -Ligand exchange with dimercaptosuccinic acid on single-core NPs (SC-DMSA). For DMSA ligand exchange, a standard procedure was used.51 In a typical experiment, ethanol (20 mL) was added to a volume of SC dispersed in hexane containing a mass of Fe3O4 of 50 mg. The mixture was sonicated and then placed on a magnet to separate the liquid from the black solid residue of nanoparticles. The residue was washed with more ethanol (5 x 10 mL) following the same procedure, until the discarded liquid had a clean appearance. The remaining black residue was dispersed in toluene (20 mL) and the dispersion added to a solution of DMSA (90 mg) in dimethyl sulfoxide (5 mL). The resulting black suspension was then shaken in a laboratory tube rotator. After 2 days, SC-DMSA nanoparticles were precipitated as a black powder stuck to the glass tube and the liquid phase was transparent and pale yellow. The liquid was discarded and the nanoparticles were washed with ethanol (4 x 10 mL), sonicating and centrifuging (RCF = 7500). The final black solid was air dried and redispersed in distilled water. KOH 1 M was added to increase the pH to 10 and HNO3 0.01 M was used to lower the pH to 7. The dispersion was then placed in a cellulose membrane and dialyzed for 5 days in distilled water, to remove any excess of unreacted DMSA and any other small impurities that may be present in the dispersion without being attached to the nanoparticles. Journal Name PEG conjugation reaction has been based on a previous work described in the literature.37 Amine- functionalized PEG was attached to SC-DMSA or MC-Cit via an ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)-mediated coupling reaction using the polymer O,O′-bis(2- aminoethyl)PEG, 2000 Da (Figure 3S). PEGylation reaction was carried out in a refrigerated ultrasonic bath; an aqueous solution containing 10 mg SC-DMSA or MC-Cit was mixed with the PEG derivative (4 mg). The total amount of EDC (1 mg) was divided into five aliquots, one of which was added every 1 h, and the fifth 4 h after the previous addition. The molar ratio of COOH groups/amine groups/EDC was 1:1.5:1; pH was adjusted to 6, and the mixture was sonicated (4 h, 25 °C), followed by extensive dialysis. scale Accepted Manuscript Nanoscale Accepted Manuscript Synthesis of iron oxide multicore nanoparticles (MC). The synthesis of iron oxide nanoparticles has been based on a previous work described in the literature50 but the experimental procedure and the concentration of NaAc have been varied. Typically, 2.62 mmol iron(III) chloride were dissolved with ultrasound in 109 mL of ethylene glycol. Then, 140 mmol PVP40 were added slowly under vigorously magnetic stirring (>1000 rpm) and mild heating until completely dissolved. Then, 26.2 mmol of NaAc·3H2O were added to the solution. The mixture was sealed in a Teflon-lined autoclave (125 mL) and maintained at 200 °C for 16 h for solvothermal crystallization, following cooling inside oven. The precipitated solid product was washed with ethanol and distilled water through centrifugation (RCF = 7500) several times. Nanoscale Accepted Manuscri Nanoscale Accepted Manu Nanoparticles synthesis Nanoparticles synthesis 10 \| J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Page 11 of 15 Page 11 of 15 This journal is © The Royal Society of Chemistry 20xx Nanoscale Nanoscale View Article Online DOI: 10.1039/C7NR06020C View Article Online DOI: 10.1039/C7NR06020C Histological evaluation When the embryos reached the appropriate stage, they were fixed in MEMFA (3.7 % Formaldehyde, 1X MEM salts and DEPC H2O) overnight at 4ºC. Samples were washed in PBS, dehydrated and kept in 100% ethanol. To embed embryos in wax, they were directly washed in a 65 ºC oven first in histoclear, then in 1:1 histoclear:wax and finally wax 30 min to 1 h each wash. Then the embryos were and placed in molds with wax. Embryos were sectioned using a microtome generating 10 µm slices. Slices were then hydrated in water and dried to be analyzed by Scanning electron microscopy. Cell culture Caco-2 cells (HTB-37) and HepG2 (HB-8065) were obtained from American Type Culture Collection (Manassas, VA, USA) and stored in liquid nitrogen. Hep G2 (human hepatocellular J. Name., 2013, 00, 1-3 \| 11 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Nanoscale e do not adjust margins Nanoscale Page 12 of 15 li Page 12 of 15 i Nanoscale View Article Online DOI: 10.1039/C7NR06020C ARTICLE Nanoscale Nanoscale microplate reader at 550 nm. Cell viability (%) was calculated as [(A − B)/A × 100], where A and B are the absorbance of control and treated cells, respectively. Values represent mean ± SD (n = 3). carcinoma) and Caco-2 (human colorectal adenocarcinoma) cells were cultured as mono-layers in Dulbecco’s modified Eagle medium supplemented with 2% penicillin-streptomycin and 10 % fetal bovine serum, in a humidified incubator (37 °C, 5% CO2). For toxicity experiments, cells were seeded in 96-well plates (approximately 1 x 104 cells/well, 200 μL/well). For iron uptake experiments, Caco-2 cells between passages 30-36 were seeded onto collagen-coated 12-well plates (Bio-Greiner, UK) at a density of 2 x 105 cells/well suspended in 1 mL of supplemented DMEM which was replaced every 2 days. Cells were used on confluence at days 13-15 post-seeding. In order to ensure a low basal media iron levels, 24 hours prior to the initiation of the nanoparticles treatments, the DMEM medium was replaced by Eagle’s minimum essential medium (MEM) without foetal bovine serum supplemented with 10 mmol/L PIPES [piperazine-N, N’ –bis-(2-ethanesulfonic acid)], 26.1 mM NaHCO3, 19.4 mmol/L glucose, 1 % antibiotic-antimycotic solution, 11 µmol/L hydrocortisone, 0.87 µmol/L insulin, 0.02 µmol/L sodium selenite (Na2SeO3), 0.05 µmol/L triiodothyronine and 20 µg/L epidermal growth factor.53 The day of the experiment, the nanoparticles were diluted in the low-iron MEM to obtain a 40 µg/mL final iron concentration and subsequently Caco-2 cells were exposed for 24 hours with the treatments. Ferritin formation was measured 24 h after treatment. Cells were rinsed with Milli-Q (18.2 MΩ) H2O and subsequently lysed by scraping in 100 μL (12-well plates) of CelLytic M (Sigma-Aldrich, Gillingham, UK). Cell lysates were kept on ice for 15 min and stored at −80 ◦C. For analysis, samples were thawed and centrifuged at 14,000× g for 15 min. Cellular debris was discarded and the supernatant containing the proteins was analysed for ferritin using the Spectro Ferritin ELISA assay (Ramco Laboratories Inc., Stafford, TX, USA). In vivo test Published on 08 December 2017. Downloaded by University of East Anglia Library on 13/12/2017 10:14:31. All experiments were performed in compliance with the relevant laws and institutional guidelines at the University of East Anglia. The research has been approved by the local ethical review committee according to UK Home Office regulations. Xenopus laevis embryo toxicity assays were carried out as described before54,55. Briefly, adult females were primed with PMSG (Pregnant Mare’s Serum Gonadotropin) and induced with Chorulon. Eggs were obtained manually and fertilized in a petri dish by adding male sperm (male testis incubated with 2mL 1 x MMR (Marc’s modified ringers) + 8 mL FBS). The fertilized embryos were dejellied via cysteine, and the embryos were plated in BSA-coated petri dishes covered with 0.1 x MMR. Embryos were left at 23 ºC until they reached stage 38, and then plated in 24-well plates (7 embryos/well) in 0.1 x MMR medium containing the nanoparticles and incubated at 23 ºC. Non-treated embryos were used as control. The mortality and the morphological changes of the embryos were recorded every 24 h until embryos reached stage 45. scale Accepted Manuscript Nanoscale Accepted Manuscript Nanoscale Accepted Manuscri Nanoscale Accepted Manu Magnetic characterization Cell viability was determined using the standard 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay 24 h after exposure to NP. Cells were seeded in 96-well plates at (approximately 1 x 104 cells/well, 200 μL/well). In total 60 wells were seeded per plate (6 rows x 10 columns) as the outer wells were left empty to avoid errors due to evaporation. Each row was used as a replicate (3 wells/condition) and serial dilutions went across the columns of the plate. Cells were left to grow until 70-80 % confluency. NP-containing medium was removed after 24 h, cells were rinsed three times with PBS, and MTT solution in medium (final MTT concentration 50 μg/mL) was added and incubated (2 h, 37 °C). The MTT solution was removed without disturbing cells, 0.2 mL/well of DMSO and 0.025 mL/well of Sorensen buffer were added, the plates were shaken gently to dissolve formazan crystals, and the absorbance was read on a The resulting freeze-dried samples were placed into gelatin capsules for their magnetic characterization in a Quantum Design MPMS-XL SQUID magnetometer with an AC susceptibility option. The measurements were performed with AC amplitude of 0.41 Oe, in the temperature range between 1.8 and 300 K and at a frequency of 11 Hz. Cell culture The ferritin concentration in the samples was determined using a microplate reader at an excitation wavelength of 500 nm according to the manufacturer’s protocol. Ferritin concentrations were normalized to total cell protein using the Pierce Protein BCA protein assay (ThermoFisher Scientific, Loughborough, UK). References Groups of 7 embryos were snap frozen in liquid nitrogen. RNA was extracted using High Pure RNA isolation kit (Roche) and 1 µg of RNA was taken to synthetize cDNA using Maxima First Strand cDNA synthesis kit (Thermofisher). RT-PCR was performed using SYBER Green detection method. Primers were designed targeting both copies of X. laevis genes. Gapdh have been used as a control. 1 Roger, J.; Pons, J. N.; Massart, R.; Halbreich, A.; Bacri, J. C. Some Biomedical Applications of Ferrofluids. Eur Phys J AP 1999, 5, 321-325. 1 Roger, J.; Pons, J. N.; Massart, R.; Halbreich, A.; Bacri, J. C. Some Biomedical Applications of Ferrofluids. Eur Phys J AP 1999, 5, 321-325. 2 Schutt, W.; Gruttner, C.; Hafeli, U.; Zborowski, M.; Teller, J.; Putzar, H.; Schumichen, C. Applications of Magnetic Targeting in Diagnosis and Therapy-Possibilities and Limitations: A Mini-Review. Hybridoma 1997, 16, 109-117. 3 Mejias, R.; Perez-Yague, S.; Gutierrez, L.; Cabrera, L. I.; Spada, R.; Acedo, P.; Serna, C. J.; Lazaro, F. J.; Villanueva, A.; Morales Mdel, P.; et al. Dimercaptosuccinic Acid-Coated Magnetite Nanoparticles for Magnetically Guided in Vivo Delivery of Interferon Gamma for Cancer Immunotherapy. Biomaterials 2011, 32, 2938-2952. 3 Mejias, R.; Perez-Yague, S.; Gutierrez, L.; Cabrera, L. I.; Spada, R.; Acedo, P.; Serna, C. J.; Lazaro, F. J.; Villanueva, A.; Morales Mdel, P.; et al. Dimercaptosuccinic Acid-Coated Magnetite Nanoparticles for Magnetically Guided in Vivo Delivery of Interferon Gamma for Cancer Immunotherapy. Biomaterials 2011, 32, 2938-2952. Primers Sequence 5’-3’ fth1_F tggagtaacaccctggaagc fth1_R aggatcaaccttgtcggatg tf_F agaaagggcaagtgggtttt tf_R tctggcaaagtgacaacagc dmt1_F cagaggatgaaacgcactca dmt1_R atcctgccactgatccagac hepcidin_F aaatcaaccccaatctgctg hepcidin_R gtttgttgattgccgaaggt hmox1_F ggagacctctcaggtggaca hmox1_R atggagttcatacgggaacg sod2_F tgtgcaggctcagtgtttgt sod2_R gctgcagagcaccataatca gsr_F gcaaagaggagaaggtggtg gsr_R cggaggaagtcggatgaata cat1_F cttctgcccagatgcttttc cat1_R gapdh_F gapdh_R agttgcccagagcgacttta ctttgatgctgatgctggaa gaagaggggttgacaggtga scale Accepted Manuscript Nanoscale Accepted Manuscript Nanoscale Accepted Manuscri 4 Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Elst, L. Vander; Muller, R. N. Magnetic Iron Oxide Nanoparticles: Synthesis, Stabilization, Vectorization, Physicochemical Characterizations, and Biological Applications. Chem Rev 2008, 108, 2064-2110. Nanoscale Accepted Manu 5 Alexiou, C.; Arnold, W.; Klein, R. J.; Parak, F. G.; Hulin, P.; Bergemann, C.; Erhardt, W.; Wagenpfeil, S.; Lübbe, A. S. Locoregional Cancer Treatment with Magnetic Drug Targeting. Cancer Res 2000, 60, 6641-6648. 6 Jordan, A.; Scholz, R.; Wust, P.; Fähling, H.; Felix, R. Magnetic Fluid Hyperthermia (MFH): Cancer Treatment with AC Magnetic Field Induced Excitation of Biocompatible Superparamagnetic Nanoparticles. J Magn Magn Mater 1999, 201, 413-419. 7 Weissleder, R.; Nahrendorf, M.; Pittet, M. J. References Imaging Macrophages with Nanoparticles. Nat Mater 2014, 13, 125- 138. 8 Crichton, R. Intracellular Iron Metabolism and Cellular Iron Homeostasis. In Inorganic Biochemistry of Iron Metabolism. John Wiley & Sons, Ltd, 2002; pp. 167-190. Iron content analysis Groups of 7 embryos were pooled. Animals were weighed and lyophilized 72 hours in a Telstar lyoquest lyophilizer, and the iron content was measured by Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES) in an Optima 2100 DV from PerkinElmer, after acid digestion, with concentrated HNO3 during 1 h; or kept freeze-dried for magnetic characterization, respectively. The sample manipulation was 12 \| J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Page 13 of 15 Page 13 of 15 Nanoscale View Article Online DOI: 10.1039/C7NR06020C View Article Online DOI: 10.1039/C7NR06020C View Article Online DOI: 10.1039/C7NR06020C Journal Name ARTICLE ARTICLE performed using disposable plastic material to avoid ferromagnetic contamination. assistance, especially that of Bertrand Leze from the SEM service of the University of East Anglia. assistance, especially that of Bertrand Leze from the SEM service of the University of East Anglia. Conflicts of interest 9 Crichton, R. Intracellular Iron Storage and Biomineralization. In Inorganic Biochemistry of Iron Metabolism. John Wiley & Sons, Ltd, 2002; pp. 133–165. There are no conflicts to declare. 10 Balakumaran, A.; Pawelczyk, E.; Ren, J.; Sworder, B.; Chaudhry, A.; Sabatino, M.; Stroncek, D.; Frank, J. A.; Robey, P. G. Superparamagnetic Iron Oxide Nanoparticles Labeling of Bone Marrow Stromal (Mesenchymal) Cells Does Not Affect Their “Stemness.” PLoS One 2010, 5, 1-8. Acknowledgements M. Marín-Barba has been supported by the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7 under REA grant agreement number 607142 (DevCom). This work was supported by 7-People Framework – Marie Curie Industry and Academia Partnerships & Pathways scheme (DNA-TRAP project, grant agreement nr. 612338), the European Commission Framework Program 7 (NanoMag project, NO 604448) and by the Spanish Ministry of Economy and Competitiveness (Mago project, No. MAT2014- 52069-R). H. Gavilán has carried out this work while undertaking a doctoral program on Advanced Chemistry at the Complutense University of Madrid. L. Gutiérrez acknowledges financial support from the Ramón y Cajal subprogram (RYC- 2014-15512). E. Lozano-Velasco acknowledges support from Marie Curie fellowship (705089-MIR-CHROM-C). FTIR spectroscopy and thermogravimetric and chemical analysis were carried out in the support laboratories of Instituto de Ciencia de Materiales de Madrid (ICMM/CSIC). Authors acknowledge the facilities and the scientific and technical 11 Geppert, M.; Hohnholt, M. C.; Nürnberger, S.; Dringen, R. Ferritin up-Regulation and Transient ROS Production in Cultured Brain Astrocytes after Loading with Iron Oxide Nanoparticles. Acta Biomater 2012, 8, 3832-3839. 12 Hohnholt, M. C.; Geppert, M.; Dringen, R. Treatment with Iron Oxide Nanoparticles Induces Ferritin Synthesis but Not Oxidative Stress in Oligodendroglial Cells. 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J Drug Target 2015, 23, 768-774. 24 Gutierrez, L.; Costo, R.; Gruttner, C.; Westphal, F.; Gehrke, N.; Heinke, D.; Fornara, A.; Pankhurst, Q. A.; Johansson, C.; Veintemillas-Verdaguer, S.; et al. Synthesis Methods to Prepare Single- and Multi-Core Iron Oxide Nanoparticles for Biomedical Applications. Dalton Trans 2015, 44, 2943-2952. 41 Xie, J.; Xu, C.; Kohler, N.; Hou, Y.; Sun, S. Controlled PEGylation of Monodisperse Fe3O4 Nanoparticles for Reduced Non-Specific Uptake by Macrophage Cells. Adv Mater 2007, 19, 3163-3166. 42 Hanini, A.; Schmitt, A.; Kacem, K.; Chau, F.; Ammar, S.; Gavard, J. Evaluation of Iron Oxide Nanoparticle Biocompatibility. Int J Nanomedicine 2011, 6, 787-794. 25 Silva, A. K. A.; Espinosa, A.; Kolosnjaj-Tabi, J.; Wilhelm, C.; Gazeau, F. Medical Applications of Iron Oxide Nanoparticles. In Iron Oxides; Wiley-VCH Verlag GmbH & Co. KGaA, 2016; pp. 425-472. 43 Waldvogel-Abramowski, S.; Waeber, G.; Gassner, C.; Buser, A.; Frey, B. M.; Favrat, B.; Tissot, J.-D. Physiology of Iron Metabolism. Transfus Med Hemother 2014, 41, 213-221. 26 Torchilin, V. Acknowledgements Tumor Delivery of Macromolecular Drugs Based on the EPR Effect. Adv Drug Deliv Rev 2011, 63, 131-135. 44 Elgrabli, D.; Dachraoui, W.; De Marmier, H.; Ménard-Moyon, C.; Bégin, D.; Bégin-Colin, S.; Bianco, A.; Alloyeau, D.; Gazeau, F. Intracellular Degradation of Functionalized Carbon Nanotube / Iron Oxide Hybrids Is Modulated by Iron via Nrf2 Pathway. Sci Rep 2017, 7, 40997. 27 Dutz, S.; Kettering, M.; Hilger, I.; Müller, R.; Zeisberger, M. Magnetic Multicore Nanoparticles for Hyperthermia- influence of Particle Immobilization in Tumour Tissue on Magnetic Properties. Nanotechnology 2011, 22, 265102. 28 Kolosnjaj-Tabi, J.; Lartigue, L.; Javed, Y.; Luciani, N.; Pellegrino, T.; Wilhelm, C.; Alloyeau, D.; Gazeau, F. Biotransformations of Magnetic Nanoparticles in the Body. Nano Today 2016, 11, 280-284. 45 Eisenstein, R. S.; Munro, H. N. Translational Regulation of Ferritin Synthesis by Iron. Enzyme 1990, 44, 42-58. 46 Balas, M. R.; Din Popescu, I. M.; Hermenean, A.; Cinteză, O. L.; Burlacu, R.; Ardelean, A.; Dinischiotu, A. Exposure to Iron Oxide Nanoparticles Coated with Phospholipid-Based Polymeric Micelles Induces Biochemical and Histopathological Pulmonary Changes in Mice. Int J Mol Sci 2015, 16, 29417-29435. 29 Lázaro, F. J.; Gutiérrez, L.; Abadía, A. R.; Romero, M. S.; López, A. Biological Tissue Magnetism in the Frame of Iron Overload Diseases. J Magn Magn Mater 2007, 316, 126-131. 30 Gutierrez, L.; Abadia, A. R.; Romero, M. S.; Quintana, C.; Morales, M. P.; Patino, C.; Arranz, R. Bioinorganic Transformations of Liver Iron Deposits Observed by Tissue Magnetic Characterisation in a Rat Model. J Inorg Biochem 2006, 100, 1790-1799. 47 Mejías, R.; Gutiérrez, L.; Salas, G.; Pérez-Yagüe, S.; Zotes, T. M.; Lázaro, F. J.; Morales, M. P.; Barber, D. F. 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Name., 2013, 00, 1-3 \| 15 Acknowledgements Controlled Synthesis of Uniform Magnetite Nanocrystals with High-Quality Properties for Biomedical Applications. J Mater Chem 2012, 22, 21065. 50 Sun, Q.; Ren, Z.; Wang, R.; Chen, W.; Chen, C. Magnetite Hollow Spheres: Solution Synthesis, Phase Formation and Magnetic Property. J Nanoparticle Res 2011, 13, 213-220. 51 Jun, Y.; Huh, Y.-M.; Choi, J.; Lee, J.-H.; Song, H.-T.; KimKim; Yoon, S.; Kim, K.-S.; Shin, J.-S.; Suh, J.-S.; et al. Nanoscale Size Effect of Magnetic Nanocrystals and Their Utilization for Cancer Diagnosis via Magnetic Resonance Imaging. J Am Chem Soc 2005, 127, 5732-5733. oscale Accepted Manuscript Nanoscale Accepted Manuscript 52 Martina, M.-S.; Fortin, J.-P.; Ménager, C.; Clément, O.; Barratt, G.; Grabielle-Madelmont, C.; Gazeau, F.; Cabuil, V.; Lesieur, S. Generation of Superparamagnetic Liposomes Revealed as Highly Efficient MRI Contrast Agents for in Vivo Imaging. J Am Chem Soc 2005, 127, 10676-10685. 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https://openalex.org/W2103024092	https://www.scielo.br/j/rsbmt/a/5hn7CnpjRRT9ctsMgmq6YPH/?lang=en&format=pdf	English	null	Training-related accidents during teacher-student-assistance activities of medical students	Revista da Sociedade Brasileira de Medicina Tropical	2,004	cc-by	3,029	1. Faculdade de Medicina da Universidade Federal de Minas Gerais. 2. Departamento de Clínica Médica da Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, MG. Address to: Dr. Enio R.P. Pedroso. DCM/FM/UFMG. Av. Alfredo Balena 190/4° andar, Santa Efigênia, 30130-100 Belo Horizonte, MG, Brasil. Tel: 55 31 3248-9400 e-mail: enio@medicina.ufmg.br Recebido para publicação em 29/4/2002 Aceito em 16/7/2004 ARTIGO/ARTICLE ARTIGO/ARTICLE Revista da Sociedade Brasileira de Medicina Tropical 37(5):405-408, set-out, 2004 ABSTRACT A survey was done to determine the most common hospital accidents with biologically contaminated material among students at the Medical College of the Federal University of Minas Gerais. Six hundred and ninety-four students (between fifth and twelfth semesters of the college course) answered the questionnaire individually. Three-hundred and forty-nine accidents were reported. The accident rate was found to be 33.9% in the third semester of the course, and increased over time, reaching 52.3% in the last semester. Sixty-three percent of the accidents were needlestick or sharp object injuries; 18.3% mucous membrane exposure; 16.6% were on the skin, and 1.7% were simultaneously on the skin and mucous membrane exposure. The contaminating substances were: blood (88.3%), vaginal secretion (1.7%), and others (9.1%). The parts of the body most frequently affected were: hands (67%), eyes (18.9%), mouth (1.7%), and others (6.3%). The procedures being performed when the accidents occurred were: suture (34.1%), applying anesthesia (16.6%), assisting surgery (8.9%), disposing of needles (8.6%), assisting delivery (6.3%), and others (25.9%). Forty-nine percent of those involved reported the accident to the accident control department. Of these 29.2% did not receive adequate medical assistance. Eight percent of those involved used antiretroviral drugs and of these 86% discontinued the treatment on receiving the ELISA method applied to the patient (HIV- negative); 6.4% discontinued the treatment due to its side-effects; and 16% completed the treatment. Key-words: Human Immunodeficiency Virus. Medical students. Needlestick injuries. Aids. words: Human Immunodeficiency Virus. Medical students. Needlestick injuries. Aids. RESUMO Este estudo transversal foi realizado para avaliar acidentes com material biologicamente contaminado entre estudantes de medicina da Universidade Federal de Minas Gerais. Foi aplicado um questionário individual de resposta espontânea e sigilosa, em 694 (43,4%) estudantes do quinto ao décimo segundo semestres do curso médico. Foram relatados 349 acidentes, com aumento progressivo de 33,9 até 52,3% entre os estudantes do quinto ao décimo primeiro períodos, respectivamente. Cerca de 63% dos acidentes foram com agulha ou objeto cortante; 18,3% afetando mucosas; 16,6% a pele; 1,7% simultaneamente a pele e a mucosa. Os contaminantes principais foram sangue (88,3%) e secreção vaginal (1,7%). As regiões do corpo mais freqüentemente afetadas foram as mãos (67%), olhos (18,9%), boca (1,7%) e outras (6,3%). Os procedimentos realizados no momento em que o acidente ocorreu foram sutura (34,1%), administração de anestesia (16,6%), participação em cirurgia como observador (8,9%), punção de veia com agulha (8,6%), observação de parto (6,3%) e outros (25,9%). O setor de biossegurança da instituição foi procurado por 49% dos acidentados para a busca de providências pertinentes à cada caso. Destes estudantes, 29,2% não receberam assistência médica considerada adequada e esclarecedora para sua situação. Cerca de 8 % dos estudantes receberam antiretrovirais, destes 86% descontinuaram o tratamento com o resultado do exame ELISA rápido feito no paciente fonte (VIH-negativo); 6,4% descontinuaram o tratamento devido aos efeitos colaterais das drogas usadas e 16% completaram corretamente o tratamento. es: Virus da Imunodeficiência Humana. Estudantes de medicina. Lesões provocadas por agulhas. Sida. Palavras-chaves: Virus da Imunodeficiência Humana. Estudantes de medicina. Lesões provocadas po 405 Reis JMB et al Circumstances in Brazil oblige medical students to participate in activities that ordinarily only professionals would do in many countries. In their eagerness to learn more, students may also get involved in extracurricular activities too early. Such cases are poorly supervised. Some Brazilian researchers have investigated HIV infection among hospital workers, but none specifically deal with students5 8. These peculiarities highlight the need to evaluate the risk of medical students contracting diseases from patients in the hospital during their teacher-student-assistance work. regarding the type of accident, contaminating material, body part affected, hospital setting where the accident occurred, and subsequent procedures that were undertaken. It was possible to mark more than one option as an answer to the questions about the use of personal protection equipment (barrier use) and about the factors that contributed to the accidental exposure. RESULTS Several reports evaluate accidental HIV exposure7 8 10 11 12 14. The research difficulties include recall bias, the limited scope of the survey, the chance of obtaining inaccurate information, and the subjective nature of the responses. Some retrospective studies have been conducted, interviewing students that reported exposure to potentially contaminated material8 11. Others used a daily questionnaire to overcome recall bias14. Still others, including this study, distributed questionnaires to a sample number of students7 10 12. In spite of the difficulties involved in each research method they can all provide valuable information. The total number interviewed represented a significant sample of the population. The rate at which students between fifth and twelve semesters of the medical course suffered accidents with potentially blood-contaminated material was found to be 34.2% (236/691) (Table 1). The rates according to each semester are given in Table 2. The total number of accidents reported by 236 students was 349: 63.3% (221/349) were needlestick or sharp object injuries; 18.3% (64/349) mucous membrane exposure; 16.6% (58/349) on the skin; and 1.7% (6/349) simultaneously on the skin and mucous membrane. This study had the objective of evaluating the risk of medical students to accidental exposure to potentially infected material in a hospital setting during their undergraduate training activities and their access to biosafety measures whenever necessary. The hospital setting which presented the highest number of accidents was the small wounds sector, with 42.1% of the cases (Table 3). The contaminating materials were: blood, 88.3% (308/ 349) of the accidents; vaginal secretion, 1.7% (6/349); nasal RESUMO It was asked if the student reported the accident to the medical center and what procedures they followed. The sample calculated was based on an estimate of the accident rate. Each semester of the medical course required its own estimate. These estimates, set by a previous study13. Calculations were done using Epi Info program2 to determine the minimum sample necessary to establish statistical significance for each semester. The 8% margin of error was adopted. This information is shown in Table 1. The risk of transmitting human immunodeficiency virus (HIV) as a result of a single percutaneous exposure to contaminated material is considered low. The Centers for Disease Control estimated this risk to be 0.4%9. The fact that every health professional is continuously exposed increases the risk factor. Based on previously published seroconversion rates, estimated numbers of accidents, and estimated number of HIV-positive patients, it is possible to calculate the annual contamination index for students. This fluctuates between 27 and 46 per 100,000 individuals10. Personal accidents, the leading cause of death in this age group, has an equivalent death toll. The sample obtained was analyzed by the Epi Info program2. Those interviewed as well as those who declined to participate were guaranteed their identities would not be disclosed. 80 students were participating in health related rural area programs and did not participate in this study MATERIAL AND METHODS Students were studied at the Medical College/UFMG (between fifth and twelve semesters). Multiple-choice questionnaires were used, distributed individually by the researchers just before theoretical classes for each level. The form was completed spontaneously and handed back at the end of the class period. All the research was done from October to December, 1999. To prevent anyone from participating twice, one of the opening questions asked whether the student had already answered the questionnaire, in which case it was excluded. Table 1 - The calculated sample of students for each semester of the medical course at the Medical College of the Federal University of Minas Gerais showing the estimated of the accident rate with potentially contaminated material in hospital setting during 1999. Table 1 - The calculated sample of students for each semester of the medical course at the Medical College of the Federal University of Minas Gerais showing the estimated of the accident rate with potentially contaminated material in hospital setting during 1999. Table 1 - The calculated sample of students for each semester of the medical course at the Medical College of the Federal University of Minas Gerais showing the estimated of the accident rate with potentially contaminated material in hospital setting during 1999. Semester of the Total number Estimated Necessary Total of those medical course of students accident rate (%) sample interviewed Fifth 172 3 16 58 Sixth 172 3 16 66 Seventh 156 15 51 87 Eighth 164 25 67 107 Ninth 166 45 78 113 Tenth 162 50 78 113 Eleventh 82 60 53 61 Twelfth 169 65 78 86 Total 1,243 - 437 691 *80 students were participating in health related rural area programs and did not participate in this study The questionnaire was made up of two parts: the first consisted of a question about whether the student had already suffered an accident with biologically contaminated material; the second part was only answered by students who had in fact been involved in such an accident. In the second part, students were asked to answer a specific questionnaire about each accident they recalled, 406 Revista da Sociedade Brasileira de Medicina Tropical 37(5):405-408, set-out, 2004 Table 2 - Accident rate by semester of the medical course with potentially blood-contaminated material in hospital setting at the Medical College of the Federal University of Minas Gerais during 1999. MATERIAL AND METHODS Hospital Setting Number of accidents Percentage Small wounds sector 147 42.1 Maternity 78 22.3 Surgery unit 46 13.2 Out-patient surgery unit 36 10.4 Intensive care unit 13 3.7 Patient ward 12 3.4 First aid unit 6 1.7 Others 11 3.2 Total 349 100.0 Factors contributing to accidents Number of accidents Percentage Carelessness 156 45.3 Lack of experience 108 30.9 Carelessness of others 101 28.9 Lack of personal protection equipment 49 14.4 Long work hours 43 12.3 Poor instructions 31 8.9 Inadequate facilities 19 5.4 Other factors 17 4.9 discontinued the treatment after receiving the blood test results of the patient involved; 6.4% (2/31) discontinued the treatment due to its side effects; 16% (5/31) complied with the treatment completely. discontinued the treatment after receiving the blood test results of the patient involved; 6.4% (2/31) discontinued the treatment due to its side effects; 16% (5/31) complied with the treatment completely. secretion, 1.1% (4/349); bloodless pus, 1.1% (4/349); others, 6.9% (24/349). The most affected body parts were: hands, 67% (234/349); eyes, 18.9% (66/349); mouth, 1.7% (6/349); eyes and mouth, 2% (7/349); others 6.3% (22/349). Four percent (14/349) of the accidents occurred while the student was not using personal protection equipment. The frequency with which gloves, gowns, masks, and goggles were used is shown in Table 4. MATERIAL AND METHODS Table 5 - Procedures which involved the most frequent accidental exposures with potentially blood-contaminated material in hospital setting at the Medical College of the Federal University of Minas Gerais during 1999. Procedures Number of accidents Procedure rate (%) Suturing 119 33.8 Applying local anesthesia 58 16.6 Assisting surgery 31 8.9 Recapping needle 30 8.6 Assisting delivery 22 6.3 Handling needle case 12 3.4 Handling knife 6 1.7 Others 69 19.8 Did not respond 2 0.6 Total 349 19.7 Number of accidents/ Semester of the students (NA) Number of students Accident medical course absolute % interviewed (NI) Rate (NA/NI X 100) Fifth 2 0.8 58 3.4 Sixth 6 2.5 66 9.1 Seventh 12 5.1 87 13.8 Eighth 27 11.4 107 25.2 Ninth 49 20.9 113 43.4 Tenth 60 25.4 113 53.1 Eleventh 35 14.8 61 57.4 Twelfth 45 19.1 86 52.3 Table 3 - The hospital settings which presented the highest number of accidents with potentially blood contaminated material at the Medical Table 3 - The hospital settings which presented the highest number of accidents with potentially blood-contaminated material at the Medical College of the Federal University of Minas Gerais during 1999. Table 6 - Factors contributing to 349 accidents with potentially blood- contaminated material in hospital setting at the Medical College of the Federal University of Minas Gerais during 1999 as reported by the students. Table 6 - Factors contributing to 349 accidents with potentially blood- contaminated material in hospital setting at the Medical College of the Federal University of Minas Gerais during 1999 as reported by the students. Table 6 - Factors contributing to 349 accidents with potentially blood- contaminated material in hospital setting at the Medical College of the Federal University of Minas Gerais during 1999 as reported by the students. DISCUSSION The accident rate for medical students with potentially contaminated material is high, although it is lower than that of medical interns10. About one third of the population surveyed were involved in accidents. During the final semesters of the course, more than half of the students suffered some kind of accident involving blood. The data collected here are similar to those observed at the University of California, USA, where 65% of the medical students had been involved in some kind of accident by the end of the course11. Blood transmitted viruses, such as HIV, hepatitis B, and hepatitis C, are not uncommon in such cases. This demonstrates the great ethical responsibility of medical teaching institutions. Table 4: Use of personal protection equipment during 349 accidents with potentially blood-contaminated material in hospital setting at the Medical College of the Federal University of Minas Gerais during 1999. Equipment Frequency of the use of personal protection Percentage Gloves 313 89.7 Gowns 226 64.8 Masks 172 49.3 Goggles 115 33.0 None 14 4.0 Suturing accounted for 34% (119/349) of the accidents and was the most frequent form of exposure to accidents. The rate at which other procedures resulted in accidents is given in Table 5. The impact of training, awareness programs, and supervision to reduce accident risk has been analyzed by some studies11 14. In spite of determined effort by the institution to train students to avoid accidents during teacher-student activities, the University of California continued to have a high accident rate among students3 11, due to factors that are inherent to medical learning. Natural student anxiety as they treat patients is one of the factors that goes beyond the control of precautionary training. Extensive training on mannequins, emotional support, and extra teacher attention during teacher- Table 6 shows that the greatest factor contributing to accidents was carelessness, with 44.7% (156/349). Forty-nine percent (173/349) of the accidents were reported to the health center. Of these, 57% considered they did not receive adequate assistance. Nine percent (31/349) of those involved in accidents used a antiretroviral drugs. Seventy seven percent (24/31) 407 Reis JMB et al student assistance activities could play a decisive role in reducing the risk of accidental HIV exposure. Revista da Escola de Enfermagem da Universidade de São Paulo 32:262- 272, 1998. Revista da Escola de Enfermagem da Universidade de São Paulo 32:262- 272, 1998. 6. Hermann RC. DISCUSSION Risks of HIV exposure to medical students and health care personnel. Journal of American Medical Association 264: 1187-1192, 1990 Despite these questions, the medical curriculum should prepare students without exposing them to unacceptable risks. The medical teaching institution should guarantee adequate instruction to the pupils as well as working toward this objective in other ways. Forbidding students to have contact with HIV positive patients is considered unethical and a poor teaching method3. Post-accident prophylaxis, however has been proven to reduce the risk of HIV contamination1 4, and should, in such circumstances, be considered a priority and made easily available, including technical and emotional support, as well as medical follow up. 7. Keita-Perse O, Pradier C, Rosenthal E, Altare J, Cassuto JP, Dellamonica P. Hospital medical students: a population at risk for accidental exposure to blood. Presse Medicale 27: 1723-1726, 1998. 8. Machado AA, Costa JC, Gir E, Moriya TM, Figueiredo FC. Risco de infecçäo pelo vírus da imunodeficiência humana (HIV) em profissionais de saúde. Revista de Saúde Pública 26: 54-56, 1992. 9. Marcus R. Surveillance of health care workers exposed to blood from patients infected with the human immunodeficiency virus. New England Journal of Medicine 319: 1118-1123, 1998. 10. O’Neill TM, Abbott AV, Radecki SE. Risk of needlesticks and occupational exposures among residents and medical students. Archives of Internal Medicine 152:1451-1456, 1992. REFERENCES 11. Osborn HE, Papadakis MA, Gerberding JL. Occupational exposures to body fluids among medical students. A 7-year longitudinal study. Annals of Internal Medicine 130:45-51, 1999. 1. Cardo DM, Culver DH, Ciesielski JL. Srivastava PU, Marcus R. A case-control study of HIV seroconversion in health care workers after percutaneous exposure. New England Journal of Medicine 337:1485-1490, 1997. 12. Resnic SF, Noerdlinger MA. Occupational Exposure Among Medical Students and House Staff at a New York City Medical Center. Archives of Internal Medicine 155:75-80, 1995. 2. Dean AG, Dean JA, Coulombier D. Centers for Disease Control and Prevention (CDC). Epi Info version 6,04, 1994. 2. Dean AG, Dean JA, Coulombier D. Centers for Disease Control and Prevention (CDC). Epi Info version 6,04, 1994. 13. Soares ES, Salles JA, Prado RS, Pedroso ERP. Avaliação dos comportamentos de risco de estudantes de medicina em relação à síndrome de imunodeficiência adquirida. In: Resumos do XXXV Congresso da Sociedade Brasileira de Medicina Tropical, 1999. Revista da Sociedade Brasileira de Medicina Tropical 32 (supl I): 350, 1999. 3. Federman DD. Protecting the future of Medicine – from themselves. Annals of Internal Medicine 130:66-67, 1999. 3. Federman DD. Protecting the future of Medicine – from themselves. Annals of Internal Medicine 130:66-67, 1999. 4. Gerberding JL. Management of occupational exposure to blood-borne viruses. New England Journal of Medicine 332:444-451, 1995. 4. Gerberding JL. Management of occupational exposure to blood-borne viruses. New England Journal of Medicine 332:444-451, 1995. 14. Wong ES, Stotka JL. Are Universal Precautions Effective in Reducing the Number of Occupational Exposures Among Health Care Workers? Journal of American Medical Association 265:1123-1128, 1991. 5. Gir E, Costa FPP, Silva AM. A enfermagem frente a acidentes de trabalho com material potencialmente contaminado na era do HIV. 5. Gir E, Costa FPP, Silva AM. A enfermagem frente a acidentes de trabalho com material potencialmente contaminado na era do HIV. 408
https://openalex.org/W2999569007	https://wwwnc.cdc.gov/eid/article/26/2/pdfs/19-0721.pdf	English	null	Neutralizing Antibodies against Enteroviruses in Patients with Hand, Foot and Mouth Disease	Emerging infectious diseases	2,020	cc-by	7,110	Author affiliations: Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam (L.A. Nguyet, T.T. Thanh, N.T.T. Hong, L.N.T. Nhu, H.M.T. Van, N.T.H. Ny, N.T. Anh, D.D.K. Han, L. Thwaites, H. Clapham, G. Thwaites, D.Q. Ha, H.R. van Doorn, L.V. Tan); Children’s Hospital 1, Ho Chi Minh City (L.N.T. Nhan, T.H. Khanh, N.T. Hung); Children’s Hospital 2, Ho Chi Minh City (H.M. Tuan, H.L. Viet, D.C. Viet); University of Medicine and Pharmacy, Ho Chi Minh City (H.M. Tuan, V.Q. Huy); Pasteur Institute, Ho Chi Minh City (H.Q. Cuong, N.T.T. Thao); University of Oxford, Oxford, UK (L. Thwaites, H. Clapham, G. Thwaites, H.R. van Doorn); Hospital for Tropical Diseases, Ho Chi Minh City (N.V.V. Chau) Neutralizing Antibodies against Enteroviruses in Patients with Hand, Foot and Mouth Disease Lam Anh Nguyet, Tran Tan Thanh, Le Nguyen Thanh Nhan, Nguyen Thi Thu Hong, Le Nguyen Truc Nhu, Hoang Minh Tu Van, Nguyen Thi Han Ny, Nguyen To Anh, Do Duong Kim Han, Ha Manh Tuan, Vu Quang Huy, Ho Lu Viet, Hoang Quoc Cuong, Nguyen Thi Thanh Thao, Do Chau Viet, Truong Huu Khanh, Louise Thwaites, Hannah Clapham, Nguyen Thanh Hung, Nguyen Van Vinh Chau,Guy Thwaites, Do Quang Ha, H. Rogier van Doorn, Le Van Tan (EV-A71), have increasingly been reported world wide, including in the United States and Europe (3– 5). Although HFMD is a mild infection in most cases, severe clinical complications (e.g., central nervous system involvement as brainstem encephalitis) may happen and can be fatal (1,6). However, no antiviral drugs are available to the affected patients, including those with severe clinical phenotypes. (EV-A71), have increasingly been reported world wide, including in the United States and Europe (3– 5). Although HFMD is a mild infection in most cases, severe clinical complications (e.g., central nervous system involvement as brainstem encephalitis) may happen and can be fatal (1,6). However, no antiviral drugs are available to the affected patients, including those with severe clinical phenotypes. Hand, foot and mouth disease (HFMD) is an emerging infection with pandemic potential. Knowledge of neutral izing antibody responses among its pathogens is essen tial to inform vaccine development and epidemiologic research. We used 120 paired-plasma samples collected at enrollment and >7 days after the onset of illness from HFMD patients infected with enterovirus A71 (EV-A71), coxsackievirus A (CVA) 6, CVA10, and CVA16 to study cross-neutralization. For homotypic viruses, seroposi tivity increased from <60% at enrollment to 97%–100% at follow-up, corresponding to seroconversion rates of 57%–93%. Seroconversion for heterotypic viruses was recorded in only 3%–23% of patients. All plasma samples from patients infected with EV-A71 subgenogroup B5 could neutralize the emerging EV-A71 subgenogroup C4. Collectively, our results support previous reports about the potential benefit of EV-A71 vaccine but highlight the necessity of multivalent vaccines to control HFMD. Hand, foot and mouth disease (HFMD) is an emerging infection with pandemic potential. Knowledge of neutral izing antibody responses among its pathogens is essen tial to inform vaccine development and epidemiologic research. We used 120 paired-plasma samples collected at enrollment and >7 days after the onset of illness from HFMD patients infected with enterovirus A71 (EV-A71), coxsackievirus A (CVA) 6, CVA10, and CVA16 to study cross-neutralization. DOI: https://doi.org/10.3201/eid2602.190721 RESEARCH RESEARCH Neutralizing Antibodies against Enteroviruses in Patients with Hand, Foot and Mouth Disease For homotypic viruses, seroposi tivity increased from <60% at enrollment to 97%–100% at follow-up, corresponding to seroconversion rates of 57%–93%. Seroconversion for heterotypic viruses was recorded in only 3%–23% of patients. All plasma samples from patients infected with EV-A71 subgenogroup B5 could neutralize the emerging EV-A71 subgenogroup C4. Collectively, our results support previous reports about the potential benefit of EV-A71 vaccine but highlight the necessity of multivalent vaccines to control HFMD. y HFMD is caused by various serotypes of entero virus A of the family Picornaviridae. Of these, EV- A71, coxsackievirus A (CVA) 6, CVA10, and CVA16 are the most common pathogens isolated from pa tients with clinically suspected HFMD, with CVA6 being increasingly reported (7–9). In Vietnam, our re cent report showed that of 1,547 patients with HFMD enrolled in a clinical study, EV-A71 was detected in 24.4%, followed by CVA6 (21.8%), CVA16 (10.8%), and CVA10 (7.9%). Other enteroviruses detected spo radically included CVA4 (1.7%), CVA12 (1.4%), and CVA2 (0.6%) (10). Infection with EV-A71 has received more attention because it frequently causes severe HFMD, especially in recent outbreaks recorded in the Asia–Pacific region since 1997 (6,11). Consequently, inactivated monovalent vaccines for EV-A71 have been successfully developed and licensed in China (12–14). The use of those vaccines, however, has been voluntary and restricted within mainland China. S ince 1997, hand, foot and mouth disease (HFMD) has emerged as a serious childhood infection in the Asia–Pacific region (1,2) with the potential of spread ing to other parts of the world. Indeed, HFMD epi demics, especially those caused by enterovirus A71 S in h y Because the viruses causing HFMD are diverse, ongoing efforts exist to develop multivalent vac cines, especially those including antigens of the aforementioned common serotypes (15). Results from these preclinical studies using animal models showed a lack of cross-reactivity among EV-A71, CVA6, CVA10, and CVA16 (16,17). There is, how ever, scarce information about to what extent hu man infection with 1 HFMD-causing enterovirus serotype can elicit (cross-)neutralizing antibodies DOI: https://doi.org/10.3201/eid2602.190721 298 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 Neutralizing Antibodies in Patients with HFMD against homotypic and heterotypic enterovirus se rotypes. Patient Enrollment and Data Collection We screened all patients <12 years of age who came to outpatient departments or were admitted to inpatient wards of CH1, CH2, or HTD with a clinical diagnosis of HFMD and, if outpatients, an illness of <3 days for enrollment in our study. We excluded any patient for whom the attending physician believed another diag nosis was more likely. Neutralizing Antibodies against Enteroviruses in Patients with Hand, Foot and Mouth Disease Such data are of paramount importance to support the development of intervention strate gies (including vaccines) and the design of epide miologic research on surveillance and transmission dynamics of HFMD and will contribute to the ex panded knowledge about host–pathogen and patho gen–pathogen interaction of this emerging clinical problem. We aim to fill the existing gaps in knowl edge about seropositivity and (cross-)neutralization elicited as a consequence of human infection by EV- A71, CVA6, CVA10, and CVA16, the 4 most com mon serotypes responsible for the ongoing epidemic of HFMD worldwide, especially in Asia. (myoclonus observed by medical staff or history of myoclonus and lethargy or pulse >130 bpm), and grade 2B2 (ataxia, cranial nerve palsies, limb weak ness, nystagmus, persistent high fever, or pulse >150 bpm). Grade 3 involves autonomic dysfunction with sweating, hypertension, tachycardia, and tachypnea. Grade 4 is for disease with additional cardiopulmo nary compromise with pulmonary edema or shock syndrome (18). Patients with grade 2B1 or above are considered to have severe HFMD, and often require intravenous immunoglobulin administration. Settings The clinical and patient data used in this study were derived from an ongoing clinical study of HFMD that has been conducted at Children’s Hospital (CH) 1, CH2, and the Hospital for Tropical Diseases (HTD) in Ho Chi Minh City, Vietnam, since 2013 (6,8). These hospitals are tertiary referral centers for children with HFMD in Ho Chi Minh City and southern Vietnam, covering a catchment population of >40 million. Determination of Enterovirus Serotype and EV-A71 Subgenogroup We determined enterovirus serotype using a combi nation of PCR and sequencing approaches (19–21). In brief, we first extracted viral RNA from throat/rec tal swab specimens. We then used a 1-step multiplex real-time reverse transcription PCR assay to simulta neously detect all enterovirus serotypes and EV-A71 (19). We then tested all specimens positive for entero virus serotype or EV-A71 to further identify specific enterovirus serotypes or EV-A71 subgenogroups, using a combination of viral protein (VP) 1 PCR and sequencing of the obtained PCR amplicon (18,20,21). Finally, we analyzed the obtained VP1 sequences us ing a previously described online tool to determine enterovirus serotype or EV-A71 subgenogroup (22). Selection of Patients and Plasma Sample for Microneutralization Assay From this study, we selected a convenience sample of 120 patients (30 per serotype CVA6, CVA10, CVA16, and EV-A71) who had plasma samples collected at enrollment and follow-up and were available for im munological response analysis. In addition, for as sessment of antigenic difference between subgeno group B5 (circulating in Vietnam during 2013–2015) and C4 (circulating in Vietnam in 2018), we included 1 available follow-up plasma sample collected from a fatal case, in which the patient was infected with sub genogroup C4 during the 2018 outbreak (6). y We collected information regarding demograph ics, clinical signs/symptoms, clinical grades, treat ments, laboratory tests, length of hospital stay, and outcomes. In addition, for enterovirus serotype de termination, we sampled acute throat and rectal swabs at enrollment and collected a plasma sample from each participant at enrollment and 7–14 days after enrollment. Microneutralization Assay July 2013–March 2017. Male patients were predomi nant (female/male ratio 45/75). Thirteen (10.8%) pa tients had severe HFMD (grade 2B1 or above); 119 pa tients (99.2%) made a complete recovery. Following the onset of illness, 50% of the patients were admitted to hospital within 1 day (range 0–5 days) and were enrolled in the clinical study within 2 days (range 0–6 days). All second/follow-up plasma samples in cluded for analysis were collected at day >7 (median 9 days, (range 7–17) after the illness onset. We performed the microneutralization as previously described (23). In brief, we first inactivated plasma samples at 56°C for 30 min, and we then diluted the samples in serial ratios from 1:8 to 1:512 in mainte nance medium (Sigma-Aldrich, https://www.sig maaldrich.com). Accordingly, the lower limit of de tection of the assay was 1:8 and the upper limit was 1:512. Next, we incubated plasma dilutions with an equal volume of 100 times the median culture of in fectious dose (TCID50) of the virus at 37°C for 1 hour, and then transferred them into a 96-well plate pre- coated with human rhabdomyosarcoma (RD) cells (American Type Culture Collection, https://www. atcc.org). The plate was then incubated in a 5% car bon dioxide incubator at 37°C. Cells were observed daily for cytopathic effects. The antibody titer of the sample was determined by the highest plasma dilu tion that prevented cytopathic effects in 50% of the wells. We tested each dilution in quadruplicate and included negative and positive controls in each ex periment. We defined seropositivity as neutralizing antibody titer >1:8. We defined seroconversion as a change from seronegativity to seropositivity, or at least a 4-fold rise in the neutralizing antibody titer be tween enrollment and follow-up time points. y g Of the 30 patients infected with EV-A71, detailed information about subgenogroup was successfully generated for 13 patients, who were all positive for subgenogroup B5. Of the patients with EV-A71 infec tions, 20% had severe clinical phenotypes (grade 2B1 or above), whereas severe outcome was recorded in 3.3% of patients infected with CVA6, 6.7% of patients infected with CVA10, and 13.3% of patients infected with CVA16 (Table 1). Mouth lesion distributions and C-reactive protein levels were statistically different among serotypes. RESEARCH RESEARCH Seropositivity and Seroconversion We compiled the results of seropositivity testing at enrollment (baseline) and follow-up (Table 2). Al though 60% (18/30) of patients infected with EV-A71 had specific antibodies to EV-A71 at the measured level (>1:16) or above in their blood samples, most of the patients (>70%) infected with CVA6, CVA10, or CVA16 had no specific antibodies to the infecting vi ruses at enrollment. The proportion of patients with antibodies against heterotypic viruses ranged from 7% (2/30) of CVA6 patients having neutralizing anti bodies against EV-A71 to 57% (17/30) of EV-A71 pa tients having antibodies against CVA10. We performed statistical analyses of clinical data us ing IBM SPSS Statistics 23 (IBM Corp., https://www. ibm.com). We compared categorical variables using a χ2 test or Fisher exact test and compared continu ous variables using the Mann-Whitney U-test, 1-way analysis of variance (ANOVA) test, or Kruskal-Wallis test. We tested the difference in neutralizing antibody titers between samples obtained at enrollment and follow-up using the Wilcoxon matched-pairs signed rank test, available in Prism 5.04 (GraphPad Software, https://www.graphpad.com). The institutional review boards of CH1, CH2, and HTD, as well as the Oxford Tropical Research Eth ics Committee (OxTREC), approved the study. We obtained written informed consent from a parent or guardian of each enrolled patient. g g At follow-up (>7 days after the onset of ill ness), seropositivity for homotypic viruses reached 97%–100% in all patient groups; comparisons for se ropositivity rates at enrollment and follow-up were all significant (p<0.001) (Table 2), corresponding to seroconversion rates of 57% (17/30) for EV-A71, 77% (23/30) for CVA16, 83% (25/30) for CVA10, and 93% (28/30) for CVA6 (Table 2). We found no difference in antibody responses (seropositivity and serocon version) between the groups of patients who were positive for EV-A71 subgenogroup B5 and those from whom EV-A71 subgenogroup was not available (data not shown). Microneutralization Assay Otherwise, there were considerable similarities between groups of patients who were infected with EV-A71, CVA6, CVA10, or CVA16 in terms of clinical characteristics and outcome, as well as blood biochemistry parameters (Table 1), in agree ment with a previous report (8). Results Baseline Characteristics of Patients HFMD Clinical Grade Classification We isolated representative samples of CVA10, CVA16, and EV-A71 (including EV-A71 subgenogroup B5 in 2013 and C4 in 2018) used for microneutralization as say from patients with HFMD who were enrolled in the clinical study (7,8,10). We obtained a CVA6 isolate from the virus archive of Pasteur Institute in Ho Chi Minh City. For EV-A71, unless specified, all neutral ization experiments were carried out using EV-A71 subgenogroup B5. According to the Vietnamese Ministry of Health, HFMD is clinically divided into 4 major grades. Grade 1 is assigned to patients with mouth ulcers or vesicles/papules on hands, feet, or buttocks, with or without mild fever (<39°C). Grade 2 is fur ther divided into grade 2A (central nervous system [CNS] involvement, myoclonus reported by parents or caregivers only, fever >39°C or ataxia), grade 2B1 299 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 202 Neutralizing Antibodies in Patients with HFMD Neutralizing Antibodies in Patients with HFMD Table 1. Baseline characteristics and clinical outcome of the patients included in study of patients with hand, foot and mouth disease, Vietnam* Characteristics All, N = 120 EV-A71, N = 30 CVA6, N = 30 CVA10, N = 30 CVA16, N = 30 p value† Demographics Sex ratio, F/M 45/75 8/22 8/22 14/16 15/15 0.12 Median age, mo (range) 16.2 (1.8–59) 16.7 (4.9–58.6) 16.4 (5.3–59) 14.7 (1.8–41.4) 19 (5.8–46.5) 0.11 Median day of illness from onset (range) To hospital admission 1 (0–5) 2 (0–4) 1 (0–3) 1 (0–3) 1 (0–5) 0.058 To enrollment in study 2 (0–6) 2 (0–4) 1.5 (0–3) 2 (0–6) 2 (0–6) 0.802 To collection of second plasma 9 (7–17) 9 (7–12) 9 (7–14) 9 (7–14) 9 (8–17) 0.211 Median day of hospitalization‡ 3 (1–12) 4 (2–10) 3 (1–8) 3 (1–6) 4 (2–12) 0.3 Inpatient/outpatient ratio 77/43 17/13 18/12 25/5 17/13 0.08 Clinical characteristics, no. Neutralizing Antibodies in Patients with HFMD ‡Inpatients only s and clinical outcome of the patients included in study of patients with hand, foot and mouth disease, for CVA16 in 10%. Five patients became seronega tive for heterotypic viruses at follow-up (Figures 1, 2; Appendix Figures 1, 2, https://wwwnc.cdc.gov/ EID/article/26/2/19-0721-App1.pdf). The difference in the proportion of patients who were seropositive for heterotypic viruses was not sta tistically significant between the enrollment and fol low-up time points (Table 2). At follow-up, serocon version for heterotypic viruses was recorded in only 3% (1/30) to 23% (7/30) of the patients, with compa rable rates across serotypes (Table 2). For example, of the 30 patients infected with CVA16, seroconversions for CVA6 were recorded in 10%, seroconversions for CVA10 in 7%, and seroconversions for EV-A71 in 7%. Similarly, of the 30 patients infected with EV-A71, seroconversions for CVA6 were recorded in 13%, se roconversions for CVA10 in 3%, and seroconversions Baseline Characteristics of Patients l d h b l h We compiled the baseline characteristics and clinical outcome of 120 patients included for analysis of neu tralizing antibody responses to all 4 enterovirus sero types (EV-A71, CVA6, CVA10, and CVA16) (Table 1). All patients were enrolled in the clinical study during 300 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 Neutralizing Antibodies in Patients with HFMD (%) Fever 87 (72.5) 24 (80) 17 (56.7) 24 (80) 17 (56.7) 0.16 Cough 26 (21.7) 6 (20) 2 (6.7) 8 (26.7) 10 (33.3) 0.06 Runny nose 21 (17.5) 6 (20) 3 (10) 5 (16.7) 7 (23.3) 0.63 Vomiting 25 (20.8) 7 (23.3) 5 (16.7) 5 (16.7) 8 (26.7) 0.77 Diarrhea 14 (11.7) 3 (10) 3 (10) 3 (10) 5 (16.7) 0.89 Drowsiness 6 (5) 2 (6.7) 0 2 (6.7) 2 (6.7) 0.6 Irritability 14 (11.7) 5 (16.7) 2 (6.7) 5 (16.7) 2 (6.7) 0.46 Myoclonus 30 (25) 10 (33.3) 3 (10) 9 (30) 8 (27) 0.13 Sweating 4 (3.3) 1 (3.3) 1 (3.3) 1 (3.3) 1 (3.3) 1 Lethargy 2 (1.7) 0 0 2 (6.7) 0 0.24 Conjunctivitis 1 (0.8) 0 1 (3.3) 0 0 1 Rash 106 (88.3) 30 (100) 30 (100) 17 (56.7) 19 (63.3) 0 Mouth lesion 111 (92.5) 28 (93.3) 23 (76.7) 30 (100) 30 (100) 0.001 Limb weakness 4 (3.3) 1 (3.3) 1 (3.3) 1 (3.3) 1 (3.3) 0.80 Median pulse, bpm (range) 120 (96–180) 120 (96–180) 120 (100–167) 125 (100–165) 120 (100–170) 0.632 Median blood pressure, mm Hg (range) Systolic 90 (75–128) 90 (75–100) 90 (80–120) 90 (80–120) 90 (80–128) 0.661 Diastolic 60 (40–80) 55 (50–62) 55 (40–80) 60 (50–80) 60 (50–77) 0.551 Blood biochemistry results, median (range) Leukocyte count,  109 cells/L 13.1 (1–50.6) 13.6 (7.8–50.6) 12.5 (1–25.9) 14.3 (5–24.7) 12.4 (4.5–24.4) 0.638 Neutrophils, % 51 (2.1–92.7) 51.2 (19.7–72.8) 52.7 (7.7–92.7) 47.6 (2.1–76.3) 50.9 (24.2–86.1) 0.658 Lymphocytes, % 37.1 (3.7–80.2) 37.1 (18.4–65.5) 33.9 (3.7–80.2) 37.6 (17.6–71.2) 36.5 (6.1–60.3) 0.846 Platelet count,  109/L 322.5 (96–597) 360 (189–522) 341.5 (150–513) 297 (96–452) 293.5 (175–597) 0.086 Glucose, mg/dL 107 (0–212) 112 (62–170) 108.5 (0–154) 108.5 (68–212) 98.5 (51–164) 0.324 C-reactive protein, mg/dL 12.4 (0–102) 4.1 (0–100) 13.7 (0–102) 23.3 (3–58) 9.9 (0–39) <0.001 Clinical grade, no. (%) Mild 107 (89.2) 24 (80) 29 (96.7) 28 (93.3) 26 (86.7) 0.2 Severe 13 (10.8) 6 (20) 1 (3.3) 2 (6.7) 4 (13.3) IVIg administration, no. (%) 8 (6.7%) 3 (10) 1 (3.3) 1 (3.3) 3 (10) 0.5 Outcome, no. (%) Full recovery 119 (99.2) 30 (100) 29 (96.7) 30 (100) 30 (100) 0.17 Incomplete recovery 1 (0.8) 0 1 (3.3) 0 0 CV, coxsackievirus; EV, enterovirus; IVIg, intravenous immunoglobulin. †Results of statistical analyses comparing individual groups of patients infected with EV-A71, CVA6, CVA10, or CVA16. Seropositivity versus Illness Day at Enrollment and Patient Agei We found a significant difference in illness days at enrollment between the groups of patients who were seropositive by neutralization testing for any homo typic virus and those who were negative, median day of illness 2 (range 1–6 days) versus 1 (0–6 days) (p<0.001) (Figure 3). Despite the small sample size, 301 g Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 RESEARCH nd seroconversion in plasma from hand, foot and mouth disease patients infected with EV-A71, CVA6, ses Vietnam Table 2. Seropositivity and seroconversion in plasma from hand, foot and mouth disease patients infected with EV-A71, CVA6, CVA10, and CVA16 viruses, Vietnam* Virus Immunity status Virus, no. (%) samples CVA6 CVA10 CVA16 EV-A71 CVA6 Seropositivity At enrollment 2 (7) 10 (33) 5 (17) 2 (7) At follow-up 30 (100) 9 (30) 9 (30) 3 (10) p value <0.001 1.0 0.36 1.0 Seroconversion 28 (93) 1 (3) 7 (23) 2 (7) CVA10 Seropositivity At enrollment 4 (13) 9 (30) 3 (10) 3 (10) At follow-up 6 (20) 29 (97) 4 (13) 5 (17) p value 0.73 <0.001 1.0 0.71 Seroconversion 4 (13) 25 (83) 3 (10) 2 (7) CVA16 Seropositivity At enrollment 7 (23) 10 (33) 8 (27) 5 (17) At follow-up 9 (30) 11 (37) 30 (100) 7 (23) p value 0.77 1.0 <0.001 0.75 Seroconversion 3 (10) 2 (7) 23 (77) 2 (7) EV-A71 Seropositivity At enrollment 5 (17) 17 (57) 6 (20) 18 (60) At follow-up 7 (23) 14 (47) 8 (27) 30 (100) p value 0.75 0.61 0.76 <0.001 Seroconversion 4 (13) 1 (3) 3 (10) 17 (57) n = 30 for each virus. p values reflect the results of statistical analysis comparing the seropositive rates between the 2 time points (enrollment and follow- up) of the corresponding enterovirus serotypes. CV, coxsackievirus; EV, enterovirus. subgroup analysis demonstrated a similar associa tion between illness day and seropositivity at enroll ment among patients infected with EV-A71 and those infected with CVA6 (Appendix Figure 3). We found no difference in age (months) between the groups of patients with and without neutralizing antibodies at enrollment (data not shown). To further shed light on the kinetics of neutralizing an tibody titers over the course of illness, we plotted and compared the neutralizing antibody titers against ho motypic and heterotypic enterovirus serotypes at en rollment and follow-up time points (Table 3; Figures 1, 2; Appendix Figures 1, 2). Figure 1. Kinetics of neutralizing antibody titers in plasma samples collected at enrollment and follow- up from patients infected with EV-A71 in study of patients with hand, foot and mouth disease, Vietnam. A) CVA6 (p = 0.073); B) CVA10 (p = 0.347); C) CVA16 (p = 0.250); D) EV-A71 (p<0.001). CV, coxsackievirus; EV, enterovirus. Neutralizing Antibodies in Patients with HFMD titers to homotypic enteroviruses at follow up were subgenogroup C4 which emerged in 2018 and caused Figure 2. Kinetics of neutralizing antibody titers in plasma samples collected at enrollment and follow-up from patients infected with CVA6 in study of patients with hand, foot and mouth disease, Vietnam. A) CVA6 (p<0.001); B) CVA10 (p = 0.915); C) CVA16 (p = 0.021); D) EV-A71 (p = 0.5). CV, coxsackievirus; EV, enterovirus. Figure 2. Kinetics of neutralizing antibody titers in plasma samples collected at enrollment and follow-up from patients infected with CVA6 in study of patients with hand, foot and mouth disease, Vietnam. A) CVA6 (p<0.001); B) CVA10 (p = 0.915); C) CVA16 (p = 0.021); D) EV-A71 (p = 0.5). CV, coxsackievirus; EV, enterovirus. Figure 2. Kinetics of neutralizing antibody titers in plasma samples collected at enrollment and follow-up from patients infected with CVA6 in study of patients with hand, foot and mouth disease, Vietnam. A) CVA6 (p<0.001); B) CVA10 (p = 0.915); C) CVA16 (p = 0.021); D) EV-A71 (p = 0.5). CV, coxsackievirus; EV, enterovirus. subgenogroup C4, which emerged in 2018 and caused a large outbreak of >130,000 hospitalizations and 17 deaths in Vietnam (6), we performed a complemen tary analysis using 6 follow-up plasma samples from the aforementioned group of patients infected with subgenogroup B5. Subsequently, all the included plasma samples could neutralize 2018 subgenogroup C4, and there was no difference in neutralizing an tibody titers against the EV-A71 subgenogroups C4 and B5 (Figure 4). Likewise, the only available follow- up plasma sample collected from a patient infected with 2018 subgenogroup C4 had a neutralizing anti body titer of 1:512 against both subgenogroups. titers to homotypic enteroviruses at follow-up were significantly higher than those measured at enrollment (p<0.001; Table 3). All patients at follow-up had anti body titers against homotypic viruses ranging from 32 to 512, well above the protective level as defined from vaccine trials (12–14) (Appendix Figures 1, 2). Neutralizing antibody titers against heteroge neous serotypes measured at the 2 time points were not statistically different, with most patients (15/50– 27/30 [50%–90%)]) having antibody titers below the assay cutoff (i.e., below the protective level). There was, however, a significant difference in antibody ti ter against CVA16 between the 2 time points among patients infected with CVA6 (p = 0.021; Table 3). Discussion Despite the public health threat of HFMD, scarce in formation exists for pathogen–pathogen and host– pathogen interactions, from the immunity perspec tive, to inform the development and implementation of intervention strategies, especially vaccines, and the design of epidemiologic research on disease surveil lance and transmission dynamics. Here we report on the seropositivity, seroconversion, and neutraliza tion in HFMD patients infected with EV-A71, CVA6, Neutralizing Antibodies in Patients with HFMD Sub analysis did not show that samples positive for CVA6 were more likely to be positive for CVA16 or any other serotypes than samples that were negative for CVA6 (Appendix Table 1). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 Seropositivity versus Illness Day at Enrollment and Patient Agei Overall, the antibody 302 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 ( ) pp g ) y Figure 1. Kinetics of neutralizing antibody titers in plasma samples collected at enrollment and follow- up from patients infected with EV-A71 in study of patients with hand, foot and mouth disease, Vietnam. A) CVA6 (p = 0.073); B) CVA10 (p = 0.347); C) CVA16 (p = 0.250); D) EV-A71 (p<0.001). CV, coxsackievirus; EV, enterovirus. Figure 1. Kinetics of neutralizing antibody titers in plasma samples collected at enrollment and follow- up from patients infected with EV-A71 in study of patients with hand, foot and mouth disease, Vietnam. A) CVA6 (p = 0.073); B) CVA10 (p = 0.347); C) CVA16 (p = 0.250); D) EV-A71 (p<0.001). CV, coxsackievirus; EV, enterovirus. Figure 1. Kinetics of neutralizing antibody titers in plasma samples collected at enrollment and follow- up from patients infected with EV-A71 in study of patients with hand, foot and mouth disease, Vietnam. A) CVA6 (p = 0.073); B) CVA10 (p = 0.347); C) CVA16 (p = 0.250); D) EV-A71 (p<0.001). CV, coxsackievirus; EV, enterovirus. Figure 1. Kinetics of neutralizing antibody titers in plasma samples collected at enrollment and follow- up from patients infected with EV-A71 in study of patients with hand, foot and mouth disease, Vietnam. A) CVA6 (p = 0.073); B) CVA10 (p = 0.347); C) CVA16 (p = 0.250); D) EV-A71 (p<0.001). CV, coxsackievirus; EV, enterovirus. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 302 Neutralizing Antibodies in Patients with HFMD RESEARCH RESEARCH CVA10, or CVA16, the 4 most common enterovirus serotypes responsible for the ongoing HFMD epi demic in Vietnam and the Asia–Pacific region over the past few decades. CVA10, or CVA16, the 4 most common enterovirus serotypes responsible for the ongoing HFMD epi demic in Vietnam and the Asia–Pacific region over the past few decades. Figure 3. Association between antibody response (seropositive) and illness days at enrollment (p<0.001) in study of patients with hand, foot and mouth disease, Vietnam. There were 82 patients with antibody titers below assay cutoff, and 38 patients with antibody titers above assay cutoff. p In terms of seropositivity, our results showed that antibody response against homotypic viruses at or above the titer of the protective level developed quickly after the onset of illness, with seropositivity for homotypic viruses changing from <60% at day 0–6 after illness onset (at enrollment) to 97%–100% at follow-up (7–19 days after the onset of illness). We could find no existing data obtained from natural in fection to compare with our results. However, results obtained from phase 3 vaccine trials have shown that at day 56 after the administration of the first 2 doses of inactivated EV-A71 vaccine, 98.5–99.9% of the vol unteers had neutralizing antibody against EV-A71 at titers of >1:16 (12–14). Collectively, these data expand our knowledge about immunogenicity elicited as a consequence of EV-A71 vaccination and natural in fection. Coxsackievirus vaccine development has not gone beyond animal experiments; thus, no similar data exist for CVA6, CVA10, or CVA16 (15,17). Figure 3. Association between antibody response (seropositive) and illness days at enrollment (p<0.001) in study of patients with hand, foot and mouth disease, Vietnam. There were 82 patients with antibody titers below assay cutoff, and 38 patients with antibody titers above assay cutoff. ( ) In contrast to the observed data for homotypic vi ruses, seropositive rates for heterotypic viruses were recorded in <57% of the patients during the course of illness. Furthermore, at follow-up, only a small pro portion (3%–23%) of the patients had seroconverted for heterotypic viruses, suggesting that cross-neutral ization among EV-A71, CVA6, CVA10, and CVA16 is absent or occurs in only a small proportion of pa tients (24,25). Antigenic Difference between Subgenogroup B5 and Emerging Subgenogroup C4 To assess the extent to which infection with sub genogroup B5 circulating during 2013–2015 could elicit cross-neutralizing antibody responses against 303 rging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 p values reflect the results of statistical analysis comparing the geometric mean titers between the 2 time points (enrollme corresponding enterovirus serotypes. CV, coxsackievirus; EV, enterovirus; HFMD, hand, foot and mouth disease. RESEARCH It cannot, however, be ruled out that these seropositive and seroconverstion rates, espe cially among CVA6 patients, were attributable to pre vious exposure or co-infection with other serotypes (e.g., CVA16 in the case of CVA6 patients), which may have been undetected by PCR. Our data sup port a recent report about recurrent HFMD episodes resulting from reinfection with heterotypic serotypes in China (9) and the absence of cross-neutralization among these 4 enterovirus serotypes observed in vac cine studies (13,16,17). As such, multivalent vaccines are needed to control HFMD. EV-A71 exists as a single serotype but is geneti cally divided into several genogroups (e.g., A, B, and C) and subgenogroups (e.g., C1–C5 and B1–B5). In Vietnam, HFMD has been a major public health concern since 2011, causing an average of 80,000 hospitalizations per year. The switches between pre dominant EV-A71 subgenogroups have been well documented; C4 was responsible for the 2011–2012 Table 3. Geometric mean titers of neutralizing antibodies against enteroviruses in study of patients with hand, foot and mouth disease, Vietnam* Virus Samples Geometric mean titer (95% CI) of neutralizing antibodies CVA6 CVA10 CVA16 EV-A71 CVA6 Enrollment 4.9 (3.6–6.7) 13.3 (6.8–26.1) 6.9 (4.3–11.3) 5.0 (3.6–7.0) Follow-up 151.4 (113.0–202.8) 13.0 (6.4–26.2) 10.8 (5.6–20.8) 6.0 (3.8–9.7) p value <0.001 0.83 0.021 0.50 CVA10 Enrollment 6.2 (3.9–9.7) 10.1 (5.7–17.7) 5.3 (3.8–7.2) 5.9 (3.8–9.7) Follow-up 8.7 (4.8–16.1) 245.5 (181.1–332.7) 6.3 (4.0–10.1) 7.6 (4.3–13.8) p value 0.063 <0.001 0.098 0.50 CVA16 Enrollment 9.0 (5.0–16.0) 13.6 (6.9–26.7) 10.5 (5.7–18.6) 7.4 (4.3–12.9) Follow-up 10.3 (5.7–18.7) 14.6 (7.5–28.5) 141.2 (104.547–191.0) 9.8 (5.1–18.7) p value 0.25 1.0 <0.001 0.098 EV-A71 Enrollment 6.6 (4.2–10.4) 23.2 (11.5–46.7) 7.6 (4.4–13.0) 37.7 (17.9–79.4) Follow-up 9.4 (5.3–16.7) 18.8 (10.0–35.4) 10.1 (5.4–18.7) 295.1 (260.6–334.2) p value 0.073 0.31 0.25 <0.001 p values reflect the results of statistical analysis comparing the geometric mean titers between the 2 time points (enrollment and follow-up) of the corresponding enterovirus serotypes CV coxsackievirus; EV enterovirus; HFMD hand foot and mouth disease Table 3. Geometric mean titers of neutralizing antibodies against enteroviruses in study of patients with hand, foot and mouth disease, p values reflect the results of statistical analysis comparing the geometric mean titers between the 2 time points (enrollment and follow-up) of the corresponding enterovirus serotypes. CV, coxsackievirus; EV, enterovirus; HFMD, hand, foot and mouth disease. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 304 Neutralizing Antibodies in Patients with HFMD Figure 4. RESEARCH The underlying mechanism that determines the emergence of certain subgeno groups in specific localities remains a puzzle; it may be a consequence of a complex interplay among the pathogen, the hosts, and public health response, of which antigenic evolution might play a role (26,27). The fact that all serum samples from subgenogroup B5—infected patients collected before 2018 could neutralize the 2018 C4 virus suggests that immuni ty developed as a consequence of natural infection with subgenogroup B5 could provide protection against subgenogroup C4. However, the extent to which waning immunity (28), as observed from vac cine trials, may influence the long-term protection and overall population immunity, in turn resulting in possible reinfection, which has previously been reported in China (9), as well as disease emergence, remains unknown. The underlying mechanism de termining the emergence of subgenogroup C4 in Vietnam in 2018 warrants further research. outbreak (18) followed by the predominance of B5 during 2013–2015 (10) and the reemergence of C4 in 2018 (6). Of note, the emergence of C4 in 2018 resulted in a severe outbreak that caused >130,000 hospital izations and 17 deaths. The underlying mechanism that determines the emergence of certain subgeno groups in specific localities remains a puzzle; it may be a consequence of a complex interplay among the pathogen, the hosts, and public health response, of which antigenic evolution might play a role (26,27). The fact that all serum samples from subgenogroup B5—infected patients collected before 2018 could neutralize the 2018 C4 virus suggests that immuni ty developed as a consequence of natural infection with subgenogroup B5 could provide protection against subgenogroup C4. However, the extent to which waning immunity (28), as observed from vac cine trials, may influence the long-term protection and overall population immunity, in turn resulting in possible reinfection, which has previously been reported in China (9), as well as disease emergence, remains unknown. The underlying mechanism de termining the emergence of subgenogroup C4 in Vietnam in 2018 warrants further research. Acknowledgments We thank Le Kim Thanh for her logistics support. We are indebted to the patients who participated in this study. This study was funded by the Wellcome Trust of Great Britain (grant nos.106680/B/14/Z and 204904/Z/16/Z). About the Author Ms. Nguyet is a research assistant at Oxford University Clinical Research Unit in Ho Chi Minh City, Vietnam. She is particularly interested in the application of classical virology methods as virus isolation and neutralization to study viruses responsible for emerging infections (hand, foot and mouth disease) in Vietnam and the region. RESEARCH Neutralizing antibody titers (in binary logarithm) against subgenogroups C4 and B5 in follow-up plasma samples collected from 6 patients infected with EV-A71 subgenogroup B5 (p = 1.0) in study of patients with hand, foot and mouth disease, Vietnam. Information about years of collection is shown alongside the EV- A71 subgenogroup on the x-axis. EV, enterovirus. in the Asia–Pacific region, whereas >20 enterovirus serotypes have been reported to be associated with HFMD in the region. Furthermore, because of the unavailability of plasma samples, we were not able to informatively assess the antigenic relationship be tween EV-A71 subgenogroups responsible for major HFMD outbreaks in Vietnam since 2011 (C4 and B5) with proper sample size. Likewise, our syndromic hospital-based surveillance may have missed atypical HFMD cases. Together with the convenience sample used, these limitations may lower the level of gener alizability of the obtained results to some extent. In addition, we were unable to obtain long-term follow- up blood samples from HFMD cases after hospital discharge. Therefore, evaluation of antibody kinetics and the waning antibody profiles of the natural infec tion beyond the sampling period of the current study remain unknown. In summary, because human infection with 1 HFMD-causing enterovirus serotype can elicit neu tralizing antibodies against homotypic viruses, our results support previous reports about the potential benefit of monovalent EV-A71 vaccine in reducing the incidence of EV-A71–associated HFMD (29). The data also emphasize the requirement for multivalent vaccines to control HFMD. Our results offer evidence that is essential for the development of intervention strategies, especially multivalent vaccines, and the design of seroepidemiologic studies. Figure 4. Neutralizing antibody titers (in binary logarithm) against subgenogroups C4 and B5 in follow-up plasma samples collected from 6 patients infected with EV-A71 subgenogroup B5 (p = 1.0) in study of patients with hand, foot and mouth disease, Vietnam. Information about years of collection is shown alongside the EV- A71 subgenogroup on the x-axis. 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https://openalex.org/W2032123938	https://bmcproc.biomedcentral.com/counter/pdf/10.1186/1753-6561-6-S2-S3	English	null	Comparison of analyses of the XVth QTLMAS common dataset III: Genomic Estimations of Breeding Values	BMC proceedings	2,012	cc-by	4,579	© 2012 Le Roy 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. Abstract Background: The QTLMAS XVth dataset consisted of pedigree, marker genotypes and quantitative trait performances of animals with a sib family structure. Pedigree and genotypes concerned 3,000 progenies among those 2,000 were phenotyped. The trait was regulated by 8 QTLs which displayed additive, imprinting or epistatic effects. The 1,000 unphenotyped progenies were considered as candidates to selection and their Genomic Estimated Breeding Values (GEBV) were evaluated by participants of the XVth QTLMAS workshop. This paper aims at comparing the GEBV estimation results obtained by seven participants to the workshop. Methods: From the known QTL genotypes of each candidate, two “true” genomic values (TV) were estimated by organizers: the genotypic value of the candidate (TGV) and the expectation of its progeny genotypic values (TBV). GEBV were computed by the participants following different statistical methods: random linear models (including BLUP and Ridge Regression), selection variable techniques (LASSO, Elastic Net) and Bayesian methods. Accuracy was evaluated by the correlation between TV (TGV or TBV) and GEBV presented by participants. Rank correlation of the best 10% of individuals and error in predictions were also evaluated. Bias was tested by regression of TV on GEBV. Results: Large differences between methods were found for all criteria and type of genetic values (TGV, TBV). In general, the criteria ranked consistently methods belonging to the same family. Conclusions: Bayesian methods - A<B<C<Cπ - were the most efficient whatever the criteria and the True Value considered (with the notable exception of the MSEP of the TBV). The selection variable procedures (LASSO, Elastic Net and some adaptations) performed similarly, probably at a much lower computing cost. The TABLUP, which combines BayesB and GBLUP, generally did well. The simplest methods, GBLUP or Ridge Regression, and even worst, the fixed linear model, were much less efficient. recently made operational by SNP chips which provide tens of thousands genotypes per individual. The seminal paper of Meuwissen et al. [2] presented a few statistical approaches of these Genomic Estimated Breeding Values (GEBV). A large literature followed, describing and com- paring various methods. Comparison of analyses of the XVth QTLMAS common dataset III: Genomic Estimations of Breeding Values Pascale Le Roy1,2, Olivier Filangi1,2, Olivier Demeure1,2, Jean-Michel Elsen3 From 15th European workshop on QTL mapping and marker assisted selection (QTLMAS Rennes, France. 19-20 May 2011 Le Roy et al. BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 Le Roy et al. BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 PROCEEDINGS Open Access Correspondence: Pascale.LeRoy@rennes.inra.fr 1INRA, UMR1348 PEGASE, Domaine de la Prise, 35590 Saint-Gilles, France Full list of author information is available at the end of the article Computation of the true genotypic and breeding values “True” genetic values of the candidates to selection were calculated from simulated QTL genotypes information. Two values were calculated for each candidate. Firstly, a True Genotypic Value (TGV) defined as the sum of the 5 chromosomal genotypic values corresponding to the candidate genotypes at each of these chromosomes. The TGV of candidate i, TGVi depends on its QTL geno- types (g1 i , g2 i ...g5 i ) and on the QTL effects (aj) given in the Table 1 of Elsen et al. [8]: Variable selection procedures were proposed to better fit this biological knowledge. In [2], a stepwise proce- dure, including a QTL detection step through single segments regression analyses, was envisaged in the least square framework. The efficiency for genomic evaluation of more advanced penalized regression approaches were evaluated, like sparse PLS [4], LASSO [5] or Elastic Net [6], which all allow the vast majority of loci to have null regression coefficients. On the other hand, Bayesian methods were proposed to take into account the between chromosome segments variances heterogeneity. In BayesA [2], each chromosome segment is given its own variance, all segments contribut- ing to the variability. This last hypothesis is made free with other Bayesian techniques which assume that only a fraction π of the segments carry QTL: BayesB keeps the between segments variance heterogeneity, while BayesC considers a single variance for the active segments. In BayesCπ, the proportion π is estimated from the data [7]. TGVi = 5 j=1 aj(gj i) Secondly, the expectation of the genotypic value of candidate’s progenies was calculated, according to the same principle, i.e. as a sum of chromosomal genotypic values. It depends on the QTL genotypes of the candi- date, on the QTL effects and on the frequencies of QTL genotypes in the population, i.e. the QTL genotypes probabilities of the mate of the candidate. This breeding value was noted TBVi: The QTLMAS XVth dataset consisted of the pedigree, marker genotypes and quantitative trait performances of animals with a sib family structure [8]. Pedigree and geno- types concerned 3,000 progenies among those 2,000 were phenotyped. The trait was regulated by 8 QTLs which dis- played additive, imprinting or epistatic effects. The 1,000 unphenotyped progenies were considered as candidates to selection. Simulated data The simulated data set was described by Elsen et al. [8]. Briefly, the population comprised 3,000 individuals born from 20 sires and 200 dams , i.e. 10 dams per sire. Within each family, 10 progenies were assigned phenotypes and marker genotypes and 5 were assigned only marker geno- types. A total of 10,000 SNPs carried by 5 chromosomes of 1 Morgan each were simulated. Eight QTLs were simulated: one quadri-allelic additive QTL with a large effect on Chr1, two linked QTLs in phase on Chr2, two linked QTLs in repulsion on Chr3, one imprinted QTL on Chr4 and two interacting QTLs on Chr5. Random noise was added giving an heritability coefficient of 0.30. Background In 1990, Lande and Thompson [1] defined a two steps marker assisted selection procedure. Firstly, apparent effects of markers were estimated in a reference popula- tion. Secondly, during n generations, breeding values of candidates to selection were calculated from these esti- mated effects giving a so called Molecular Scores. These ideas, which founded the genomic selection, were more These methods could be classified according to the assumption made concerning the variance of chromosome segments effects. The simplest assumption, assumed in BLUP methodology [2] or Ridge Regression [3], is that the variance of these effects is equal for all chromosome * Correspondence: Pascale.LeRoy@rennes.inra.fr 1INRA, UMR1348 PEGASE, Domaine de la Prise, 35590 Saint-Gilles, France Full list of author information is available at the end of the article Le Roy et al. BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 Page 2 of 6 The marker density, linkage disequilibrium (LD) and minor allele frequency (MAF) were similar to real life parameters. segments. However, this hypothesis is not consistent with classical genetic prior, and observations, that only a few chromosome segments contain QTL, with various extent of their effects, while most chromosome segments do not contain QTL. Computation of the true genotypic and breeding values Participants of the XVth QTLMAS workshop were invited to predict GEBV of these 1,000 individuals and to send to the organizers the description of their meth- ods and results before the meeting. This paper aims at comparing the GEBV estimations obtained by participants to the workshop. Comparing the results obtained by the different groups should provide insight into determining which method is best fitted to analyze this kind of data set. TBVi = 5 j=1 n gj=1 prob(gj/gj i) ∗aj(gj) where the gj are the n QTL possible genotypes on the chromosome j (n=(10,9,9,4,9) for the chromosomes 1 to 5 respectively), prob(gj/gj i) is the probability of the geno- type gj for the progeny of the candidate i given the can- didate’s genotype and aj (gj) is the genotypic value associated to that QTL genotype (Table 1 in [8]). Con- cerning the QTL6 on chromosome 4, which was imprinted, candidates were considered as those which give the allele expressed by their progeny. Methods The participants were sent the TGV and TBV only after the meeting. Methods used by the participants The participants estimated Genomic Estimated Breeding Values, noted GEBV in the following, and sent them, with a short description of the methods used, to organizers before the meeting. A total of 27 methods were studied by the participants (table 1). Most of them belong to the three categories which were presented in the introduction: (i) (G)BLUP methods - including Ridge regression [9], GBLUP describing dominance effect [10] and TABLUP [11] where the genomic matrix includes information about the SNP effect variance (here estimated using BayesB) [12] Le Roy et al. BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 Page 3 of 6 Table 1 Methods used by the participants to the XVth QTLMAS workshop First author Label Method Description Shariati BayesS_1 2 steps (all SNP) First step: a GBLUP giving estimation of SNP effects. Groups of size 150, 75 (SPNa) or 50 (SNPb) are made assembling SNP of similar effect. Second step: BayesA with all or a limited (1500 or 450) number of SNP and a unique SNP effect variance per group. BayesS_2 2 steps (1500 SNP) BayesS_3 2 steps-Bayes (450 SNPa) BayesS_4 2 steps-Bayes (450 SNPb) Ogutu RR Ridge regression GBLUP_O GBLUP Qualified Ridge Regression BLUP by the authors LASSO_O LASSO LASSO_ad Adaptative LASSO Following Zou [21], data-driven weights are added to the penalty to force LASSO to be consistent EN Elastic net EN_ad Adaptative EN Mixture of adaptative lasso and EN Wang BayesA_W BayesA BayesB_W BayesB BayesCπ_W BayesCπ TABLUP TABLUP In the genomic matrix, loci IBD probability estimations are weighted by their effect variance estimated from BayesB [11] GBLUP_W GBLUP Mucha AM Animal model All models are estimating haplotypes effects. Haplotypes are obtained using the PHASE software [18]. RM1 and RM2 differ by the estimation of the haplotype effect variance FM Fixed effect RM1 Random model 1 RM2 Random model 2 Zeng GBLUPa_Z GBLUP1 Additive effect only GBLUPd_Z GBLUP2 Additive and dominance effect BayesB _W BayesB BayesCπ_W BayesCπ Usai LASSO_Uc LASSO-LARS classic The penalty is describes as ∑\|bj\|≤t. In the LASSO-LARS classic, the t parameter is the average number of active SNP in 1000 simulations. Methods used by the participants In strategy 1, the number which occurred more than 5% of the times and in strategy 2, which minimized a selection criteria LASSO_Uc1 LASSO-LARS strategy 1 LASSO_Uc2 LASSO-LARS strategy 2 Schurink BayesZ BayesZ Similar to BayesCπ, with a Bernoulli prior for π Table 1 Methods used by the participants to the XVth QTLMAS workshop Similar to BayesCπ, with a Bernoulli prior for π (ii) Selection variable procedures - LASSO and Elastic Net, including adaptative versions which aim at forcing the LASSO to be consistent, i.e. to correctly estimate the sub- set of zero coefficients with a probability tending to 1 [9,13] and (iii) Bayesian approaches [12,10] - including the BayesZ [14,15] and a new two-steps Bayes procedure intermediate between the BayesA or B (one variance for (ii) Selection variable procedures - LASSO and Elastic Net, including adaptative versions which aim at forcing the LASSO to be consistent, i.e. to correctly estimate the sub- set of zero coefficients with a probability tending to 1 [9,13] and (iii) Bayesian approaches [12,10] - including the BayesZ [14,15] and a new two-steps Bayes procedure intermediate between the BayesA or B (one variance for each SNP) and the BayesC (a single variance for the active SNP), with a grouping of SNP based on their effect esti- mated with a GBLUP [16]. This method will be given the “BayesS” acronym in the following. Mucha et al. [17] used simple linear models (fixed or random) with the idea of estimating haplotype rather than SNP effects, the haplo- types being inferred with the PHASE software [18]. Le Roy et al. Methods used by the participants BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 Page 4 of 6 Table 3 Comparison of True Breeding Values estimations First author Label r rank bias MSE Shariati BayesS_1 0.84 0.48 0.33 12.92 BayesS_2 0.84 0.47 0.33 12.94 BayesS_3 0.83 0.49 0.33 13.37 BayesS_4 0.82 0.49 0.32 13.62 Ogutu RR 0.83 0.52 0.45 5.55 GBLUP_O 0.81 0.51 0.39 8.23 LASSO_O 0.87 0.55 0.43 6.44 LASSO_ad 0.88 0.60 0.37 9.94 EN 0.87 0.52 0.44 5.86 EN_ad 0.81 0.48 0.33 12.01 Wang BayesA_W 0.86 0.61 0.38 9.07 BayesB_W 0.89 0.66 0.38 9.12 BayesCπ_W 0.88 0.65 0.39 9.00 TABLUP 0.88 0.64 0.36 10.88 GBLUP_W 0.77 0.48 0.46 4.98 Mucha AM 0.59 0.37 0.40 5.93 FM 0.47 0.44 0.13 43.01 RM1 0.70 0.34 0.68 2.54 RM2 0.70 0.34 0.65 2.68 Zeng GBLUPa_Z 0.82 0.50 0.40 7.61 GBLUPd_Z 0.81 0.49 0.40 7.59 BayesB _W 0.89 0.66 0.38 9.22 BayesCπ_W 0.89 0.66 0.39 8.84 Usai LASSO_Uc 0.86 0.53 0.45 5.66 LASSO_Uc1 0.86 0.62 0.37 9.54 LASSO_Uc2 0.87 0.55 0.43 6.48 Schurink BayesZ 0.87 0.63 0.39 5.20 (r=Pearson correlation between TBV and GEBV, rank=rank correlation of the best 10% TBV, bias = regression coefficient between TBV and GEBV, MSE= mean squared error of prediction of TBV by GEBV) Results They are presented in table 2 (TGV) and 3 (TBV). The ranking is nearly the same for those two values. Table 2 Comparison of True Genomic Values estimations First author Label r rank bias MSE Shariati BayesS_1 0.86 0.53 0.89 7.51 BayesS_2 0.86 0.53 0.89 7.52 BayesS_3 0.86 0.53 0.88 7.85 BayesS_4 0.85 0.55 0.87 8.00 Ogutu RR 0.85 0.54 1.19 8.44 GBLUP_O 0.90 0.52 1.11 5.55 LASSO_O 0.92 0.63 1.09 4.67 LASSO_ad 0.92 0.62 1.02 4.30 EN 0.92 0.62 1.23 4.96 EN_ad 0.90 0.40 0.97 5.48 Wang BayesA_W 0.92 0.65 1.06 4.15 BayesB_W 0.93 0.70 1.05 3.66 BayesCπ_W 0.93 0.70 1.06 3.63 TABLUP 0.91 0.68 0.97 4.59 GBLUP_W 0.78 0.37 1.20 11.71 Mucha AM 0.61 0.36 1.06 17.57 FM 0.49 0.32 0.35 43.17 RM1 0.70 0.38 1.77 16.65 RM2 0.71 0.38 1.69 16.30 Zeng GBLUPa_Z 0.82 0.53 1.04 8.94 GBLUPd_Z 0.81 0.52 1.04 9.46 BayesB _W 0.93 0.71 1.05 3.63 BayesCπ_W 0.94 0.72 1.07 3.41 Usai LASSO_Uc 0.92 0.62 1.25 5.04 LASSO_Uc1 0.90 0.64 1.02 5.30 LASSO_Uc2 0.92 0.63 1.09 4.66 Schurink BayesZ 0.90 0.60 1.06 5.20 r=Pearson correlation between TGV and GEBV, rank=rank correlation of the best 10% TGV, bias = regression coefficient between TGV and GEBV, MSEP= mean squared error of prediction of TGV by GEBV. Table 2 Comparison of True Genomic Values estimations (r=Pearson correlation between TBV and GEBV, rank=rank correlation of the best 10% TBV, bias = regression coefficient between TBV and GEBV, MSE= mean squared error of prediction of TBV by GEBV) Comparison criteria Table 3 Comparison of True Breeding Values estimations Results (GEBV as given by the participants) were com- pared based on 4 criteria. For each criteria, the two True Values (TGV and TBV) were considered. Accuracy of GEBV was calculated as the Pearson’s correlation between the TV and the GEBV. Ability to identify the best individuals was assessed from the Spearman’s rank correlation between the TV and the GEBV in the top 10% of TV. Bias was assessed from the linear regression coefficient (named also the regression slope) of the TV on the GEBV. Finally, mean squared error of prediction was calculated on GEBV and TV centered on zero. Author details 1INRA, UMR1348 PEGASE, Domaine de la Prise, 35590 Saint-Gilles, France. 2Agrocampus OUEST, UMR1348 PEGASE, 65 rue de St Brieuc, 35042 Rennes, France. 3INRA UR0631 SAGA, chemin de borde rouge, BP 52627, 31326 Castanet-Tolosan, France. Conclusions Even if all tendencies were observed for both groups of correlations, the correlations between GEBV and the TBV were always lower than the correlations between GEBV and TGV. These last correlations were always lower than the former. The very general tendency is a better ranking of the Bayesian methods, in the alphabetic order (A<B<C<Cπ) whatever the criteria and the True Value considered (with the notable exception of the MSEP of the TBV). The Selection variable procedures (LASSO, Elastic Net and some adaptations) performed similarly, probably at a much lower computing cost. The TABLUP, which com- bines BayesB and GBLUP, generally did well. The sim- plest methods, GBLUP or Ridge Regression, and even worst, the fixed linear model, were much less efficient. The approach followed by Mucha et al. [17] to incorpo- rate haplotype information was not efficient. List of abbreviations used l l d l List of abbreviations used SNP: Single Nucleotide Polymorphism; QTL: Quantitative Trait Locus; MAF: Minor Allele Frequency; LD: Linkage Disequilibrium; GEBV: Genomic Estimated Breeding Value; TBV: True Breeding Value; TGV: True Genomic Value; LASSO: Least Absolute Shrinkage and Selection Operators; EN: Elastic Net; MSEP: Mean Squared Error of Prediction; GBLUP: Genomic Best Linear Unbiased Prediction. The rank correlation between GEBV and TBV is gen- erally lower than the GEBV-TGV one, with some excep- tions (GBLUP [12], animal and fixed models [17], adaptative EN [9]). Mean squared error of prediction (MSEP) The results are still very consistent with the other cri- teria. The Bayesian techniques (excluding BayesS) and the selection variable techniques (LASSO or EN) gave the more precise estimations of the TGV. TABLUP was in the same range. The GBLUP and BayesS performed Authors’ contributions OD and OF collected and processed the data files. PLR analyzed the data. PLR and JME wrote the manuscript. All authors contributed to the ideas and methods, and read and approved the manuscript. Accuracy The Pearson correlation between GEBV and the TV were consistent within type of technique used. The range was large, from 0.49 (GEBV- TGV correlation, 0.47 for GEBV-TBV) for the Mucha et al. [15] fixed effect model to 0.94 (GEBV- TGV correlation, 0.89 for GEBV-TBV) for the Zeng et al. [10] BayesCπ. The highest correlations were obtained with the Bayesian approaches, with a very good performance of BayesCπ [10,12] which overper- formed BayesZ, a similar approach based on an alterna- tive prior. The very limited number of QTL simulated in the dataset, a situation far from the BayesZ prior, is a possible explanation for this difference. The same argu- ment could explain the lower performance of the BayesS [16], where SNP effects are assembled in groups of simi- lar effects. The TABLUP, which mixes BayesB estimation and GBLUP, was intermediate between the “classical” and the new Bayesian approaches of Shariati et al. [16]. Le Roy et al. BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 Le Roy et al. BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 Page 5 of 6 not as well and Mucha et al. haplotypes models [17] did very badly. The variable selection procedures can work nearly as well as the BayesB or C, in particular the LASSO and Elastic net [9,13]. However the adaptative Elastic Net did not give the expected improvement. The MSEP of the TBV were quite different and were above or under the TGV MSEP depending on the method. The more precise estimation was given by the Mucha et al. [17] random model. LASSO, EN and GBLUP were satisfy- ing, while the Bayesian approaches (in particular the BayesS) provided high Mean Squared Error of Prediction. The GBLUP performances were more variable with a very low correlation given by the Mucha et al. [17] version based on haplotypes, and higher values for the Zeng et al. [10] and Ogutu et al. [9] proposals. Finally, the fixed effect linear model was far below all other methods. Regression coefficient (or regression slope) Unbiased estimators are supposed to have a regression coefficient of 1. Most of the regression coefficients observed were in the range 0.85-1.25. The ranking of the Bayesian techniques were consistently correct, while the coefficient were more variable for the other approaches. Three of the methods proposed by Mucha et al. [15] clearly gave biased estimations (the fixed and both random models). Acknowledgements This article has been published as part of BMC Proceedings Volume 6 Supplement 2, 2012: Proceedings of the 15th European workshop on QTL mapping and marker assisted selection (QTL-MAS). The full contents of the supplement are available online at http://www.biomedcentral.com/bmcproc/ supplements/6/S2. Rank correlation As compared to the Pearson’s correlation, this criteria, which illustrates how methods can capture the best indivi- duals, shows a similar range (0.32 to 0.72, i.e. 0.4 points of correlation between extreme situations), i.e. the fixed model and the BayesCπ. Globally the classification between groups of methods is the same: Bayes methods outperformed the selection variable approaches, the GBLUP family arrived last in the classification. The only exception was the TABLUP which was positioned between the two first groups. However, within some groups, differ- ences were exacerbated. This was particularly true for the Bayes group, where the ranking BayesCπ > BayesB > BayesA > BayesZ > BayesS was preserved, and even more for the Ogutu et al. [9] selection variable, with a very low correlation observed for the adaptative Elastic Net. Nota- bly the random model proposed by Mucha et al. [17] fell in the worst positions with this criterion. These observations are consistent with the results pre- sented in the previous analyses of QTLMAS data [19-21], even if the genetic architecture simulated was restricted to a quite limited (8) number of QTL. It may be that this oligogenic situation did not work in favor of methods probably more suited to highly polygenic cases, such as BayesS [16] or BayesZ [15]. References Ogutu JO, Schulz-Streeck T, Piepho HP: Genomic selection using regularized linear regression models: ridge regression, lasso, elastic net and their extensions. In Proceedings of the XVth QTLMAS Workshop: 19-20 May 2011; Rennes France Demeure O, Elsen JM, Filangi O, Le Roy P 2012. 10. Zeng J, Pszczola M, Wolc A, Strabel T, Fernando RL, Garrick DJ, Dekkers JCM: Genomic Breeding Value Prediction and QTL mapping of QTLMAS2011 data using Bayesian and GBLUP methods. In Proceedings of the XVth QTLMAS Workshop: 19-20 May 2011; Rennes France Demeure O, Elsen JM, Filangi O, Le Roy P 2012. 11. Zhang Z, Liu J, Ding X, Bijma P, de Koning DJ, Zhang Q: Best Linear Unbiased Prediction of Genomic Breeding Values Using a Trait-Specific Marker-Derived Relationship Matrix. PLoS ONE 2010, 5(9):e12648. 12. Wang CL, Ma PP, Zhang Z, Ding XD, Liu JF, Fu WX, Weng ZQ, Zhang Q: Comparison of five methods for genomic breeding value estimation for the common dataset of the 15th QTL-MAS Workshop. In Proceedings of the XVth QTLMAS Workshop: 19-20 May 2011; Rennes France Demeure O, Elsen JM, Filangi O, Le Roy P 2012. 13. Usai MG, Carta A, Casu S: Alternative strategies for selecting subsets of predicting SNPs by LASSO-LARS procedure. In Proceedings of the XVth QTLMAS Workshop: 19-20 May 2011; Rennes France Demeure O, Elsen JM, Filangi O, Le Roy P 2012. 14. Janss LLG: Bayz manual version 2.03 Leiden, The Netherlands, Janss Biostatistics; 2010. 15. Schurink A, Janss LLG, Heuven HCM: Bayesian Variable Selection to identify QTL affecting a simulated quantitative trait. In Proceedings of the XVth QTLMAS Workshop: 19-20 May 2011; Rennes France Demeure O, Elsen JM, Filangi O, Le Roy P 2012. 16. Shariati MM, Sorensen P, Janss L: A two step Bayesian approach for genomic prediction of breeding values. In Proceedings of the XVth QTLMAS Workshop: 19-20 May 2011; Rennes France Demeure O, Elsen JM, Filangi O, Le Roy P 2012. 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: 17. References 1. Lande R, Thompson R: Efficiency marker assisted selection in the improvement of quantitative traits. Genetics 1990, 124:743-756. 1. Lande R, Thompson R: Efficiency marker assisted selection in the improvement of quantitative traits. Genetics 1990, 124:743-756. 1. Lande R, Thompson R: Efficiency marker assisted selection in the improvement of quantitative traits. Genetics 1990, 124:743-756. improvement of quantitative traits. Genetics 1990, 124:743-756. 2. Meuwissen THE, Hayes BJ, Goddard ME: Prediction of Total Genetic Value using genome-wide dense marker maps. Genetics 2001, 157:1819-1829. 3. Piepho HP: Ridge regression and extensions for genome wide selection in maize. Crop Science 2009, 49:1165-1176. 2. Meuwissen THE, Hayes BJ, Goddard ME: Prediction of Total Genetic Value using genome-wide dense marker maps. Genetics 2001, 157:1819-1829. 22. Zou H: The adaptive lasso and its oracle properties. Journal of the American Statistical Association 2006, 101:1418-1429. 22. Zou H: The adaptive lasso and its oracle properties. Journal of the American Statistical Association 2006, 101:1418-1429. 3. Piepho HP: Ridge regression and extensions for genome wide selection in maize. Crop Science 2009, 49:1165-1176. doi:10.1186/1753-6561-6-S2-S3 Cite this article as: Le Roy et al.: Comparison of analyses of the XVth QTLMAS common dataset III: Genomic Estimations of Breeding Values. BMC Proceedings 2012 6(Suppl 2):S3. 4. Colombani C, Legarra A, Croiseau P, Guillaume F, Fritz S, Ducrocq V, Robert- Granié C: Application of PLS and Sparse PLS regression in genomic selection. In Proceedings of the 9th World Congress of Genetics Applied to Livestock Production: 1-6 August 2010; Leipzig Germany Gesellschaft für Tierzuchtwissenschaften 2010, 0439. 5. Usai MG, Goddard ME, Hayes BJ: LASSO with cross-validation for genomic selection. Genet Res 2009, 91:427-436. 6. Croiseau P, Colombani C, Legarra A, Guillaume F, Fritz S, Baur A, Dassonneville R, Patry C, Robert-Granié C, Ducrocq V: Improving genomic evaluation strategies in dairy cattle through SNP selection. In Proceedings of the 9th World Congress of Genetics Applied to Livestock Production: 1-6 August 2010; Leipzig Germany Gesellschaft für Tierzuchtwissenschaften 2010, 0360. 7. Habier D, Fernando RL, Kizilkaya K, Garrick DJ: Extension of The Bayesian alphabet for genomic selection. In Proceedings of the 9th World Congress of Genetics Applied to Livestock Production: 1-6 August 2010; Leipzig Germany Gesellschaft für Tierzuchtwissenschaften 2010, 0468. 8. Elsen JM, Tesseydre S, Filangi O, Le Roy P, Demeure O: XVth QTLMAS: simulated dataset. In Proceedings of the XVth QTLMAS Workshop: 19-20 May 2011; Rennes France Demeure O, Elsen JM, Filangi O, Le Roy P 2012. 9. Competing interests Published: 21 May 2012 Le Roy et al. BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 Le Roy et al. BMC Proceedings 2012, 6(Suppl 2):S3 http://www.biomedcentral.com/1753-6561/6/S2/S3 Page 6 of 6 21. Lund M, Sahana G, de Koning D-J, Su G, Carlborg Ö: Comparison of analyses of the QTLMAS XII common dataset. I: Genomic selection. BMC Proceedings 2009, 3(Suppl 1):S1. 22. Zou H: The adaptive lasso and its oracle properties. Journal of the American Statistical Association 2006, 101:1418-1429. doi:10.1186/1753-6561-6-S2-S3 Cite this article as: Le Roy et al.: Comparison of analyses of the XVth QTLMAS common dataset III: Genomic Estimations of Breeding Values. BMC Proceedings 2012 6(Suppl 2):S3. 21. Lund M, Sahana G, de Koning D-J, Su G, Carlborg Ö: Comparison of analyses of the QTLMAS XII common dataset. I: Genomic selection. BMC Proceedings 2009, 3(Suppl 1):S1. References Mucha A, Wierzbicki H: Linear models for breeding values prediction in haplotype-asssited selection - an analysis of QTLMAS Workshop 2011 Data. In Proceedings of the XVth QTLMAS Workshop: 19-20 May 2011; Rennes France Demeure O, Elsen JM, Filangi O, Le Roy P 2012. 18. Stephens M, Smith N, Donnelly P: A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics 2001, 68:978-989. 19. Pszczola M, Strabel T, Wolc A, Mucha S, Szydlowski M: Comparison of analyses of the QTLMAS XIV common dataset. I: genomic selection. BMC Proceedings 2011, 5(Suppl 3):S1. 20. Bastiaansen J, Bink M, Coster A, Maliepaard C, Calus M: Comparison of analyses of the QTLMAS XIII common dataset. I: genomic selection. BMC Proceedings 2010, 4(Suppl 1):S1.
https://openalex.org/W2887700645	https://aip.scitation.org/doi/pdf/10.1063/1.5042797	English	null	Thermal stress distribution in a laser array structure selectively grown on V-groove-patterned silicon	AIP advances	2,018	cc-by	5,978	RESEARCH ARTICLE \| AUGUST 07 2018 Thermal stress distribution in a laser array structure selectively grown on V-groove-patterned silicon Haiying Yin; Jun Wang  ; Zeyuan Yang; Zhuo Cheng; Wei Wang; Yongqing Huang; Xiaomin Ren AIP Advances 8, 085007 (2018) https://doi.org/10.1063/1.5042797 AIP Advances 8, 085007 (2018) https://doi.org/10.1063/1.5042797 RESEARCH ARTICLE \| AUGUST 07 2018 Thermal stress distribution in a laser array structure selectively grown on V-groove-patterned silicon Haiying Yin, Jun Wang,a Zeyuan Yang, Zhuo Cheng, Wei Wang, Yongqing Huang, and Xiaomin Ren State Key Laboratory of Information Photonics and Optical Communications, Beijing University of Posts and Telecommunications, Beijing 100876, China (Received 5 June 2018; accepted 28 July 2018; published online 7 August 2018) The thermal stress due to the thermal expansion mismatch could induce crystallo- graphic defects such as buckling and cracking and degrade device performance. In this paper, the thermal stress distribution in a laser array structure selectively grown on V-groove-patterned Si substrates was investigated by two-dimension ﬁnite-element method. Surprisingly, unexpected results are observed that the top of the InGaAs active layer and the most region of the InP cap layer are in compression, which is far different from the thermal stress distribution in planar structures. Two mechanisms have been proposed and modeled to explain the difference—(i) the width of uncoalesced layers is smaller than that of the Si substrate, which causes thermal stress to change in epitaxial layers, and (ii) thermal stress in the InGaAs and InP layers is affected by the V-groove structure. The results show that whether or not the epitaxial layers are coalesced has signiﬁcant effect on the thermal stress distribution. The effect of the height of the V- groove, the height and the width of the SiO2 mask on the thermal stress distribution was also studied. It is found that the height of V-groove and the height of SiO2 mask play a critical role in the stress distribution. These ﬁndings are useful for the optimal designs for the laser array and provide an important step towards the realization of photonic integration circuits on silicon. © 2018 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/1.5042797 24 October 2024 04:45:05 aAuthor to whom correspondence should be addressed. Electronic mail: wangjun12@bupt.edu.cn Articles You May Be Interested In Local strain, defects, and crystallographic tilt in GaN(0001) layers grown by maskless pendeo-epitaxy from x-ray microdiffraction J. Appl. Phys. (December 2004) Spatially resolved distribution of dislocations and crystallographic tilts in GaN layers grown on Si(111) substrates by maskless cantilever epitaxy Design of micro-nano grooves incorporated into suspended GaN membrane for active integrated optics AIP Advances (November 2018) 24 October 2024 04:45:05 AIP ADVANCES 8, 085007 (2018) I. INTRODUCTION Low-defect growth of GaAs,15 InP,16,17 and InGaAs18 compounds on pre-patterned Si substrates was achieved using SAG. Moreover, V-groove-patterned on-axis Si (001) substrates were found to effectively eliminate the APBs.19 It is essential to eliminate the APBs in order to integrate highly performing III–V semiconductors on the current Si standard process plat- form. In addition, using V-groove-patterned substrates, many optoelectronic devices are fabricated. It is worth mentioning that long-wavelength lasers on Si have been achieved, which is essential to the long-awaited missing piece of Si photonics: integrated laser sources on Si. Wang et al. achieved room-temperature InP distributed feedback laser array directly grown on silicon by using V-groove patterned Si substrates.5 And O-band DFB laser array directly grown on (001) silicon has been reported.6 With the laser array structures they used, the dislocations induced by lattice mismatch can be effectively conﬁned to the region near the InP/Si interface, and the APBs were effectively eliminated by the V-groove-patterned on-axis Si (001) substrates. 24 October 2024 04:45:05 However,theinﬂuenceofthermalstressresultedfromthermalexpansionmismatch,whencooling from the growth temperature to room temperature, has not been paid attention by them. In fact, stress free is desirable for the heteroepitaxy of the ﬁlms, since the stress not only affects the quality of thin ﬁlms due to the formation of cracks, but also deteriorates the performance and lifetime of devices fabricated on the epilayers. Moreover, a few reports have been found that the thermal stress of the patterned structure is far different and complicated from that of unpatterned structure by ﬁnite-element method.20–23 While the patterned substrates in these studies are some special patterned substrates, such as the sawtooth- patterned Si substrate which may not be effective for reducing dislocation, rather than the most widely used and promising V-groove Si substrates. In addition, the size of the structures in these studies are usually in micron dimensions, while the current structures are all in nanometers, which may result in different thermal stress distribution. Furthermore, the structures used in these studies are all sim- ple patterned structure, not including the complete device structures. In fact, it is very necessary to study the thermal stress distribution of the device, especially the laser array structure selectively grown on V-groove-patterned silicon. On the one hand, the achievement of long-wavelength laser arrays on Si is essential to the long-awaited missing piece of Si photonics: integrated laser sources on Si. I. INTRODUCTION Integrating III–V compound semiconductors on silicon-based platform to achieve high perfor- mance and energy-efﬁcient optoelectronic device such as solar cells,1,2 photodetectors3,4 and lasers5,6 has attracted great interest in the past several years. III-V compound semiconductors integrated onto Si chips not only would take advantage of direct-bandgap III-V compound semiconductors as a light emitter, but also can leverage the well-established and advanced manufacturing processes developed initially by the electronics industry, which is considered as promising candidates for the realization of photonic integrated circuits. p g However, considerable difﬁculties which inhibit the development of the integration of III–V compound semiconductors and silicon need to be overcome. When directly growing III–V semicon- ductors on silicon, the mismatch of lattice constant, the difference of thermal expansion coefﬁcients and the different polarity between III–V materials and silicon lead to a high density of crystalline defects, such as misﬁt and threading dislocations, stacking faults, twins, and anti-phase boundaries (APBs), which can greatly degrade the performance and reduce the lifetime of fabricated devices.7 So far, many planar growth methods have been proposed and taken to overcome these issues, such as the two-step or three-step growth method,8,9 thermal cycle annealing,10 buffer layer method,11 strained- layer superlattices (SLSs)12 and dislocation ﬁlter with quantum dots.13 Although these methods could © Author(s) 2018 8, 085007-1 2158-3226/2018/8(8)/085007/12 085007-2 Yin et al. AIP Advances 8, 085007 (2018) AIP Advances 8, 085007 (2018) reduce the defect density by orders of magnitude, they need to be less time-consuming before being employed in industrial manufacturing process. In addition, several planar methods are usually used together in heteroepitaxy growth, which leads to the complexity of the growth process.14 Selective area growth (SAG), as an attractive method which can effectively reduce the dislocation density in lattice-mismatched heteroepitaxy, has been demonstrated to obtain high quality materials.15–17 A noteworthy advantage of SAG is that it is typically combined with the aspect ratio trapping (ART) technique, which traps the threading dislocations due to the mismatch of lattice constant at the side- walls of the dielectric mask. In addition, compared with the planar methods, the growth can be achieved in speciﬁc regions by SAG, which is advantageous for us to achieve the preparation of various devices, such as laser arrays. Signiﬁcant successes have been achieved in direct growth of III–V/Si using SAG method com- bined with the ART technique. II. LASER STRUCTURES AND SIMULATION MODE The focus of this section is to study the thermal stress distribution in the laser array structure. The geometry of the basic unit of a laser array used in simulation is show in Fig. 1. The structure is made up of the V-groove-patterned InP/Si substrate, the SiO2 mask, the InGaAs active layer and the InP cap layer. Note that a study of the effect of the parameters of the V-groove is done in the next section, in which case the V-groove height of 250 nm and the width of the V-groove of 125 nm are also analyzed. The aspect ratio of groove is supposed to be constant in the following studies. The height and width of the SiO2 mask can also vary, but in this case, they are chosen to be 125 nm and 250 nm, respectively. The values of the InGaAs active layer and the InP cap layer thickness are 60 nm and 20 nm, respectively. The width of the InGaAs and InP layers is equal to the V-groove width. The substrate thickness is set as 20 µm because the ﬁnite element results are not affected when its thickness is thicker than 20 µm. The substrate width is set to be 20 µm, which is large enough to mimick an inﬁnite substrate. The boundary conditions are such that node A (indicated by the arrow in Fig.1) on the substrate is ﬁxed, and node B (indicated by the arrow) on the substrate is held constrained in the y direction. All other node in the structure then move freely according to the law of thermal expansion without any external mechanical constraint or any internal dislocation. Another important factor affects the ﬁnite-element calculation results is the meshing of ﬁnite elements. Taking into account the accuracy of results and the consumption of simulation time, an extremely ﬁne mesh is introduced to model for the V-groove (between the InGaAs active layer and Si substrate) and appropriate mesh size is modeled for other regions. For example, maximum element size of 5 nm and minimum element size of 2 nm are used for the V-groove. Mesh of the V-groove consists of 168592 elements and 6720 boundary elements. 24 October 2024 04:45:05 y The ﬁnite element simulations were carried out using a commercial software. I. INTRODUCTION On the other hand, the use of SAG method directly on standard (001) Si substrates facilitates future integration of laser array with both photonic and electronic circuits. Lastly, to the best of our knowledge, thermal stress distribution in V-groove-patterned structures has not been reported to date. Therefore, it is necessary to conduct thermal stress analysis in nanoscale V-groove pat- terned structures for the selective growth of III–V/Si. And it is important to study the thermal stress distribution in laser array structures selectively grown on V-groove-patterned silicon for their opti- mal designs to provide an important step towards the realization of photonic integration circuits on silicon. In this article, we aim to study the thermal stress distribution in the laser array structure selectively grown on V-groove-patterned silicon by ﬁnite-element method. First, we present the ﬁnite-element method for 2D model of the simpliﬁed Ge grown on the patterned Si substrate structure to examine the reliability of the procedure and methods used in simulation. Following this, we describe the modeling of a 2D model with a laser array structure. Surprisingly, unexpected results are observed that the top of the InGaAs active layer and the most region of the InP cap layer are in compression which is far different from the thermal stress distribution in planar structures. Two mechanisms we have proposed AIP Advances 8, 085007 (2018) 085007-3 Yin et al. and modeled to explain this difference. Lastly, an investigation of the effect of parameters of the V-groove on thermal stress is carried out. II. LASER STRUCTURES AND SIMULATION MODE In the following calculations, the substrate and the epitaxial layers are assumed stress free at growth temperature of 600 ◦C, and thermal stress distribution are calculated at 20 ◦C. Values of Young’s modulus E, Poisson’s ration ν, and thermal expansion coefﬁcient α are given in Table I.24 The parameters of InGaAs are derived from those of GaAs and InAs on the assumption of its linearity for the composition.25 Isotropy FIG. 1. Geometry of the basic unit of a laser array used for the FE calculation. FIG. 1. Geometry of the basic unit of a laser array used for the FE calculation. TABLE I. Material parameters used in simulation. Materials Elastic Modulus (GPa) Poisson’s ratio Coefﬁcient of thermal expansion (10-6/K) Si 170.0 0.28 2.6 SiO2 70.0 0.17 0.5 InP 60.7 0.35 4.6 InAs 50.5 0.35 4.5 Ge 103 0.26 5.9 TABLE I. Material parameters used in simulation. AIP Advances 8, 085007 (2018) 085007-4 Yin et al. 085007-4 of the elastic constants is assumed. It is important to note that all our results shown in the legend of ﬁgure and in the subsequent stress distribution ﬁgures are in MPa, unless mentioned otherwise. A. Veriﬁcation of simulation procedure In order to examine the reliability of the procedure and method used in our simulation, we ﬁrst calculated the thermal stress distribution of the patterned substrate structure in Ref. 26. The schematic 2D geometry is shown in Fig. 2. Material parameters used in simulation are list in Table I. The SiO2 trench width and pitch are 200 nm and 400 nm respectively. The thickness of Ge epilayer and SiO2 mask are 1 um and 60 nm respectively. Temperature excursion of 600 K is calculated. The boundary conditions are such that node A (indicated by the arrow in Fig. 2) on the substrate is ﬁxed, and node B (indicated by the arrow in Fig. 2) on the substrate is held constrained in the vertical direction. The meshing of the 2D geometry is quite easy, as it is not a complex geometry. Appropriate mesh size is introduced for the model. For example, maximum element size and minimum element size for the SiO2 mask are 9 nm and 6 nm, respectively. Mesh of the SiO2 mask consists of 14066 domain elements and 1974 boundary elements. Figure 3 demonstrates the comparison of stress distribution for Ge grown on the patterned GoS substrate between Ref. 26 and our results. It can be seen that the overall agreement of stress distribution is achieved. There are only small deviations in stress value between Fig. 3(a) and (b). For example, the maximum stress occurred in Ge near the top corner of the SiO2 mask (indicated by arrows in Fig. 3(a)) described in Ref. 26 is about ∼1.8×108 Pa, and the maximum stress we calculated is about ∼1.9×108 Pa. The comparison shows differences in the results within 5%. Thus, we can conﬁrm that the procedure and method used in our simulation is reliable. B. Thermal stress distribution of the laser array structure In this section we will discuss the thermal stress distribution on the laser array structure. The thermal stress distribution for the 2D model in Fig. 1 was calculated as shown in Fig. 4. The thermal stress values along the lines AB, BC, and CD in Fig. 4 are shown in Fig. 5. It is important to note that two different thermal stress values are obtained for point B and point C, since they are located in the interface of two different materials. It is found from Fig. 4 that the stress of most of the region in the V-groove below the InGaAs layer are in tensile (positive), since the thermal expansion coefﬁcient of InP is greater than that of Si. While the compressive stress (negative) is observed in a narrow region at the bottom of the V-groove (indicated by the arrow in Fig. 4). It has been found from our in-depth analysis that this is due to the extremely narrow width at the bottom of the V-groove. In addition, one can notice the stress at the top of the V-groove near the InGaAs layer changes from tension to compression along the upward direction of line AB, which is consistent with the observations of Lingunis et al.21 In their study for thermal stress in the square-patterned GaAs/Si structure, tensile stress in the middle of the GaAs layer and compressive stress in the top of the GaAs layer have been observed. Figure 5(a) shows the stress values alone line AB in the V-groove. The stress values of point A1 and A2 are 0. we can observe FIG. 2. Geometry of Ge grown laterally on the patterned GoS substrate (Ge on Si). FIG. 2. Geometry of Ge grown laterally on the patterned GoS substrate (Ge on Si). AIP Advances 8, 085007 (2018) 085007-5 Yin et al. ( ) FIG. 3. Comparison of the thermal stress distribution for Ge grown on the patterned GoS substrate between (a) Ref. 26 and (b) our results. 24 October 2024 04:45:05 IG. 3. Comparison of the thermal stress distribution for Ge grown on the patterned GoS substrate between (a) Ref. 26 and ) our results. hat the region between point A and point A1 is subjected to compression, and the compressive stress ecreases monotonically with the distance from point A. It is clear seen from Fig. 5(a) that thermal stresses are tensile between point A1 and A2. B. Thermal stress distribution of the laser array structure The tensile stress ﬁrst gradually increases until it reaches the maximum value about 78 MPa. This corresponds FIG. 4. Thermal stress distribution in a laser array structure with uncoalesced InGaAs and InP layers. FIG. 4. Thermal stress distribution in a laser array structure with uncoalesced InGaAs and InP layers. 085007-6 Yin et al. AIP Advances 8, 085007 (201 AIP Advances 8, 085007 (2018) 085007-6 Yin et al. 7-6 Yin et al. AIP Advance 6 Yin et al. AIP Advances 8, 0 FIG. 5. Thermal stress values calculated along (a) line AB (b) line BC and (c) line CD. ncrease of width from bottom to top of the V-groove. However, the stress th es, and ﬁnally changes from tension to compression. FIG 5 Th l t l l l t d l ( ) li AB (b) li BC d ( ) li CD FIG. 5. Thermal stress values calculated along (a) line AB (b) line BC and (c) line CD. to the increase of width from bottom to top of the V-groove. However, the stress then graduall decreases, and ﬁnally changes from tension to compression. y g p In the InGaAs active layer, the region near the V-groove is subject to tension, whereas the region ear the InP cap layer is subject to compression (see Fig. 4). More detail can be seen in Fig. 5(b), 085007-7 Yin et al. AIP Advances 8, 085007 (2018) AIP Advances 8, 085007 (2018) which shows the stress values along line BC in InGaAs active layer. The stress value of point B1 in Fig. 5(b) is 0. It can be seen that the region between point B and B1 is subjected to tension and the tensile stress decreases monotonically with the distance from point B. While the region between point B1 and C is subjected to compression and the compressive stress increases monotonously as the distance from point B1 increases. p Most of the region of the InP cap layer (except for the corner) is in compression (see Fig. 4). The stress values along line CD in InP layer can be seen in Fig. 5(c). The compressive stress along line CD increases monotonously as the distance from point C increases. It has been found from our in-depth analysis that the distribution of the compressive stress along the line CD is affected by the ratio of the InP cap layer thickness to the InGaAs layer thickness. B. Thermal stress distribution of the laser array structure In fact, the stress of the ﬁlm is greatly affected by the neighbors. If the ﬁlm is thin compared to the neighbors, the stress of the ﬁlm caused by the differential thermal expansion of the neighbors can be very large, and the change behavior of the stress is different. In this case, the thickness of the InP cap layer (20 nm) is lower than that of the InGaAs layer (60nm), the stress along line CD increases monotonously with the thickness and the maximum stress is up to 92MPa. In contrast, the stress along line CD decrease monotonically with the thickness, and the minimum stress is only 15MPa, while the thickness of the InP cap layer is larger than that of the InGaAs layer. The stress of the InP cap layer changes little, when the thickness of the InP cap layer is equal to the InGaAs layer thickness. According to the above description, we know that the top of the InGaAs layer and most of the region of the InP cap layer are subjected to compression, which is different from the stress distribution in planar structures.25,26 For example, Ge epitaxial layer is subjected to tension in planar Ge/Si structure (Ge grown on Si without the SiO2 mask).26 We consider two probable mechanisms responsible for this. The ﬁrst mechanism is that the width of the InGaAs and InP layers are smaller than that of the substrate owing to the fact that the InGaAs layer and the InP cap layer here are not coalesced. The second mechanism is that the thermal stress distribution of the InGaAs and InP layers is inﬂuenced by the V-groove structure. 24 October 2024 04:45:05 y g To explore the ﬁrst mechanism, a laser with coalesced InGaAs and InP layers is modeled, and the results of stress distribution are shown in Fig. 6. Apparent tensile stress can be observed both in InGaAs active layer and InP cap layer. The big difference of stress distribution in InGaAs and InP layers in Fig. 4 and Fig. 6 reveals that whether or not the epitaxial layers are coalesced has great inﬂuence on the stress distribution. This is agreement with the ﬁrst mechanism. We will discuss the effect of V-groove parameters on the thermal stress in the following section, so the analysis of the second mechanism will also be carried out in the following section. B. Thermal stress distribution of the laser array structure Previous experimental results showed that the quality of epitaxial layers are signiﬁcantly inﬂu- enced by the parameters of the V-groove structure. For example, the deeper “V-groove” might induce “aspect ratio trapping” of defects in the epitaxial layer.19,27 However, few papers have studied the FIG. 6. Thermal stress distribution in laser structure with coalesced InGaAs and InP layers. FIG. 6. Thermal stress distribution in laser structure with coalesced InGaAs and InP layers. 085007-8 Yin et al. AIP Advances 8, 085007 (201 085007-8 Yin et al. AIP Advances 8, 085007 (2018) AIP Advances 8, 085007 (2018) Yin et al. 085007-8 effect of V-groove structural parameters on thermal stress distribution. Therefore, it is necessary to study the effect of the parameters of V-groove on thermal stress. Factors such as the height of the V-groove, the height and the width of the SiO2 mask were studied. g , g 2 G. 7. Thermal stress values calculated along (a) line AB (b) line BC and (c) line CD with different heights of the V-groove. IG. 7. Thermal stress values calculated along (a) line AB (b) line BC and (c) line CD with different heights of the V-groove. 085007-9 Yin et al. 085007-9 AIP Advances 8, 085007 (2018) Figure 7 shows the effect of the height of the V-groove on the thermal stress. We can observe that the stress value at point A changes little at different V-groove height (as indicated by the arrow in Fig. 7(a)). The tensile stress increases as the height of the V-groove increases. This is consistent with the variation of stress distribution in the V-groove we described above. G. 8. Thermal stress values calculated along (a) line AB (b) line BC and (c) line CD with different heights of the SiO sk. FIG. 8. Thermal stress values calculated along (a) line AB (b) line BC and (c) line CD with different heights of the SiO2 mask. 085007-10 Yin et al. AIP Advances 8, 085007 (2018) The stress state at point B changing from compression to tension with the increase of the V-groove height can be noticed in Fig. 7(b), and the stress value at point B also changes from 22 MPa (negative) at the V-groove height of 150 nm to 20 MPa (positive) at the V-groove height of 350 nm. B. Thermal stress distribution of the laser array structure The change of the stress state at point B indicates that the height of the V-groove has groove height can be noticed in Fig. 7(b), and the stress value at point B also changes from MPa (negative) at the V-groove height of 150 nm to 20 MPa (positive) at the V-groove height 350 nm. The change of the stress state at point B indicates that the height of the V-groove has G. 9. Thermal stress values calculated along (a) line AB (b) line BC and (c) line CD with different widths of the SiO2 mask. al stress values calculated along (a) line AB (b) line BC and (c) line CD with different widths FIG. 9. Thermal stress values calculated along (a) line AB (b) line BC and (c) line CD with different widths of the SiO2 mask. 085007-11 Yin et al. AIP Advances 8, 085007 (2018) great inﬂuence on the thermal stress distribution of the InGaAs active layer, which is consistent with our second mechanism. In addition, a special point B3 can be observed (indicated by the arrow in Fig. 7(b)). The stress value at point B3 is constant at different V-groove heights. Moreover, we can notice that the change behavior of thermal stress on both side of point B3 is different. So far, no suitable reason has been found to explain this special point, and further studies are required to clarify this question. q Figure 7(c) shows the evolution of stress values along line CD at different heights of V-groove. It can be seen that increasing the height of the V-groove resulted in enhanced thermal stress. In addition, when the height of the V-groove is so small, the stress along line CD does not increase monotonically with the distance from point C and the stress changes little. However, as the height of the V-groove increases, the stress along line CD increases monotonously with the distance from point C. In fact, in this case, the V-groove acts as the virtual substrate. The height of the substrate has great inﬂuence on the stress distribution of the InP cap layer. Figure 8 shows the effect of the SiO2 mask height on the thermal stress. It can be seen from Fig. 8(a) that the compressive stress at point A increases with the height of the SiO2 mask. However, we can notice from the stress distribution above the dash-dot line in Fig. B. Thermal stress distribution of the laser array structure 8(a) that the tensile stress decreases as the height of the SiO2 mask increases. Figure 8(b) shows the stress distribution along line BC for various heights of the SiO2 mask. It is important to point out that the stress state at point B changes from tension to compression, and the stress value changes from 20 MPa (positive) at the SiO2 mask height of 80nm to1.5 MPa (negative) at the SiO2 mask height of 240 nm. This is also consistent with our second mechanism. Moreover, we observe that as the height of the SiO2 mask increases, the height of the SiO2 mask has little effect on the stress distribution along line BC. The maximum stress variation is only 1.5 MPa when the SiO2 mask height changes from 200nm to 240nm. 24 October 2024 04:45:05 Figure 8(c) shows that the compressive stress along line CD increases as the height of the SiO2 mask is increased. However, the increase in stress become smaller with the increase of the SiO2 mask height. When the SiO2 mask height is 200 nm and 240 nm, the two curves are so close to each other. Finally, we study the effect of the SiO2 mask width on thermal stress along lines AB, BC and CD. The results are shown in Figure 9. It can be seen that the width of the SiO2 mask has little effect on the thermal stress. Maximum variation occurs in the point B in InGaAs layer with a variation of about 13 MPa. Consequently, according to the calculation, the SiO2 mask is not expected to hinder the relief of the stress. IV. SUMMARY AND OUTLOOK In summary, we performed thermal stress analysis on a laser array structure selectively grown on the V-groove-patterned substrate by ﬁnite-element method. A patterned substrate structure was ﬁrst modeled to examine the reliability of the procedure and method used in simulation. Next, we investigated the thermal stress distribution in a laser array structure consist of the V-groove InP/Si substrate, the unpatterned InGaAs active layer and InP cap layer. Unexpected results are observed that the top of the InGaAs active layer and the most region of the InP cap layer are in compression, which is different from the thermal stress distribution in planar structures. Two mechanisms were proposed and modeled to explain this result. On the one hand, the width of uncoalesced layers is smaller than that of the Si substrate, which causes thermal stress to change in epitaxial layers. On the other hand, stress in the InGaAs and InP layers is affected by the V-groove structure. The results show that whether or not the epitaxial layers are coalesced has signiﬁcant effect on the thermal stress distribution and the height of V-groove and SiO2 mask has great inﬂuence on the thermal stress distribution. Although we have conducted a detailed study for these differences, we acknowledge that these mechanisms have so far only scratched the surface of these differences and more details are needed to be further studied in the future. Various factors including the height of the V-groove, the height and the width of the SiO2 mask which may affect thermal stress distribution were also studied. The results indicated that the height of V-groove and SiO2 mask has signiﬁcant effect on the stress. These ﬁndings are important for the design of laser arrays and provide an important step towards the realization of photonic integration circuits on silicon. 085007-12 Yin et al. AIP Advances 8, 085007 (2018) AIP Advances 8, 085007 (2018) AIP Advances 8, 085007 (2018) ACKNOWLEDGMENTS This work was supported by the Fund of State Key Laboratory of Information Photonics and Optical Communications (Beijing University of Posts and Telecommunications), the National Natural Science Foundation of China (Grant Nos. 61574019, 61474008, 61674020), the International Science & Technology Cooperation Program of China (Grant 2011DFR11010), the Doctoral Program of Higher Specialized Research Fund (Grant 20130005130001), and the 111Project of China (Grant B07005). 1 M. Yamaguchi, Y. Ohmachi, T. Oh’hara, Y. Kadota, M. Imaizumi, and S. 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https://openalex.org/W2116144159	https://digital.csic.es/bitstream/10261/225098/1/lattices.pdf	English	null	Lattice modulation spectroscopy of strongly interacting bosons in disordered and quasiperiodic optical lattices	Physical review. A, Atomic, molecular, and optical physics/Physical review, A, Atomic, molecular, and optical physics	2,009	cc-by	9,439	I. INTRODUCTION signatures of the theoretically predicted transition from a su- perﬂuid to the Bose-glass phase 19–21. A pioneering step in this direction was recently taken by the Florence group by using lattice modulation spectroscopy 11. This technique consists in heating an ultracold gas loaded in an optical lat- tice by periodically modulating the depth of the lattice 22–24. When perturbed in this way, the gas is driven out of equilibrium and absorbs energy. When the perturbation is switched off, and after rethermalization, the broadening of the momentum distribution around zero momentum is taken as a measure of the energy absorbed by the system during the lattice modulation 22. On the theory side, for nondisor- dered lattices, the calculation of the energy absorption rate due to the lattice modulation was ﬁrst performed analytically within linear-response theory by some of the present authors 23. These results were conﬁrmed and extended beyond lin- ear response using time-dependent density-matrix renormalization-group methods, both for the case of bosons 24 and fermions 25. More recently, for disordered optical lattices, the energy absorption rate has been numerically cal- culated using full diagonalization in small systems 26,27. Other methods that have also been discussed in the literature for detecting signatures of the effect of disorder or quasip- eriodicity on interacting boson systems in one dimension focus on the momentum distribution 16,28, the expansion dynamics in quasiperiodic potentials 15, and the dipole os- cillations in the presence of defects 29. In recent years, developments in the ﬁeld of ultracold atomic gases have considerably enlarged the possibilities for exploring the physics of strongly correlated systems 1. For instance, the study of quantum phase transitions, a subject of continuous theoretical interest, has been strongly stimulated by the experimental observation of the superﬂuid to Mott insulator transition using optical lattices 2. Indeed, ultra- cold atom systems offer us an unprecedented control over the system parameters, which can allow us to ultimately under- stand the physics of very hard problems such as the phase diagram of the Hubbard model in two dimensions 3. A particularly interesting and fertile arena is the study of disordered ultracold atoms. Random potentials can be intro- duced in a controlled way by laser beams generating speckle patterns 4–9 or by loading in an optical lattice a mixture of two kinds of atoms, one heavy and one light. DOI: 10.1103/PhysRevA.80.033625 DOI: 10.1103/PhysRevA.80.033625 PHYSICAL REVIEW A 80, 033625 2009 PHYSICAL REVIEW A 80, 033625 2009 Lattice modulation spectroscopy of strongly interacting bosons in disordered and quasiperiodic optical lattices G. Orso,1 A. Iucci,2,3 M. A. Cazalilla,4,5 and T. Giamarchi3 1Laboratoire Physique Théorique et Modèles Statistiques, Université Paris Sud, Bat. 100, 91405 Orsay Cedex, France nstituto de Física la Plata (IFLP)–CONICET and Departamento de Física, Universidad Nacional de La Plata, CC 67, 1900 La Plata Argentina 3 3DPMC-MaNEP, University of Geneva, 24 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland 4Centro de Física de Materiales (CSIC-UPV/EHU), Ediﬁcio Korta, Avenida de Tolosa 72, 20018 San Sebastián, Spain 5Donostia International Physics Center (DIPC), Manuel de Lardizábal 4, 20018 San Sebastián, Spain Received 21 July 2009; published 30 September 2009 3DPMC-MaNEP, University of Geneva, 24 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland 4Centro de Física de Materiales (CSIC-UPV/EHU), Ediﬁcio Korta, Avenida de Tolosa 72, 20018 San Sebastián, Spain 5Donostia International Physics Center (DIPC), Manuel de Lardizábal 4, 20018 San Sebastián, Spain Received 21 July 2009; published 30 September 2009 We compute the absorption spectrum of strongly repulsive one-dimensional bosons in a disordered or quasiperiodic optical lattice. At commensurate ﬁlling, the particle-hole resonances of the Mott insulator are broadened as the disorder strength is increased. In the noncommensurate case, mapping the problem to the Anderson model allows us to study the Bose-glass phase. Surprisingly, we ﬁnd that a perturbative treatment in both cases, weak and strong disorders, gives a good description at all frequencies. In particular, we ﬁnd that the infrared-absorption rate in the thermodynamic limit is quadratic in frequency. This result is unexpected since for other quantities, like the conductivity in one-dimensional systems, perturbation theory is only applicable at high frequencies. We discuss applications to recent experiments on optical lattice systems and, in particular, the effect of the harmonic trap. PACS numbers: 03.75.Lm, 71.23.k, 61.44.Fw PACS numbers: 03.75.Lm, 71.23.k, 61.44.Fw ©2009 The American Physical Society II. ENERGY ABSORPTION: LINEAR-RESPONSE THEORY For weak perturbations, corresponding to J J, the en- ergy absorption rate E˙ can be calculated using the linear- response theory. The general formula has been ﬁrst derived in Ref. 23 and in the presence of disorder it takes the form In the thermodynamic limit, both the number N of bosons and the length M of the chain diverge. The ratio =N/M is instead ﬁnite and corresponds to the ﬁlling factor, i.e., the average number of bosons per lattice site. We must distin- guish between two physically different situations. For incom- mensurate ﬁllings 1, the system has gapless excitations. Thus, a good approximation to the absorption rate at fre- quencies U can be obtained by formally taking U→+ and mapping the resulting hard-core bosons to noninteracting fermions using a Jordan-Wigner transformation see, e.g., Ref. 30. The resulting single-particle problem can be eas- ily solved for a given choice of i and Vho and the energy absorption obtained. On the other hand, for unit ﬁlling =1, the ground state is a Mott insulator with a gap. In this case, the elementary excitations are particles and holes 23 and, in a homogeneous system, the absorption can only occur at frequencies U. In the absence of disorder, the response of the system to the lattice modulation has been computed using degenerate perturbation theory within the subspace of particle and hole excitations 23. The same methods can be generalized to deal with disordered case, as shown below. E˙ = 1 2J0 2 Im−K, 4 4 where K is the Fourier transform of the retarded corre- lation function Kt=−itKt,K0 of the hopping operator K=−jbj+1 † bj+H.c., being t the step function. In Eq. 4, the bar means average over different disorder Sec. IV or quasiperiodic Sec. III realizations. In general, the calculation of the correlation function in Eq. 4 is a complicated many-body problem. However, in the limit of strong repulsion where U J,, calculations are considerably simpliﬁed by the fact that we can accurately restrict ourselves to work within a subspace of the total Hil- bert space, whose detailed structure depends on the ﬁlling. In the case of an incommensurate ﬁlling i.e., 1, this sub- space can be described in terms of noninteracting fermion states that is, Slater determinants. II. ENERGY ABSORPTION: LINEAR-RESPONSE THEORY For commensurate ﬁlling i.e., unit ﬁlling, =1, we can restrict ourselves to the sub- space with one particle and one hole excitation, as described below. The resulting general picture of the energy absorption is depicted in Fig. 1. The low frequency 0 W, where W max4J,2 is the effective bandwidth, is only present for incommensurate ﬁllings, 1. A second distribution is cen- tered at frequency =U and comes from particle-hole exci- tations generating doubly occupied sites. The width of this absorption line is also given by W. I. INTRODUCTION In this work, we extensively compare two cases aj uniformly distributed in−,, 2 bj = cos2j, irrational, 3 FIG. 1. Sketch of the full absorption spectrum at incommensu- rate ﬁlling. Here, Wmax4J,2 is the effective bandwidth. The peak at U stems from particle-hole excitations, whereas the low-frequency absorption appears as a consequence of the forma- tion of a Bose glass at incommensurate ﬁlling. 2 3 bj = cos2j, irrational, where measures the strength of the disorder. From the experimental point of view, speckle patterns and lattice con- taining heavy atom impurities can be modeled by case a, whereas quasiperiodic potentials obtained by superimposing two optical potentials with incommensurate periodicities d1/d2= are described by case b. The paper is organized as follows. In Sec. II, we present the general formalism needed to calculate the absorption rate for incommensurate and unit ﬁlling. In Secs. III and IV, we present our analytical and numerical results obtained for the random and the quasiperiodic potential, respectively. In Sec. V, we discuss the effects of a trapping potential. Finally in Sec. VI, we provide our conclusions. A derivation of the general formula see Eq. 9 for the absorption rate valid for weak disorder is given in the Appendix. In this work, we assume that the hopping amplitude J in Eq. 1 is modulated periodically in time according to Jt =J+J cos t, where is the modulation frequency. We cal- culate the energy absorption rate within linear-response theory, which is valid for weak lattice modulations J J. We shall restrict ourselves to the strongly repulsive re- gime of Eq. 1, where the on-site repulsion is large com- pared to both the hopping amplitude and the disorder strength U J,. We ﬁrst discuss systems in the thermo- dynamic limit by setting Vj ho=0. Effects of the harmonic trapping potential will be discussed later, in Sec. V. We also set =1 to simplify the notation. I. INTRODUCTION When the heavy atoms become randomly localized in the lattice, they will act as impurities for the lighter atoms 10. Another available technique is to superimpose two optical lattices with incommensurate periodicities, thus, generating a quasi- periodic potential 11. The quasiperiodic lattices as well as the speckle patterns have been recently used in the experi- mental efforts to observe the effects of Anderson localization in dilute Bose gases expanding in highly elongated traps 12,13. Since Anderson localization is a single-particle ef- fect, the next logical step is to study the interplay of disorder and interactions in strongly interacting ultracold atomic sys- tems 14–17. The latter may be accessible by tuning inter- particle interactions using Feshbach resonances 18 or by loading the atoms in sufﬁciently deep optical lattices 9 and/or strongly conﬁning them to low dimensions in tight traps 1. Let us consider a one-dimensional disordered Bose gas described by the following Hamiltonian: H = −J j bj+1 † bj + H.c. + U 2 j njnj −1 + j j + Vj honj, 1 1 where bj denotes the boson annihilation operator at sites j, nj=bj †bj being the local density. Here J and U are the usual where bj denotes the boson annihilation operator at sites j, nj=bj †bj being the local density. Here J and U are the usual In the context of the efforts described above, one of the experimental challenges is to observe in ultracold gases clear 033625-1 1050-2947/2009/803/03362510 ©2009 The American Physical Society PHYSICAL REVIEW A 80, 033625 2009 ORSO et al. 0 W W U ω F FIG. 1. Sketch of the full absorption spectrum at incommensu- rate ﬁlling. Here, Wmax4J,2 is the effective bandwidth. The peak at U stems from particle-hole excitations, whereas the low-frequency absorption appears as a consequence of the forma- tion of a Bose glass at incommensurate ﬁlling. parameters of the Bose-Hubbard model corresponding to the tunneling rate and the on-site repulsion U0. The last term in the right-hand side of Eq. 1 accounts for the pres- ence of both the harmonic trap Vj ho and the disorder potential j. 0 W W U ω F j The distribution of the on-site energies j in Eq. 1 de- pends on the speciﬁc choice of the random or pseudoran- dom potential. B. Unit ﬁlling K = j j + 1j + jj + 1 2. 8 We next turn our attention to the commensurate case with =1. In the strong-coupling regime U J,, the system is deep in the Mott insulator phase, corresponding to exactly one particle per site. In Ref. 23, it was shown that for clean systems the absorption rate is zero at low frequencies and exhibits a narrow peak of width J centered about =U. In this section, we consider the broadening of such peak due to a disorder or quasiperiodic potential i. 8 In the previous expressions and j are the eigenvalues and eigenfunctions of H, respectively. In Eq. 7, f =exp−/T+1−1 is the Fermi-Dirac distribution func- tion at a temperature T and chemical potential . The latter is ﬁxed by the normalization condition =f. At zero temperature, the only relevant processes in Eq. 7 corre- spond to transitions from an occupied level with energy T=0 to an unoccupied level T=0. In par- ticular, for unit ﬁlling the absorption 7 vanishes, consis- tently, with the fact that a Mott insulator can only absorb at much higher frequencies U. In the previous expressions and j are the eigenvalues and eigenfunctions of H, respectively. In Eq. 7, f =exp−/T+1−1 is the Fermi-Dirac distribution func- tion at a temperature T and chemical potential . The latter is ﬁxed by the normalization condition =f. At zero temperature, the only relevant processes in Eq. 7 corre- spond to transitions from an occupied level with energy T=0 to an unoccupied level T=0. In par- ticular, for unit ﬁlling the absorption 7 vanishes, consis- tently, with the fact that a Mott insulator can only absorb at much higher frequencies U. In order to obtain the energy absorption rate within linear response, we ﬁrst use the spectral decomposition of the cor- relation function K in terms of the exact eigenstates of the unperturbed Hamiltonian. This yields the following ex- pression for the energy absorption: E˙ = J0 2 2 n nK02 + E0 −En, 11 In the absence of disorder i.e., for i=0 in Eq. 6, the hopping modulation commutes with the Hamiltonian H and, therefore, the absorption rate vanishes to all orders, even beyond linear response. A. Incommensurate ﬁlling For ﬁlling 1 and large on-site repulsion J, U, we take the hard-core limit U→+. Bosons are then mapped onto noninteracting spinless fermions via the Jordan-Wigner transformation, 033625-2 LATTICE MODULATION SPECTROSCOPY OF STRONGLY… PHYSICAL REVIEW A 80, 033625 2009 cj = exp i k=1 j−1 nk bj, 5 From Eq. 8, we ﬁnd Kjj=1 if j and j are nearest neighbor and zero otherwise. The absorption rate 7 thus reduces to 5 E˙ = J0 2 2 r=1 j=0 L−1 fj −fj+r + j −j+r. where cj satisfy fermionic commutation relations cj,cj=0 and cj,cj †=1. Under the above transformation, the Hamil- tonian 1 is mapped onto the single-particle Hamiltonian j 10 10 H = −J j cj+1 † cj + H.c. + j jcj †cj, 6 6 In Sec. III, we shall explicitly compare the results obtained using exact numerical diagonalization with the above results obtained both in the limit of weak 9 and strong 10 disor- ders. Finally, it is important to emphasize that Eq. 7 does not account for particle-hole excitations, which are relevant at much higher frequencies U. These excitations become particularly important at unit ﬁlling =1, when the strongly repulsive Bose gas becomes a Mott insulator and the absorp- tion at low frequency U predicted by Eq. 7 vanishes because there are no empty sites i.e., holes in the ground state. The contribution to the absorption from particle-hole excitations at unit ﬁlling will be discussed next. where the on-site repulsion U has disappeared and the hop- ping operator K in Eq. 4 is now given by K=−jcj+1 † cj +H.c.. Notice that the mapping of observables that are non- local in space is far less trivial, an example is the momentum distribution studied in Ref. 28 for disordered hard-core bosons in one dimension. After some algebra, the absorbtion rate 4 becomes E˙ = J0 2 2 , Kf −f + −, 7 where the matrix K is deﬁned as where the matrix K is deﬁned as B. Unit ﬁlling In the above expression, this is re- ﬂected in that, for i=0, the eigenstates of H become plane waves, kjeikj, with energy dispersion k=−2J cos k k being the lattice momentum. Thus, the matrix 8 is diago- nal, i.e., Kkk=4k,kcos2 k, which, together with the factor fk−fk in Eq. 7 makes E˙ vanish. 11 where n are the eigenstates of the original Hamiltonian 1 with energies En, and 0=1,1, ... ,1 is the ground state in the Fock representation corresponding to one boson per lattice site. The low-energy states are particle-hole exci- tations m, j= 1 2bm † bm+j0 corresponding to double oc- cupation at site m and an empty site m+ j. These excitations are all degenerate with energy U in the absence of tunneling and i.e., for =J=0. At weak disorder i.e., J , the absorption rate 7 can be evaluated using the perturbation theory the details can be found in the Appendix, which yields For ﬁnite values of and J, the eigenstates n can be calculated using degenerate perturbation theory by writing n=m,jfm,jm, j, where the coefﬁcients satisfy E˙ = J0 2 2M k,k Vk−k2 J2 fk −fk + k −k, 9 m,j m,jHm,jfm,j = Efm,j. 12 12 where Vk= 1 Mj=0 M−1eikjj is the Fourier transform of the dis- order potential. Equation 9 shows that the perturbation ex- pansion in disorder is well deﬁned provided the Fourier transform Vk is ﬁnite. The above matrix element and the energy absorption can be computed by taking into account that m, jK0= −2j,1, within the subspace containing just one particle and one hole. Hence, the matrix elements in Eq. 11 corre- spond to nK0=−m,r=12fm,r. Notice that a similar technique has been recently employed in Ref. 31 to calcu- Let us ﬁnally consider the so-called atomic limit, which corresponds to J yet U. In this limit, tunneling can be neglected and the eigenstates are given by mj=jm. 033625-3 PHYSICAL REVIEW A 80, 033625 2009 ORSO et al. III. RESULTS FOR A DISORDER POTENTIAL late the dynamic structure factor of a one-dimensional Mott insulator in a parabolic trap. In this section, we assume that the on-site energy i in Eq. 1 are random numbers uniformly distributed within the in- terval −,. The absorption rate 4 can thus be conve- niently recast as In the “atomic limit” where the tunneling can be ne- glected, Eq. 11 simpliﬁes considerably. Localized particle- hole excitations become the exact eigenstates n =m, j with energy E0+U+m−m+j, where E0=i=1 M i is the energy of the ground state for a given realization of i. The absorption rate 11 then takes the form E˙ = MJ0 2F, 14 14 where F is a dimensionless function that can be numerically evaluated. The results for incommensurate and commensu- rate cases are described below. E˙ = J0 2 r=1 m=1 M −U + m −m+r, 13 13 B. Unit ﬁlling 0 0.0005 0.001 0.0015 0 1 2 3 4 5 F ω/J ∆= 0.1J ν = 0.1 ν = 0.2 ν = 0.3 ν = 0.4 ν = 0.5 0 0.05 0.1 0.15 0 1 2 3 4 5 F ω/J ∆= J ν = 0.1 ν = 0.2 ν = 0.3 ν = 0.4 ν = 0.5 0 0.2 0.4 0.6 0.8 0 5 10 F ω/J ∆= 5J ν = 0.1 ν = 0.2 ν = 0.3 ν = 0.4 ν = 0.5 (b) (a) (c) FIG. 2. Color online Energy absorption rate of hard-core bosons in a random potential: the response function F see Eq. 14 is plotted versus modulation frequency for different ﬁlling factors and increasing values of disorder strength: =0.1J top panel, = J, and =5J. Calculations are done on a ring of M=500 lattice sites yielding negligible ﬁnite-size effects. The number of disorder realizations used was Nr=500. 0 0.0005 0.001 0.0015 0 1 2 3 4 5 F ω/J ∆= 0.1J ν = 0.1 ν = 0.2 ν = 0.3 ν = 0.4 ν = 0.5 0 0.05 0.1 0.15 0 1 2 3 4 5 F ω/J ∆= J ν = 0.1 ν = 0.2 ν = 0.3 ν = 0.4 ν = 0.5 (b) (a) (a) 0 0.2 0.4 0.6 0.8 0 5 10 F ω/J ∆= 5J ν = 0.1 ν = 0.2 ν = 0.3 ν = 0.4 ν = 0.5 (c) (c) (c) FIG. 2. Color online Energy absorption rate of hard-core bosons in a random potential: the response function F see Eq. 14 is plotted versus modulation frequency for different ﬁlling factors and increasing values of disorder strength: =0.1J top panel, = J, and =5J. Calculations are done on a ring of M=500 lattice sites yielding negligible ﬁnite-size effects. The number of disorder realizations used was Nr=500. A. Incommensurate ﬁlling 18, the following low-frequency behavior is obtained: y =2J1−2/4J2−1, we ﬁnd F = 2 24J4 a b d 1 −2/4J21 − + 2/4J2 , 16 F = 42min¯ , − −max−,¯ −. 19 19 16 This behavior is exhibited by the numerics, as shown in Fig. 2 lower panel, see also discussion further below. Further- more, in the low-frequency limit 19, we again recover the quadratic behavior described above on general grounds where a=max−2J,− and b=min,2J−. Here the chemical potential is related to the ﬁlling factor by = −2J cos . By expanding Eq. 16 at low frequencies, we again obtain that the quadratic behavior discussed above F = 2 6J2 22 = 2 24J4 2 sin2 . 17 F = 422. 20 20 17 Notice however that the proportionality constant is now in- dependent of the ﬁlling factor. In Fig. 4, a more detailed comparison of the numerics with the analytical result of Eq. It should be noticed that the right-hand side of Eq. 17 di- verges in the limit of vanishing lattice ﬁlling →0 because the density of states has a Van Hove singularity at zero energy in one dimension. Thus, the low ﬁlling limit, the qua- dratic behavior of Eq. 17 is only recovered at increasingly low frequencies, as shown in Fig. 2 upper panel. 0 50 100 150 200 ω/J 0 0.2 0.4 0.6 0.8 1 F ν=0.3 ν=0.5 0 200 0.03 FIG. 4. Color online Comparison between numerics symbols and analytics solid line, Eq. 19 for strong disorder. The value of the disorder is /J=100 and the ﬁlling factors are =0.3 and =0.5. The length of the chain is M=2000 and the number of disor- der realizations is Nr=2000. In the inset, we compare our numerics with the quadratic expansion 20 expected at low frequency. In both cases, the agreement is quite good. 0 50 100 150 200 ω/J 0 0.2 0.4 0.6 0.8 1 F ν=0.3 ν=0.5 0 200 0.03 In Fig. 3, we show a comparison, in the limit of weak disorder =0.01J, of the numerical results open symbols obtained using exact diagonalization with the analytical ex- pression of Eq. 16 continuous lines. The agreement is indeed very good over the entire frequency range. In the inset, it is demonstrated that numerical results are consistent with the quadratic behavior of Eq. 17, expected at low fre- quencies. A. Incommensurate ﬁlling 7 become irrel- evant, and thus any dependence on the value chemical po- tential disappears. However, at low frequencies, the absorption rate vanishes quadratically with frequency for any strength of the dis- order. To understand this, let us expand in Eq. 7 the distri- bution functions f−f−f. Taking into account that, at zero temperature, f=−−, we ﬁnd that FC2, where the constant C = 2 K − − 15 15 FIG. 3. Color online Comparison between numerics symbols and analytics Eq. 9, solid line for weak disorder. Here, /J =0.01 and we consider two ﬁlling factors =0.3 and =0.5. The length of the chain is M=2000 and the number of disorder realiza- tions is Nr=2000. The inset is a zoom of the low-frequency regime, where the absorption rate is quadratic in frequency as given by Eq. 17. FIG. 3. Color online Comparison between numerics symbols and analytics Eq. 9, solid line for weak disorder. Here, /J =0.01 and we consider two ﬁlling factors =0.3 and =0.5. The length of the chain is M=2000 and the number of disorder realiza- tions is Nr=2000. The inset is a zoom of the low-frequency regime, where the absorption rate is quadratic in frequency as given by Eq. 17. FIG. 3. Color online Comparison between numerics symbols and analytics Eq. 9, solid line for weak disorder. Here, /J =0.01 and we consider two ﬁlling factors =0.3 and =0.5. The length of the chain is M=2000 and the number of disorder realiza- tions is Nr=2000. The inset is a zoom of the low-frequency regime, where the absorption rate is quadratic in frequency as given by Eq. 17. can be evaluated numerically. From Eq. 15, it can be seen that the constant C is nonzero provided there are nonvanish- ing matrix elements K connecting two states and at the Fermi level ==. In the limit of weak disorder corresponding to J, the absorption can be evaluated using Eq. 9, where Vk2 =2/3M, as follows from the expression for the Fourier transform of the disorder potential Vk. Going to the thermo- dynamic limit and introducing the density of states and density of states, respectively. In the random potential, the latter is constant and given by ¯=1/2, and therefore ¯ =2−1. Using Eq. A. Incommensurate ﬁlling which can be readily evaluated numerically once the disorder potential is known. We emphasize that Eq. 13 assumes that the system is a Mott insulator with exactly one atom per site. This condition is easily satisﬁed in the strong-coupling re- gime J, U, where the disorder only broadens the absorp- tion spectrum without affecting the nature of the ground state. A ﬁnite response at low frequency only occurs for stronger U, where the system is close to the Bose-glass transition. In this limit, however, the random or quasiperi- odic potential changes the occupation numbers at different sites, creating holes and doublons, so the ground state is no longer given by the Fock state 0 and Eq. 13 ceases to be valid. As stated above, the absorption rate is calculated numeri- cally at zero temperature starting from Eqs. 7 and 8. In Fig. 2, we plot the frequency dependence of the response function F for different ﬁllings and increasing values of dis- order =0.1J upper panel, =J central panel, and =5J lower panel. Since, in the fermionic representation, the Hamiltonian of Eq. 6 is particle-hole symmetric, the ab- sorption rate in Eq. 7 is unchanged under the transforma- tion →1−, so we restrict our discussion to ﬁllings 1/2. A noticeable feature of Fig. 2 is that the response at high frequencies is independent of the ﬁlling factor. In this limit, the relevant processes contributing to the absorption mainly 033625-4 PHYSICAL REVIEW A 80, 033625 2009 LATTICE MODULATION SPECTROSCOPY OF STRONGLY… 0 1 2 3 4 ω/J 0 5e−06 1e−05 1.5e−05 2e−05 ν=0.5 ν=0.3 0 0.1 0.2 0 5e−08 1e−07 FIG. 3. Color online Comparison between numerics symbols and analytics Eq. 9, solid line for weak disorder. Here, /J =0.01 and we consider two ﬁlling factors =0.3 and =0.5. The length of the chain is M=2000 and the number of disorder realiza- tions is Nr=2000. The inset is a zoom of the low-frequency regime, where the absorption rate is quadratic in frequency as given by Eq. 17. 0 1 2 3 4 ω/J 0 5e−06 1e−05 1.5e−05 2e−05 ν=0.5 ν=0.3 0 0.1 0.2 0 5e−08 1e−07 involve transitions from states far below the Fermi level i.e., into empty states far above it i.e., . As a result, the Fermi-Dirac distributions in Eq. B. Unit ﬁlling The absorption rate for =1 is calculated numerically starting from Eqs. 11 and 12. The result is shown in Fig. 5 as a function of frequency for different values of the dis- order strength and J=0.01U. In Fig. 6 we plot the frequency dependence of the re- sponse function 14 for increasing values of the disorder strength . Compared to Fig. 2, we see that the absorption spectra in quasiperiodic potential exhibit a much richer struc- ture. Interestingly, the response function becomes very large and actually diverges when matches special values. Moreover, for a given ﬁlling factor, the absorption rate is nonzero only within a certain range of frequencies. In the absence of disorder =0, the absorption rate can be evaluated analytically and the dimensionless function F in Eq. 14 is given by 23 F = 2 3J sin cos−1 −U 6J , for −U 6J, 21 These features can again be understood in the limits of weak and at strong disorder, where results can be obtained analytically. In the limit J, the absorption rate can be calculated starting from Eq. 9 and taking the thermody- namic limit. This yields k=−2J cos k 21 and vanishes otherwise. The dashed line in Fig. 5 corre- sponds to our numerical result for =0, which fully agrees with the above formula. F = 2 dk 2 dk 2 Vk −k2 J2 + k −k −kk −, 24 F = 2 dk 2 dk 2 Vk −k2 J2 + k −k As the strength of disorder is increased, we see from Fig. 5 that the absorption spectrum becomes broader and progres- sively develops a trianglelike shape as the limit J is ap- proached. This behavior can be obtained analytically starting from Eq. 13. In the J limit, since the on-site energies at different lattice sites are uncorrelated, we obtain −kk −, 24 24 where the integration over momenta is restricted to 0,2. Using that the modulus square of the Fourier transform of the disorder potential is Vk2=limM→ VMk2, where F 22 − d − d −U + −, 22 22 VMk2 = 2 4M eik+k0M −1 eik+k0 −1 + eik−k0M −1 eik−k0 −1 2 , 25 25 where ¯=1/2 is the disorder-averaged density of states in the atomic limit introduced above. Upon integration, Eq. IV. QUASIPERIODIC POTENTIAL In this section, we assume that the external potential in Eq. 1 is j= cos2j 15–17, being an irrational number. This quasiperiodic potential distribution is realized experimentally by superimposing two different periodic po- tentials with incommensurate lattice periods 11. Differently from the disorder potential considered in the previous sec- tion, where all states are localized in the thermodynamic limit for an arbitrarily small amount of disorder, in the qua- siperiodic case there is a phase transition 32 at c=2J: for 2J all states are extended while for 2J all states are exponentially localized. FIG. 5. Color online Energy absorption rate in the Mott insu- lator phase for hard-core bosons in a random potential: the response function F see Eq. 14 is plotted versus modulation frequency for ﬁxed J=0.01U and increasing values of the disorder strength /U=0black dotted line,0.03,0.06,0.1. The length of the chain is M=60 and the number of disorder realizations is Nr=200. A broadening of the absorption peak is observed for increasing disorder. FIG. 5. Color online Energy absorption rate in the Mott insu- lator phase for hard-core bosons in a random potential: the response function F see Eq. 14 is plotted versus modulation frequency for ﬁxed J=0.01U and increasing values of the disorder strength /U=0black dotted line,0.03,0.06,0.1. The length of the chain is M=60 and the number of disorder realizations is Nr=200. A broadening of the absorption peak is observed for increasing disorder. FIG. 5. Color online Energy absorption rate in the Mott insu- lator phase for hard-core bosons in a random potential: the response function F see Eq. 14 is plotted versus modulation frequency for ﬁxed J=0.01U and increasing values of the disorder strength /U=0black dotted line,0.03,0.06,0.1. The length of the chain is M=60 and the number of disorder realizations is Nr=200. A broadening of the absorption peak is observed for increasing disorder. As described below, we ﬁnd that the absorption spectra of bosons in the quasiperiodic potential are remarkably differ- ent from the spectra described in Sec. III for the bosons moving on a disorder potential. 19 for the limit of strong disorder is shown. In the inset, we also compare our numerical results with the quadratic behav- ior 20 expected at low frequency. In both cases, the agree- ment is remarkably good. A. Incommensurate ﬁlling For lattice ﬁllings 1 i.e., less than a boson per site, we calculate the absorption rate numerically starting from Eqs. 7 and 8. We restrict to zero temperature and ﬁx =0.771 452 45, which is relevant to the experiments carried out by the Florence group. A. Incommensurate ﬁlling On the other hand, in the opposite limit of strong disorder J, the tunneling can be neglected. In this limit, the ab- sorption rate can be evaluated directly from Eq. 10. Taking into account that the on-site energies j at different sites are completely uncorrelated, we ﬁnd FIG. 4. Color online Comparison between numerics symbols and analytics solid line, Eq. 19 for strong disorder. The value of the disorder is /J=100 and the ﬁlling factors are =0.3 and =0.5. The length of the chain is M=2000 and the number of disor- der realizations is Nr=2000. In the inset, we compare our numerics with the quadratic expansion 20 expected at low frequency. In both cases, the agreement is quite good. FIG. 4. Color online Comparison between numerics symbols FIG. 4. Color online Comparison between numerics symbols and analytics solid line, Eq. 19 for strong disorder. The value of the disorder is /J=100 and the ﬁlling factors are =0.3 and =0.5. The length of the chain is M=2000 and the number of disor- der realizations is Nr=2000. In the inset, we compare our numerics with the quadratic expansion 20 expected at low frequency. In both cases, the agreement is quite good. F = − ¯ d¯ ¯ d¯ + −, 18 18 where ¯ and ¯ are the disorder-averaged chemical potential 033625-5 PHYSICAL REVIEW A 80, 033625 2009 ORSO et al. 0.7 0.8 0.9 1 1.1 1.2 1.3 ω/U 0 20 40 60 80 F ∆=0U ∆=0.03U ∆=0.06U ∆=0.1U J=0.01U FIG. 5. Color online Energy absorption rate in the Mott insu- lator phase for hard-core bosons in a random potential: the response function F see Eq. 14 is plotted versus modulation frequency for ﬁxed J=0.01U and increasing values of the disorder strength /U=0black dotted line,0.03,0.06,0.1. The length of the chain is M=60 and the number of disorder realizations is Nr=200. A broadening of the absorption peak is observed for increasing disorder. 0.7 0.8 0.9 1 1.1 1.2 1.3 ω/U 0 20 40 60 80 F ∆=0U ∆=0.03U ∆=0.06U ∆=0.1U J=0.01U F 222 − −U, 23 23 showing that the line shape of the absorption rate in the atomic limit is approximately triangular and vanishes at −U=2 for J . LATTICE MODULATION SPECTROSCOPY OF STRONGLY… 24, we obtain F = 2 4J2 0 2 dk 2 + k −k+k0 −kk+k0 −. 27 27 F = 0 2 dy 2 + cos y − cosy + 2 The integral in Eq. 27 can be evaluated using the identity a sin k+b cos k=c cosk+, where c=a2+b2 and tan = −a/b. Since a=sin k0 and b=1−cos k0, we obtain c =2 sink0/2 and tan =−1/tank0/2. The zeroes of the function in Eq. 27 occur at k=k=−arccos/2Jc. Hence, we obtain − cos y + cos y −. 29 29 The integral in Eq. 29 becomes the integral in Eq. 27 after a change of variable k=y+. We thus obtain The integral in Eq. 29 becomes the integral in Eq. 27 after a change of variable k=y+. We thus obtain F = 2 4J2 /2 2Jc2 −2 r= −r + r, 28 F = /2 c2 −2 r= + cos kr − cos kr −, 28 where r=kr−=−2J cos kr−. Equation 28 shows that the absorption is ﬁnite only in a range of frequencies given by the conditions r 0 and r+0, where r itself de- pends on through the dependence of kr. Moreover, the response diverges for =2Jc, provided this frequency value is allowed for a given ﬁlling factor. Since c=2 sin =1.315 76, the divergence occurs at =2.631 5J, as found numerically and shown in Fig. 6 upper panel. 30 showing that the behavior of the absorption rate at strong quasiperiodic potential can be obtained from Eq. 28 by simply replacing 2J with . LATTICE MODULATION SPECTROSCOPY OF STRONGLY… PHYSICAL REVIEW A 80, 033625 2009 0 1 2 3 4 ω/J 0 0.001 0.002 0.003 0.004 0.005 F ν=0.1 ν=0.2 ν=0.3 ν=0.4 ν=0.5 ∆=0.1J 0 1 2 3 4 5 ω/J 0 0.2 0.4 0.6 0.8 F ν=0.1 ν=0.2 ν=0.3 ν=0.4 ν=0.5 ∆=J 0 4 8 12 16 20 ω/J 0 0.4 0.8 1.2 1.6 2 F ν=0.1 ν=0.2 ν=0.3 ν=0.4 ν=0.5 ∆=10J (b) (a) (c) FIG. 6. Color online Energy absorption rate of hard-core bosons in a quasiperiodic potential with =0.771 452 45: the response function F see Eq. 14 is plotted versus modulation frequency for different ﬁlling factors and increasing values of disorder strength: =0.1J top panel, =J, and =10J. Calculations are done on a ring of M=1200 lattice sites yielding almost negligible ﬁnite-size effects. The number of disorder realization is Nr=500. 0 1 2 3 4 ω/J 0 0.001 0.002 0.003 0.004 0.005 F ν=0.1 ν=0.2 ν=0.3 ν=0.4 ν=0.5 ∆=0.1J 0 1 2 3 4 5 ω/J 0 0.2 0.4 0.6 0.8 F ν=0.1 ν=0.2 ν=0.3 ν=0.4 ν=0.5 ∆=J (b) (a) 0 4 8 12 16 20 ω/J 0 0.4 0.8 1.2 1.6 2 F ν=0.1 ν=0.2 ν=0.3 ν=0.4 ν=0.5 ∆=10J (c) (c) FIG. 6. Color online Energy absorption rate of hard-core bosons in a quasiperiodic potential with =0.771 452 45: the response function F see Eq. 14 is plotted versus modulation frequency for different ﬁlling factors and increasing values of disorder strength: =0.1J top panel, =J, and =10J. Calculations are done on a ring of M=1200 lattice sites yielding almost negligible ﬁnite-size effects. The number of disorder realization is Nr=500. Vk2 = 2 2 n=− + k + k0 + 2n + k −k0 + 2n. 26 peaks as shown in the central panel of Fig. 6. These peaks gradually disappear as increases and becomes much larger than J. In this regime, however, the energy absorption spec- trum becomes indeed rather similar to the case of weak qua- siperiodic potential, as can seen by comparing the upper and lower panels of Fig. 6. This peculiar effect can be explained analytically starting from Eq. 10, which applies in the atomic limit. Introducing the variable y=2n, in the ther- modynamic limit, we obtain Inserting Eq. 26 into Eq. 24, we obtain Inserting Eq. 26 into Eq. B. Unit ﬁlling 22 yields and k0=2, for M→, the right-hand side of Eq. 25 becomes a series of delta functions 033625-6 LATTICE MODULATION SPECTROSCOPY OF STRONGLY… B. Unit ﬁlling We have obtained the absorption spectrum at unit ﬁlling numerically using Eqs. 11 and 12, for the same value of For a potential strength comparable to the tunneling rate, i.e., J, the absorption spectrum develops very sharp 033625-7 PHYSICAL REVIEW A 80, 033625 2009 ORSO et al. 0 0.5 1 1.5 2 2.5 0 5 10 15 F ω /J 0 5 10 15 20 25 0 0.5 1 1.5 2 2.5 F ω /J 0 0.05 0.1 0.15 0.2 0 1 2 3 4 F 0 1 2 3 4 5 0 0.05 0.1 0.15 0.2 F N = 100 N = 200 N = 300 N = 400 0 0.001 0.002 0.003 0.004 0.005 0 1 2 3 4 F Quasi-periodic 0 1 2 3 4 5 0 0.001 0.002 0.003 0.004 0.005 F Disordered ∆= 10J ∆= J ∆= 0.1J 0 0.002 0.004 0 1 2 F ∆= 10J ∆= J ∆= 0.1J N = 200 ∆= 0 FIG. 8. Color online Energy absorption rate of disordered and quasidisordered hard-core bosons in a parabolic trap. The response function F is plotted versus modulation frequency for different ﬁll- ings. Upper panel: =0.1J, middle panel: =J, and lower panel: =10J. The system size is M=500, the number of disorder realiza- tions is Nr=4000, and ho=2.8810−4J. The inset in the upper panel shows the low-frequency absorption in the absence of disor- der, which is ﬁnite for trapped gases. As increases, this sharp peak fragments out and is no longer visible on the scale of the bulk contribution. 0.7 0.8 0.9 1 1.1 1.2 1.3 ω/U 0 20 40 60 80 F ∆=0 U ∆=0.03U ∆=0.06U ∆=0.1U J=0.01U FIG. 7. Color online Energy absorption in the Mott insulator phase for bosons in a quasiperiodic potential with =0.771 452 45: the response function F see Eq. 14 is plotted versus modulation frequency for ﬁxed J=0.01U and increasing dis- order strength /U=0black dotted line,0.03,0.06,0.1. Here, convergence is achieved for system size L=70. As disorder in- creases, the response function broadens and changes convexity, as predicted by Eq. 32. B. Unit ﬁlling As increases, this sharp peak fragments out and is no longer visible on the scale of the bulk contribution. FIG. 8. Color online Energy absorption rate of disordered and quasidisordered hard-core bosons in a parabolic trap. The response function F is plotted versus modulation frequency for different ﬁll- ings. Upper panel: =0.1J, middle panel: =J, and lower panel: =10J. The system size is M=500, the number of disorder realiza- tions is Nr=4000, and ho=2.8810−4J. The inset in the upper panel shows the low-frequency absorption in the absence of disor- der, which is ﬁnite for trapped gases. As increases, this sharp peak fragments out and is no longer visible on the scale of the bulk contribution. We see that the shape of the absorption spectrum changes considerably as increases. For sufﬁciently weak quasiperi- odic potential 3 J, the spectrum does not become broad, but instead satellite peaks appear on the sides of the central absorption feature. For stronger quasiperiodic potentials, the central peak disappears and the spectrum develops a two hump structure. To understand these features, let us again focus on the atomic limit, where the absorption rate can be obtained analytically using Eq. 13. Introducing the variable y=2n and passing to the continuum limit, we obtain the result B. Unit ﬁlling 0 0.5 1 1.5 2 2.5 0 5 10 15 F ω /J 0 5 10 15 20 25 0 0.5 1 1.5 2 2.5 F ω /J 0 0.05 0.1 0.15 0.2 0 1 2 3 4 F 0 1 2 3 4 5 0 0.05 0.1 0.15 0.2 F N = 100 N = 200 N = 300 N = 400 0 0.001 0.002 0.003 0.004 0.005 0 1 2 3 4 F Quasi-periodic 0 1 2 3 4 5 0 0.001 0.002 0.003 0.004 0.005 F Disordered ∆= 10J ∆= J ∆= 0.1J 0 0.002 0.004 0 1 2 F ∆= 10J ∆= J ∆= 0.1J N = 200 ∆= 0 0.7 0.8 0.9 1 1.1 1.2 1.3 ω/U 0 20 40 60 80 F ∆=0 U ∆=0.03U ∆=0.06U ∆=0.1U J=0.01U FIG. 7. Color online Energy absorption in the Mott insulator phase for bosons in a quasiperiodic potential with =0.771 452 45: the response function F see Eq. 14 is plotted versus modulation frequency for ﬁxed J=0.01U and increasing dis- order strength /U=0black dotted line,0.03,0.06,0.1. Here, convergence is achieved for system size L=70. As disorder in- creases, the response function broadens and changes convexity, as predicted by Eq. 32. FIG. 7. Color online Energy absorption in the Mott insulator phase for bosons in a quasiperiodic potential with =0.771 452 45: the response function F see Eq. 14 is plotted versus modulation frequency for ﬁxed J=0.01U and increasing dis- order strength /U=0black dotted line,0.03,0.06,0.1. Here, convergence is achieved for system size L=70. As disorder in- creases, the response function broadens and changes convexity, as predicted by Eq. 32. =0.771 452 45. The result is plotted in Fig. 7 for ﬁxed J/U=0.01 and increasing values of the quasiperiodic poten- tial strength . The dashed line corresponds to the clean case =0, where the absorption rate is given by Eq. 21. FIG. 8. Color online Energy absorption rate of disordered and quasidisordered hard-core bosons in a parabolic trap. The response function F is plotted versus modulation frequency for different ﬁll- ings. Upper panel: =0.1J, middle panel: =J, and lower panel: =10J. The system size is M=500, the number of disorder realiza- tions is Nr=4000, and ho=2.8810−4J. The inset in the upper panel shows the low-frequency absorption in the absence of disor- der, which is ﬁnite for trapped gases. VI. CONCLUSIONS For J, we formally expand the right-hand side of Eq. A2 in powers of the disorder strength Ak,k=Akk 0 +Akk 1 +Akk 2 +O3, so the matrix K takes the form In conclusion, we have investigated the energy absorbed by a disordered strongly interacting Bose gas in the presence of periodically modulated optical lattices. For ﬁlling factor less than one, the absorption rate has been calculated exactly in the hard-core limit via the Bose-Fermi mapping. For com- mensurate ﬁlling, corresponding to one boson per lattice site, the gas is a Mott insulator and can only absorb energy at much higher frequency on the order of the repulsive inter- action U. The disorder-induced broadening of the absorption spectrum has been calculated by restricting to the subspace of particle-hole excitations. Kk,k = Kkk 0 + Akk 0 Akk 1 + Akk 1 + Akk 2 + Akk 2 + Akk 1 2 + O3. Kk,k = Kkk 0 + Akk 0 Akk 1 + Akk 1 + Akk 2 + Akk 2 + Akk 1 2 + O3. A4 A4 Taking Eq. A3 into account, we see that the only nondiago- nal term appearing in the expansion A4 is Akk 1 2, which is on the second order in . This term can be readily evaluated from Eq. A2 by applying the ﬁrst-order perturbation theory for the eigenstates Taking Eq. A3 into account, we see that the only nondiago- nal term appearing in the expansion A4 is Akk 1 2, which is on the second order in . This term can be readily evaluated from Eq. A2 by applying the ﬁrst-order perturbation theory for the eigenstates We have performed extensive calculations comparing two different sources of disorder: a random potential, which is relevant for current experiments based on speckle patterns, and a quasiperiodic potential, which is obtained by superim- posing two optical lattices with incommensurate periods. Our results indicate that the response of the gas to the lattice modulation signiﬁcantly depends on the chosen source of randomness. k = k 0 + qk q 0Vk 0 k 0 −q 0 q 0. A5 A5 After a simple algebra, we obtain After a simple algebra, we obtain Akk 1 = k 0Vk 0 k 0 −k 0 2cos k −cos k, A6 A6 which is valid up to the linear order in . APPENDIX In this appendix, we shall derive the asymptotic formula 9 based on the perturbation theory for weak disorder. For clarity, we rewrite the general expression 7 We also see in Fig. 8 that the behavior of the absorption spectrum at frequencies close to the bandwidth crucially de- pends on whether the applied potential is truly random or quasiperiodic. Whereas the disordered case exhibits a smooth behavior in the absorption up to the bandwidth 4J where it falls to zero, the quasiperiodic one shows a sharp peak lo- cated at the bandwidth 4Jc, which resembles the divergence found in the corresponding homogeneous case. Note that the position of this peak is almost independent on the number of atoms in the tube and, therefore, the peak should be visible in a realistic experimental situation, where an average over a multiple tube setup with variable ﬁlling is performed 33. The same conclusion applies to the system with strong dis- order or quasiperiodic potential as can be observed in the lower panel in Fig. 8. For J Fig. 8, middle panel, the peak structure in the quasiperiodic case is more complex and, thus, the averaging procedure will produce some round- ing off of the peaks. Still, the absorption can be considerably larger than in the disordered case and this difference should be clearly visible. E˙ = J0 2 2 k,k Kkkfk −fk + k −k, A1 A1 using new indices k and k. Moreover, we ﬁnd convenient to introduce the matrix elements using new indices k and k. Moreover, we ﬁnd convenient to introduce the matrix elements Akk = j k j + 1kj + k jkj + 1, A2 A2 so that Kk,k=Ak,k2. k,k k,k In the absence of disorder =0, the eigenstates are plane waves k 0n=eikn/L with energy k 0=−2J cos k. Therefore, from Eq. A2, we ﬁnd Akk 0 = 2kk cos k, A3 A3 showing that the matrix A is diagonal in momentum space. Since the matrix Kkk 0 =4k,k cos2 k is also diagonal, the ab- sorption rate 7 vanishes. VI. CONCLUSIONS Finally, by using the dispersion relation k 0=−2J cos k, Eq. A6 further sim- pliﬁes yielding No. PIE 200760/007. insulator at the center of the trap cannot absorb energy at low frequency, so the only contribution comes from the outer shell, where the ﬁlling factor is less than unity. In particular, the low-frequency peak arising from edge excitations frag- ments in multiple peaks with little spectral weight compared to the response from the bulk discussed in Secs. III and IV. V. EFFECTS OF A PARABOLIC TRAP F = 2 0 2 dy 2 −U + cos y − cosy + 2. 31 F = 2 0 2 dy 2 −U + cos y − cosy + 2. In this section, we discuss the effects of a harmonic trap- ping potential Vz=mho 2 z2/2 on the absorption spectrum. Here, m is the atom mass and ho is the trapping frequency. In this case, Vj ho in Eq. 1 is nonzero and given by Vj ho =hoj−M/22, where ho=mho 2 d2/2, d being the lattice pe- riod. 31 The integral 31 can be readily evaluated using the identity a sin y+b cos y=c cosy+, where tan =−a/b and c =a2+b2. From Eq. 31 we have that c=2 sin and, therefore, We have repeated the calculations of the absorption spec- trum including Vj ho and the result is shown in Fig. 8. For a system of hard-core bosons in the absence of disorder or quasiperiodic potential, the trap favors the formation of a Mott insulator in the center surrounded by a superﬂuid re- gion at the trap edges. This gives rise to a ﬁnite absorption at low frequency see inset in the upper panel, which is related to the creation of excitations at the edge of the trap. By contrast, in a uniform system of hard-core bosons, as we have described in Sec. II, the energy absorption vanishes to all orders because the hopping operator K commutes with the Hamiltonian. F = 2 c2 − −U2 , 32 32 showing that the absorption rate diverges at the edge, where −U=c. This means that the shape of the absorption spectrum changes completely going from weak to strong quasiperiodic potential, as obtained numerically and shown in Fig. 7. Finally, in comparing Eqs. 23 and 32, we see that in a quasiperiodic potential the absorption spectrum is at least in the atomic limit narrower because c 2. Let us next consider the effect of a small amount of dis- order or a weak quasiperiodic potential. Clearly, the Mott 033625-8 PHYSICAL REVIEW A 80, 033625 2009 LATTICE MODULATION SPECTROSCOPY OF STRONGLY… No. PIE 200760/007. ACKNOWLEDGMENTS This work was partly supported by ANR under Grant No. 08-BLAN-0165-01 and by the Swiss National Fund under MaNEP and Division II. A.I. gratefully acknowledges ﬁnan- cial support from CONICET and UNLP. During the initial stage of this project, G.O. was also supported by the Euro- pean Commission under Contract No. EDUG-038970. M.A.C. was supported by MEC Spain under Grant No. FIS2007-066711-C02-02 and CSIC Spain through Grant Akk 1 = k 0Vk 0 J . A7 A7 Substituting Eq. A6 into Eq. 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https://openalex.org/W4248714794	https://zenodo.org/record/1997587/files/article.pdf	English	null	ORDINARY MEETING: 11th March, 1921	Journal of molluscan studies	1,921	public-domain	1,151	s s From these records Mr. Clarke thought valuable reports might I'e drawn up, from time to time, which would exhibit the progress of geological knowledge; and that thus a really useful work would ilL' effected by the Association. Several promises of Papers were announced. Ol'dina1'y 11feeting, 8th .1Jfarch, 1859. The Rev. Thomas Wiltshire, M.A., F.G.S., in the Chair. The Rev. Thomas Wiltshire, M.A., F.G.S., in the Chair. It was stated by Mr. Wakefield, one of the Honorary SecretaJ'ies that in consequence of the resignation of the Presidency by Toulmiu Smith, Esq., it had devolved upon the General Committee to exercise the power conferred on them by Rule XXXII. for the filling up of vacancies which might occur in the offices of the Association, awl that they had accordingly requested the Rev. Thomas Wiltshire, one of the Vice-Presidents, to accept the office of President, and that he had much pleasure in stating that that gentleman had acceded to their request. The Chair was then taken by the President. After the reading of the Minutes of the Meeting of the 8th February, The President stated that it had been considered necessary, in consequence of the urgent representations of several of the Country Members, to rescind Rule V., which excludes Country Members from the Ordinary Meetings, and to vary Rule VI. by the omission of blu- word "Town" at the beginning. The Rule so altered would then state the privileges of all the Members, whether Town or Country. The Requisition, duly signed, calling on the Secretary to convene the Meeting with a view to this and to an alteration as to the day of meeting having been read, the question as to rescinding Rule V. and the modification of Rule VI. was put to the vote and carried unani- mously. It was then put to the Meeting that Rule XXIX., as to the 6 day for the Ordinary Meetings of the Association, be altered by the substitution of the words First llIonday for Second Tuesday; which was also carried, and the Rules were declared to be amended accordingly. The following ladies and gentlemen were elected Members of the Association: - W. S. Cross, E8q.; Joseph Yellowly Watson, Esq., F.G.S.; Mr. R. C. Taylor; the Rev. J. Alloway, B.A.; Mr. G. E. Boggis ; G. R. Ritchie, Esq.; Mr. T. Bennett; John Noyes, Esq.; Mr. T. V. Robson; Mr. W. T. Sutton; Mr. John Cliff; J\l1'. \V. Myers; Mr. G. Jackson; W. Cunnington, Esq., F.G.S.; 1\11'. O. "V. Crocker; E. P. Wilkins, Esq., F.G.S.; Rev. G. B. Adam, B.A.; Richard T. Manson, Esq.; I. N. Lockyer, Esq.; Rev. J. S. Henslow,F.G.S., &c., &c., Professor of Botany in the University of Cambridge; Mr. Enoch Walker; Rev. J. S. Marsden; Mr. J. R. Gregory; Mr. Thomas Purdue; Mr. Dow; Rev. Thomas H. The Rev. Thomas Wiltshire, M.A., F.G.S., in the Chair. Brown; P. L. Simmonds, Esq., F.R.S.S.; H. Duckworth, EBq., F.G.S.; John Darlington, Esq.; Rev. L. H. Mordargue; C. F. Pocock, Esq; G. Henslow, Esq.; Rev. T. F. fitatham, M.A., F.G.S.; Mr. Cruick- shank; Mr. Michael Carty; Miss Ann Tayler Slatter; Richard J ames, Esq.; G. T. Chambers, Esq.; J. Cumming, Esq.; H. L. Her- roun, Esq.; Mr. John Lambley; P. Badcock, Esq.; James Yate Johnson, Esq.; Miss Mac Neill; W. H. Smith, EEq.; Mrs. Jane Bowles; Mr. James Taylor. The following donations to the Library, from Hyde Clarke, Esq., V.P., were announced :-" A Treatise on Copper Smelting." By Hyde Clarke, Esq. "The Progress of Mining in 1855." By J. Yellowly Watson, F.G.S. "The Recent Brachiopoda." By S. B. Sowerby, F.L.S. "The Geologist," for March, 1859. From S. J. Mackie, EBq., F.G.S. Professor J. Tennant gave a lecture on Mineralogy applied to Geology. The lecturer stated that as many as 520 species of minerals were described in one English treatise on the science; and, when any one looked at a map of the world and compared the small area of the British Isles and their mineral wealth with the extent of such terri- tories as that of Canada and Hudson's Bay, and the treasures to be there probably discovered, he must perceive the importance of some Professor J. Tennant gave a lecture on Mineralogy applied to Geology. The lecturer stated that as many as 520 species of minerals were described in one English treatise on the science; and, when any one looked at a map of the world and compared the small area of the British Isles and their mineral wealth with the extent of such terri- tories as that of Canada and Hudson's Bay, and the treasures to be there probably discovered, he must perceive the importance of some 7 acquaintance with the science of mineralogy. Australia, a few years since. was only known as containing a few sheep-walks, and as a penal settlement. In 1851, a piece of stone was received in London and placed in the Great Exhibition, where it created much sensation. It was a gold nugget. Such nuggets had been frequently picked up in Australia; stones containing the yellow metal had been built into walls and houses j but no one had, previously to this time, regarded them. The Rev. Thomas Wiltshire, M.A., F.G.S., in the Chair. Some thought the metalliferous substance to be iron-pyrites, others that it was copper-pyrites; but if these persons had been acquainted with a very simple test-a common file-they could have easily ascertained the difference between pyrites and gold. The rapid progresi:! of the colony since this discovery was familiar to every one, and the history of the gold-nugget trade would display prominently the value of observant habits. The lecturer then de- scribed the various large nuggets which had been brought into this country, the largest heing four feet two inches long by ten inches wide. This was melted and produced fine gold of the value of 6,905l. 128. gel., only twenty-one ounces of stony matter remaining. Diamonds in the rough state had been thrown aside by the gold- seekers, and many other valuable substances were frequently wasted in ignorance of their nature and properties. The most interesting part of mineralogy was crystallography, and the lecturer gave illustrations of the methods of distinguishing crys- tals by their forms, fracture, frangibility, degrees of hardness, &c. Some specimens of cryolite from Greenland, were exhibited, to show the importance of the study of mineralogy, in developing the means of cheapening useful commodities. Aluminium, at the time of the Paris Exhibition, could not be obtained for less than 4l. per ounce. It was soon afterwards offered for 2l. per ounce; but, since cryolite had been used, it was reduced to less than 158. per ounce. This was an important metal, and although hitherto only known as a curiosity in the laboratory, would probably, before long, become of the highest commercia! importance. The lecturer concluded with some statistical accounts of the values and annual produce of the chief British minerals.
https://openalex.org/W1964822542	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0022388&type=printable	English	null	Survivorship of Anopheles darlingi (Diptera: Culicidae) in Relation with Malaria Incidence in the Brazilian Amazon	PloS one	2,011	cc-by	12,443	Introduction Malaria remains one of the most important infectious diseases in the world, and has reemerged in tropical regions that experience rapid population growth [1]. The understanding of age composition of anopheline mosquito populations has been considered crucial for explaining transmission and evaluating the success of control efforts [2]. The probability of survival of the vector may be the most critical factor in the transmission cycle of arthropod-borne diseases [3–4]. A considerable number of individuals of a given vector species must survive long enough to allow sporogonic development of Plasmodium spp. for it to be an efficient vector. However, little is known of age composition and survival rates of anopheline species in the Amazon region. We have reported parous rates and methods for determining parity status, as well as dispersal, of Amazonian anophelines [5–6]. Recently, Fouque et al. [7] have observed that peak malaria incidence in the Maroni region of French Guiana was associated with higher Anopheles darlingi survival rates, the most important vector in the Amazon [8–10] but data were not statistically significant. In this paper, we present longitudinal variations in the age structure and survivorship of An. darlingi in a frontier zone of the State of Roraima, in the northern Brazilian Amazon. The Materials and Methods The protocol was approved by the ethics committee of the Oswaldo Cruz Foundation. Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: fsaito1@yahoo.com.br * E-mail: fsaito1@yahoo.com.br association of survival rates with malaria incidence was studied in detail. Abstract We performed a longitudinal study of adult survival of Anopheles darlingi, the most important vector in the Amazon, in a malarigenous frontier zone of Brazil. Survival rates were determined from both parous rates and multiparous dissections. Anopheles darlingi human biting rates, daily survival rates and expectation of life where higher in the dry season, as compared to the rainy season, and were correlated with malaria incidence. The biting density of mosquitoes that had survived long enough for completing at least one sporogonic cycle was related with the number of malaria cases by linear regression. Survival rates were the limiting factor explaining longitudinal variations in Plasmodium vivax malaria incidence and the association between adult mosquito survival and malaria was statistically significant by logistic regression (P,0.05). Survival rates were better correlated with malaria incidence than adult mosquito biting density. Mathematical modeling showed that P. falciparum and P. malariae were more vulnerable to changes in mosquito survival rates because of longer sporogonic cycle duration, as compared to P. vivax, which could account for the low prevalence of the former parasites observed in the study area. Population modeling also showed that the observed decreases in human biting rates in the wet season could be entirely explained by decreases in survival rates, suggesting that decreased breeding did not occur in the wet season, at the sites where adult mosquitoes were collected. For the first time in the literature, multivariate methods detected a statistically significant inverse relation (P,0.05) between the number of rainy days per month and daily survival rates, suggesting that rainfall may cause adult mortality. Citation: Barros FSMd, Hono´rio NA, Arruda ME (2011) Survivorship of Anopheles darlingi (Diptera: Culicidae) in Relation with Malaria Incidence in the Brazilian Amazon. PLoS ONE 6(8): e22388. doi:10.1371/journal.pone.0022388 Editor: Nirbhay Kumar Tulane University United States of America Received March 6, 2011; Accepted June 20, 2011; Published August 8, 2011 Received March 6, 2011; Accepted June 20, 2011; Published August 8, 2011 Copyright: 2011 Barros 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 Barros et al. This is an open-access article distributed unde unrestricted use, distribution, and reproduction in any medium, provided the origin Funding: The Interamerican Institute for Global Change Research, grant CRN no. 048. Fa´bio Saito Monteiro de Barros1, Nildimar Alves Hono´ rio2, Me´rcia Eliane Arruda3 1 Departamento de Zoologia, Universidade Federal de Pernambuco, Recife, Brazil, 2 Laborato´rio de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalha˜es, Fundac¸a˜o Oswaldo Cruz, Recife, Brazil, 3 Laborato´rio de Transmissores de Hematozoa´rios, Departamento de Entomologia, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, Brazil Climate and rainfall In the study area, only two seasons can be distinguished, the dry season and the rainy season. A well-defined six month-long dry period lasts from November or December to April or May, with 55–60% of the precipitation occurring from May to July [13]. A pluviometer was installed in the study site for daily monitoring of rainfall throughout the collection period, from August, 2003, to July, 2004. Annual rainfall was 1367 mm/m2. Total monthly rainfall for the collection period is shown in Table 1. Temperature and humidity were measured by digital devices during the collection periods. Mean temperature was 28.0uC (SD = 1.11), and relative humidity was around 58% (SD = 2.88), with little yearlong variation. Human malaria data If the time for egg-development is known or accounted for, the approximate duration of the gonotrophic cycle, i.e. the time elapsed from one blood-feeding to the next, would vary as a function of the time elapsed from oviposition to blood-feeding. The time from oviposition to blood-feeding can be estimated by examining the condition of the terminal portions of the ovarioles, i.e. the ovariolar stalks, after passage of the eggs [2], in females in Sella’s stage 1, 2 or 3 [16]. When the female oviposits, the eggs pass through the terminal portions of the ovarioles. These previously narrow conducts stretch to many times their original size to allow passage of eggs. In females that have recently oviposited (,24 h), the ovarioles still presents sac-like distensions due to this stretching. Within one day, the dilated portions return to their original size through progressive stages. It should be noted that there is no way of recognizing if the females have taken more than 24 h to find a host, since oviposition. To explore seasonal incidences and spatial distribution, the number of cases within the period from January, 2002, to December, 2004, was analyzed. Malaria morbidity data was collected retrospectively from the Roraino´polis Municipal Health Service. Facilities were permanently available for diagnosis of cases through microscopic examination of blood smears. Only the number of patients with malaria per month was used in the study and there was no need for storage of patient information in the hospital database, which means written consent for research was not needed. Thick films were stained with 10% Giemsa solution and examined at 61,000 under oil immersion by an expert microscopist with over 5 years of experience. Every 1–2 weeks, health agents would periodically perform thick blood smears in residents presenting non-specific acute febrile symptoms and their household contacts. All new positive malaria cases, irrespective of age, were enrolled for the study. Plasmodium vivax malaria cases were promptly treated with chloroquine and primaquine and P. falciparum with quinine or mefloquine. Cases that occurred less than 50 days after the first day of drug treatment were excluded from the study because it was unclear if they represented new cases, treatment failure or disease relapse. Mosquito Survivorship and Malaria in the Amazon lined up near the sideroad. The sideroad branches perpendicularly from the main road for 18.8 km into the rainforest. Mosquito collections Adult collections were conducted during six bimestrial collecting excursions from August, 2003 to July, 2004, including November, 2003; January, 2004; March, 2004; and May, 2004. Captures were performed in six collecting stations, during four consecutive days. Adult mosquitoes were collected on the act of landing and identified using Consoli and Lourenc¸o-de-Oliveira (1994) [14]. Residual spraying was performed irregularly and with large intervals between applications. Collections were not performed within three months of residual spraying. Bednets, which were unimpregnated, were used irregularly by only three families in the Sideroad. Collections were not performed in houses with bednets. Further methodological details have been previously described [15]. Study site The study was conducted within an agricultural frontier settlement of Roraino´polis, 300 km south of Boa Vista, the capital of the Province of Roraima, in the northern Brazilian Amazon. Climate and ecoregional characteristics of Roraima have also been previously reported [11]. Located deep inside the rainforest, most settlements in the area are recent (,ten years, at the time of study) and composed of multiple sideroads that run perpendicular to a main road, forming a characteristic fish-bone pattern. Malaria in the area is unstable and hypoendemic, predominantly due to Plasmodium vivax [12]. Sideroad 19 (00u519N, 60u219W) was chosen due to its higher malaria endemicity, in comparison with neighboring sideroads (Roraino´polis, Municipal Health Service, data not shown). Sideroad 19 is a typical secondary road composed of 66 inhabited houses. Residences are spaced at 300 m intervals and PLo August 2011 \| Volume 6 \| Issue 8 \| e22388 August 2011 \| Volume 6 \| Issue 8 \| e22388 1 PLoS ONE \| www.plosone.org Mosquito Survivorship and Malaria in the Amazon PLoS ONE \| www.plosone.org 2 August 2011 \| Volume 6 \| Issue 8 \| e22388 The cumulative number of malaria cases per month diagnosed from January 2002 to December 2004 are also shown. doi:10.1371/journal.pone.0022388.t001 Human malaria data If egg-development is assumed to take two days [17–18], the duration of the gonotrophic cycle (n), in days, can be estimated from the ratio of mosquitoes with contracted ovariolar stalks (c) and the total number collected (t) as follows: n~2z c t. This methodology has been previously used [19–21] and it will be referred to, in this article, as ‘‘Charlwood’s minimum cycle Table 1. Total monthly rainfall (mm/m2), number of wet days, from August 2003–July, 2004. Month Rainfall (mm) No. wet days Cumulative malaria cases (P. vivax) Mean daily temperatures (6C) Aug 106 19 7 27.4 Sep 103 20 10 27.8 Oct 99 18 16 28.4 Nov 85 17 20 28.8 Dec 5 8 23 29.0 Jan 23 1 25 28.9 Feb 39 4 16 28.8 Mar 2 3 21 29.0 Apr 150 8 18 28.4 May 364 11 13 26.6 Jun 278 18 10 26.4 Jul 113 20 7 26.0 Total 1367 147 186 The cumulative number of malaria cases per month diagnosed from January 2002 to December 2004 are also shown. doi:10.1371/journal.pone.0022388.t001 Table 1. Total monthly rainfall (mm/m2), number of wet days, from August 2003–July, 2004. August 2011 \| Volume 6 \| Issue 8 \| e22388 2 Mosquito Survivorship and Malaria in the Amazon method’’ because only the minimum duration of the gonotrophic cycle can be reliably determined and cycles longer than three days cannot be detected. constant densities year-round [25]. In frontier zones, dams are built for creating readily available water collections, which are used for bathing, washing, recreation and fishing. They are created by blocking small streams with raised wooden and earth barriers. Apparently, predation by introduced or native fish species in these dams is not sufficient for controlling anopheline larval densities. Mosquitoes collected within 1,000 m of the temporary river were excluded from the study. This distance was chosen based on the proposed flight range of An. darlingi mosquitoes [5], only 4.5% of mosquitoes emerging in the river would be expected to fly more than 1000 m. Detinova (1945) [2] classified ovariolar stalk distension into five categories, from A to E, where A is the most dilated and E the most contracted. In A there is ‘‘no contraction’’; B represents ‘‘first signs of contraction’’; C ‘‘noticeable contraction’’; in D ‘‘large dilatations’’ have formed; and E corresponds to ‘‘well defined dilatations’’. Types A to C were considered as representing recently oviposited females. Comparison between parous rates in different collection periods To determine if collection periods differed significantly in the proportion of the binomial parameter ‘‘mosquito parity’’, a Chi- square statistic test with Yates correction for continuity was performed using the 262 table describing the binomial variables number of nulliparous and parous females in each month of collection [27]. Statistical significance levels for multiple testing were adjusted using Bonferroni correction for multiple compar- isons, where a~ 0:05 k and k = the number of comparisons within subgroups [28]. Data for determining gonotrophic cycle duration was collected twice, in January, 2004, and July, 2004. Gonotrophic cycle duration during other dry season collections (January and May) were considered similar to that determined in January. Cycle duration during other wet season collections (August and November) were considered similar to that determined in July. Dissections were performed using a binocular dissecting microscope (406) and a fiber-optics light source. Ovarian observations were performed with a binocular conventional clinical microscope (100–1,0006), in a field laboratory. Dissections were performed immediately after identification and determina- tion of Sella’s stage and only mosquitoes dissected less than six hours after the moment of capture were considered for estimating gonotrophic cycle durations. Human malaria data Females with A, B or C type sacs were assumed to have returned to feed on the same day they had laid eggs (a 2 day cycle) while those with type D or E dilations were assumed to have delayed one day before attempting to re-feed. Therefore, c t ~ AzBzC AzBzCzDzE, i.e. corresponds to the number of recently oviposited An. darlingi females, divided by the total number of An. darlingi females captured in that collection period. Density of mosquitoes of potentially dangerous age and P. vivax sporogonic cycle duration p g y Using daily survival rates, the proportion of An. darlingi which would survive long enough for the complete development of P. vivax in the mosquito can be calculated. First the duration of the sporogonic cycle, or extrinsic incubation period, must be known. This represents the time required from the moment of infection in the mosquito to complete maturation of sporozoites in the salivary glands. The time for sporogonic development of Plasmodium spp. in An. darlingi has not been specifically investigated. It is usually presumed to vary according to temperature. For P. vivax it would be around 10 days (range 8 to 13 days) at 27–28uC [29]. The duration of the sporogonic cycle can be indirectly estimated by the Moshkovsky method [30–31] which makes use of the Blunck hyperbolic equation and the Bodenheimer formula for demon- strating the relationship between temperature and gonotrophic cycle duration. In this method, first the mean daily temperatures during the period of study must be known (Table 1). The duration of the sporogonic cycle for P. vivax corresponds to the amount of days required for the sum of degrees Celsius above the base outdoor temperature, 14.5uC, on each day, to reach 105, the degree-days. The Moshkovsky correction factor between the mosquito resting place and the outdoor temperature was considered to be 1.0. Once the duration of the sporogonic cycle (n) is known, the percentage of mosquitoes surviving long enough for complete maturation of P. vivax can be calculated by pn. Base temperatures and degree-days for P. falciparum are 16uC and 111, respectively. For P. malariae, these figures are 16uC and 144, respectively. Parous rates and survivorship Parity was determined by checking multiple parameters, such as tracheole structure, presence of yellow bodies, presence of pedicular dilations or the appearance of the ovariolar stalks. Daily survival rates (p) were calculated by Davidson’s method as p~ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ parity gp , where parity represents the ratio between the number of parous mosquitoes and the total number of females collected, and g = the duration of the gonotrophic cycle in days [22]. Ninety five percent confidence intervals (95% CI) for the proportions of parous females where obtained, as proposed by Bliss (1967) [23]. Data analysis: comparing ovariolar stalk dilations in different collections A Chi-square test was also performed on the 562 contingency table for comparing types of ovariolar stalk dilations (A to E) in two collection periods, but categories were grouped for obtaining cells .5. PLoS ONE \| www.plosone.org Mosquito Survivorship and Malaria in the Amazon Expectation of life, expectation of infective life and changes in longevity/density factors 1 per gonotrophic cycle. Dissections for studying follicular dilata- tions were performed either on the same night of capture or on the following day, in order to decrease the number of recently oviposited females. In females that have recently oviposited, i.e., types A or B ovariolar stalks where encountered, Polovodova’s technique can only be applied to follicles that have degenerated in an early stage of egg development [34]. Several of these non- functional follicles had to be visualized for a good estimate to be made. Counting follicular dilatations in these recently oviposited females is difficult and occasionally could not be performed. These mosquitoes were tentatively classified as having one or two dilatations and treated as ‘‘censored data’’ in the survival analysis procedure. Multiparous dissections were performed in two study occasions, in January and July 2004. Expectation of life was determined by 1 {loge p and expectation of infective life, i.e. the average number of days of infective life per infected mosquito, was determined as pn {loge p [32]. To determine the relative contributions of changes in density and longevity on the impact of residual insecticidal spraying, Garret-Jones & Grab (1964) [32] formulated the density factor (d) and the longevity factor (l). The density factor reflects how many times the density of females was affected and can be calculated as the ratio between pre and post-spraying expectations of life: d~ loge(ppost{spraying) loge(ppre{spraying). The longev- ity factor is the ratio of the mosquitoes’ expectation of infective lives, calculated as: l~ pn pre{spraying loge(ppre{spraying) \| loge(ppost{spraying) pnpost{spraying . The total impact of residual insecticide spraying is given by the product d6l. We postulated that the similar principles could be used to study seasonal variations of An. darlingi. Since HBR is a function of the proportion of mosquitoes biting man and the total population, it will vary with breeding rates, survival rates [33] and sampling error. Assuming sampling error was small and the proportion biting man is a species- specific constant [15] we attempted to determine the relative contribution of survival and breeding by verifying how much of the density variation was due to survival rates. Survival and density are interrelated factors and if both parameters vary concurrently, an observed decrease in parous proportion will have an expected decrease in HBR [32], because the equations imply that recruitment was kept constant. Survival curves obtained with the exponential model The fit with the highest Komolgorov-Smirnoff d statistic and P- value was the unweighted exponential model (d = 0.076; P,0.05). The exponential model was therefore adopted for further study and survival curves were obtained by a least squares fit regression of the exponential function y = bNexp(aNx), where b and a are independent parameters, as described by Service (1993) [22]. A weighted regression fit was used to account for the different sample sizes in each age group. Mosquito Survivorship and Malaria in the Amazon A reduction in daily survival rates from 0.9 to 0.8 (exemplifying on a parasite with a 12 day sporogonic cycle), should cause a 2.11 times reduction in adult mosquito density. Field measurements can measure the amount of reduction in HBR that actually occurred: if the observed equals the expected, it would be assumed that only survival rates were affected. If the measured HBR was less than expected, it would be assumed that breeding rates were negatively affected, and if HBR is more than expected, then breeding rates and recruitment would have increased. We compared the expected decreases in mosquito density to actual decreases observed in human biting rates. Changes in the longevity factor formula had to be introduced to account for varying sporogonic cycle durations from the dry to the wet season: l~ pn1 dry loge pdry \| loge pwet pn2 wet , where n1 and n2 are the sporogonic cycle durations in the dry and wet seasons, respectively. Survival and failure time analysis: Kaplan-Meier product- limit method and comparison between survival curves The probability of a mosquito surviving the duration of the sporogonic cycle varies with the Plasmodium spp. being considered. We determined sporogonic cycle durations for P. falciparum and P. malariae, for each collecting period with the Moshkovsky method. For comparing transmission with that of P. vivax, we developed a relative transmission ratio by dividing the probabilities of surviving sporogony (PSS) of each species. The ratio of ‘‘probability of surviving P. falciparum sporogony’’ divided by the ‘‘probability of surviving P. vivax sporogony’’ estimates the degree to which a mosquito population will survive to transmit falciparum malaria as compared to vivax malaria. For survival analysis, we used the ‘‘survival and failure time analysis’’ module supplied in StatisticaTM version 6.0 (StatSoft, Inc., Tulsa, OK). The survival function was estimated using the product-limit estimator method, as described by Kaplan and Meier (1958) [37]. A life table was created so that each time interval contains exactly one case. Multiplying out the survival probabilities across the ‘‘intervals’’ (i.e., for each single observa- tion) the estimates survival function (S(t)) was calculated directly from the continuous survival times as the non-parametric maximum d p likelihood estimate using S(t)~ P ti~1 i{j i{jz1 d(j) , where P denotes the multiplication (geometric sum) across all cases less than or equal to t; i is the total number of cases, d(j) is a constant that is either 1 if the j’th case is uncensored (complete), and 0 if it is censored. likelihood estimate using S(t)~ P ti~1 i{j i{jz1 d(j) , where P denotes the multiplication (geometric sum) across all cases less than or equal to t; i is the total number of cases, d(j) is a constant that is either 1 if the j’th case is uncensored (complete), and 0 if it is censored. Fitting of survival times with weighted least squares fit and survival-failure analysis Mosquitoes were grouped according to age (x) by counting the number of dilatations in the ovarioles and the number of specimens (y) in each category was determined. A regression procedure attempted fitting of the life table into four theoretical distributions (exponential, Weibull, Gompertz, and linear hazard) based on algorithms proposed by Kennedy and Gehan (1971) [35]. The logarithmic transforms of the hazard functions of all four theoretical distributions were considered linear functions of the log-trans- formed survival times. Fitting was performed using unweighted least squares goodness of fit, as well as two methods of weighted least squares, as proposed by Gehan and Siddiqui (1973) [36]. Fitting was evaluated by observing the Kolmogorov-Smirnoff goodness-of-fits statistics and P-values, using a significance level of a = 0.01. The presence of few categories and small sample sizes in older groups (.4 parous mosquitoes providing cell counts under 5) prevented distribution fitting using chi-square goodness-of-fit. PLoS ONE \| www.plosone.org Accounting for recruitment fluctuations Survival rates based on the proportion parous are only reliable when there is constant recruitment [22]. For example, if one increases the number of emerging adults, parous rates will tend to decrease, if mortality is kept constant. Larval density may also influence adult mosquito survival because high densities decrease pupal and adult size and fitness [24]. Concomitant larval studies verified that the quantity of larval habitats and larval densities were high during the dry season in a temporary river. On the contrary, larval densities were low during the wet season and the species apparently disappeared for one month in the river, presumably due to flushing away of larvae [25–26]. Meanwhile, small dams provided stable yearlong larval habitats with almost Anopheles darlingi mosquitoes of potentially dangerous age are here defined by those females surviving long enough for complete development of P. vivax to an infective stage in their salivary glands (i.e. the time needed for completion of at least one sporogonic cycle). The density of dangerously aged mosquitoes (q) can be estimated by q~HBR\|pn, where HBR = human biting rate (mosquitoes per man/night). Biting densities were directly measured using the mean number of mosquitoes retrieved in 12 hour collections and are indicated in/man/night. PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e22388 August 2011 \| Volume 6 \| Issue 8 \| e22388 3 Mosquito Survivorship and Malaria in the Amazon August 2011 \| Volume 6 \| Issue 8 \| e22388 analysis: miscellaneous tests and associations Two-tailed Mann-Whitney U tests were used for comparing the means of variables, with the wet and dry seasons as the grouping variable. Spearman’s rank correlation was used for studying the association of various variables. Forward multiple regressions were used for exploring the association of daily survival rates with meteorological variables and of log(n+1) malaria cases with adult mosquito variables. For describing pluviometry, three different parameters were compared: monthly pluviometry, the number of wet days, and degree of wetness. The number of wet days was defined as the number of days per month with more than 1 mm/m2. The degree of wetness was calculated by the product of monthly rainfall and total number of rainy days. Log transformations were performed when necessary for meeting assumptions on the normal distribution of residuals. Appearance of the ovariolar stalks after oviposition and duration of the gonotrophic cycle The appearance of ovariolar stalks after oviposition is shown in Table 2, as well as gonotrophic cycle duration. For obtaining cells .5, grouping of categories A–C and D–E was necessary, resulting in a 262 table for January and July 2004. A Yates corrected chi- square test of the table determined that the two groups differed significantly (X2 = 10.07; P,0.005). A generalized simple linear regression model was used for exploring the association of the density of dangerously aged mosquitoes with the log(n+1) malaria cases. Parous rates and survivorship of dangerously aged mosquitoes Daily survival rates, expectations of life, and expectations of infective life (for P. vivax) for An. darlingi in each collection period are presented in Table 3. For modeling percentages, the logit model was preferred over linear regression, because it is not based on any assumptions regarding the variance among collections periods. Additive logistic regression modeling was used for exploring the association of the dichotomic variable parity status (nulliparous or parous output) with the number of wet days per month. Since daily survival rates are derived from a transformation of parous percentages, a logit model was also used for associating daily survival rates with the log(n+1) malaria cases. The sporogonic cycle durations and PSS of An. darlingi for the complete development of P. vivax varied from 0.52% in August to 50.41% in January, as shown in Table 3. The proportion of mosquitoes surviving through 10 days is also given. The mean biting density of An. darlingi is indicated, as well as the estimated biting density of dangerously aged mosquitoes, which varied from 0.004 to 2.057 mosquitoes/man/night in August and January, respectively. Polovodova’s technique To confirm the accuracy of survival rates determined by Davidson’s method, survival was estimated using multiparous dissections. We performed Polovodova’s technique [2] for counting follicular stalk dilatations and determining survival rates For comparing survival curves obtained in January with July, both Breslow-Gehan’s generalized Wilcoxon test and the Cox’s F test were performed. PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e22388 4 Mosquito Survivorship and Malaria in the Amazon Mosquito Survivorship and Malaria in the Amazon Data analysis: miscellaneous tests and associations Data analysis: miscellaneous tests and associations Longitudinal variations of rainfall, daily survival rates, adult mosquito human biting rates, and density of dangerously aged mosquitoes are graphically shown in Figure 1. Rainfall appeared to be inversely related with daily survival rates, density of dangerously aged mosquitoes and malaria cases. General results (%) A (no contraction) 58 (50.88) 54 (80.6) B (first signs of contraction) 4 (3.51) 0 (0) C (noticeable contraction) 2 (1.75) 0 (0) D (large dilatations formed) 1 (0.88) 0 (0) E (well defined dilatations) 49 (42.98) 13 (19.4) Total 114 (100) 67 (100) Gonotrophic cycle duration [2+(A+B+C)/ (A+B+C+D+E)] 2.43 2.19 doi:10.1371/journal.pone.0022388.t002 November January March May July August 0.48 57.46* 9.05* 17.99 0.11 November 48.35 4.90 12.22 0.23 January 23.29 13.03 97.97 March 1.38 13.43 May 29.79 longer the sporogonic cycle of the parasite, the more it will be affected by a decrease in daily survival rates. from the other three collections, i.e. January, March and May. Also March and May had similar rates among each other, but that differed from other months. January differed from all the other months. The Chi-square statistic together with the parous proportions and Bliss 95% CI permitted identification of two groups: one comprising January, March and May; and a second group August, November and July. The first group was considered as representing the dry season collections and the second group, the wet season. May was grouped as dry season presumably because collections were performed during the first half of the month, while heavy raining started mainly after the middle half of the month. Estimated and observed density factors: determining constant or variable recruitment The longevity factor between January and July was determined as 708.63. The density factor expected by the ratio of life expectations was 6.30, which means that mosquito density was expected to have decreased 6.3 times, i.e., from 4.08 mosquitoes/ man/night, in January, to 0.65 mosquitoes/man/night in July. In July, the measured mosquito density was 0.60 mosquitoes/man/ night, which corresponds to a 6.80 times decrease, almost equal to the predicted value by life expectation ratios. This suggests that the observed changes in density were secondary to decreased survival rates alone and not decreased breeding. The data is consistent with no significant variation in recruitment between the two periods. The data also mean that variations in survival rates were over two orders of magnitude more important than variations in density, in reducing the capacity to transmit malaria. Probabilities of surviving sporogony of different Plasmodium spp pp The PSS in each collecting period, for P. falciparum and P. malariae are shown in Table 5. Also shown are the PSS ratios between these species and P. vivax. A PSS ratio of 1.0 would predict similar malaria transmission rates and ,1.0 less efficient transmission than that of P. vivax. This ratio was not constant but varied along the year, reaching minimum values (less efficient transmission) in August and July. This means that variations in mosquito survival rates affect the transmission of Plasmodium spp. differently, according to the duration of the sporogonic cycles. The doi:10.1371/journal.pone.0022388.t003 General results Yates corrected chi-square statistic test between parous rates of An. darlingi in different collection periods are shown in Table 4. The number of nulliparous and parous females were relatively similar in July, August and November, as deduced from the non- significant chi-square statistics between July–August, July–Novem- ber and August–November. However, these differed significantly A total of 2,193 An. darlingi were captured and 756 (34.47%) were dissected for determining daily survival rates and/or gonotrophic cycle duration. The number of malaria cases per month in this area is shown in Table 1. In total, 186 malaria cases of P. vivax malaria were reported. Only two P. falciparum cases were encountered and none of P. malariae. Figure 1. Davidson’s survival rates for Anopheles darlingi females dissected, number of malaria cases, log rainfall, density of adult female mosquitoes and of epidemiologically dangerous mosquitoes. Data was obtained in Sideroad 19, from different collection periods, from August, 2003, to July, 2004. Number of malaria cases were obtained from January 2002 to December 2004. doi:10.1371/journal.pone.0022388.g001 Figure 1. Davidson’s survival rates for Anopheles darlingi females dissected, number of malaria cases, log rainfall, density of adult female mosquitoes and of epidemiologically dangerous mosquitoes. Data was obtained in Sideroad 19, from different collection periods, from August, 2003, to July, 2004. Number of malaria cases were obtained from January 2002 to December 2004. doi:10.1371/journal.pone.0022388.g001 August 2011 \| Volume 6 \| Issue 8 \| e22388 PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e22388 5 Mosquito Survivorship and Malaria in the Amazon Table 4. Yates-corrected chi-square statistics between number of nulliparous and parous Anopheles darlingi in different collection periods. November January March May July August 0.48 57.46** 9.05* 17.99 0.11 November 48.35 4.90 12.22 0.23 January 23.29 13.03 97.97 March 1.38 13.43 May 29.79 Significant results are indicated: Bonferroni corrected P,0.05; Bonferroni corrected P,0.001. doi:10.1371/journal.pone.0022388.t004 Table 2. Appearance of ovariolar stalks of An. darlingi after oviposition determined in January and July 2004. Appearance of ovariolar stalks January No. (%) July No. Survival curves The number of mosquitoes in each multiparous dissection age category is summarized in Table 6. Weighted linear regression demonstrated significant fitting of the exponential curves shown in Table 3. Number of nulliparous and parous Anopheles darlingi females dissected in different collection periods, from August, 2003, to July, 2004. No. of nulliparous females dissected No. of parous females dissected Total dissected Proportion parous (Bliss 95% CI) Daily survival rates P. vivax sporogonic cycle duration, in days Probability of surviving sporogony (probability of surviving 10 days) in % Expectation of life (expectation of infective life), in days Mean An. darlingi biting density (estimated mean biting density of mosquitoes surviving .1 sporogonic cycle) in mosquitoes/man/ night Aug 53 17 70 0.25 (0.15–0.36) 0.524 8.14 0.52 (0.16) 1.55 (0.01) 0.72 (0.004) Nov 54 24 78 0.31 (0.20–0.41) 0.571 7.34 1.64 (0.37) 1.86 (0.04) 0.84 (0.014) Jan 30 114 144 0.80 (0.72–0.86) 0.911 7.29 50.41 (39.43) 10.40 (5.16) 4.08 (2.057) Mar 50 47 97 0.48 (0.38–0.59) 0.742 7.24 11.54 (5.07) 3.35 (0.39) 5.64 (0.650) May 48 65 113 0.57 (0.48–0.67) 0.812 8.68 13.88 (10.27) 4.39 (0.61) 6.24 (0.866) Jul 185 69 254 0.27 (0.24–0.31) 0.557 9.13 0.45 (0.29) 1.68 (0.01) 0.6 (0.003) Total 420 336 756 Percent parous, Davidson’s survival rates, probabilities of surviving Plasmodium vivax sporogony, expectations of life and mean An. darlingi biting densities were determined. doi:10.1371/journal.pone.0022388.t003 s and parous Anopheles darlingi females dissected in different collection periods, from August, 2003, Table 3. Number of nulliparous and parous Anopheles darlingi females dissected in different collection periods, from August, 2003, t J l 2004 Table 3. Number of nulliparous and parous Anopheles darlingi females dissected in different collection pe to July, 2004. Percent parous, Davidson’s survival rates, probabilities of surviving Plasmodium vivax sporogony, expectations of life and mean An. darlingi biting densities were determined. doi:10.1371/journal.pone.0022388.t003 August 2011 \| Volume 6 \| Issue 8 \| e22388 6 Mosquito Survivorship and Malaria in the Amazon Table 5. Sporogonic cycle durations for Plasmodium falciparum and P. malariae, probability of surviving sporogony, density of dangerously aged Anopheles darlingi and probability of surviving sporogony ratios between these species and P. vivax. Plasmodium falciparum Plasmodium malariae Sporogonic cycle duration (days) Probability of surviving sporogony (PSS) Dangerously aged An. Additive logistic model of parous rates and the number of wet days Cox’s F-Test revealed significant differences between survival rates per gonotrophic cycle in January and July (F (500, 288) = 1.92; T1 = 186.9; T2 = 207.1; P,,0.0001), with higher survival rates in January. Similarly Gehan’s Wilcoxon Test was also significant (test statistic = 27.52; P,,0.0001) for differences between these two months. Logit regression was performed using parity status as the dependant variable, the number of wet days as the independent variable, and the percent parous or nulliparous mosquitoes as the count variable. The regression equation obtained was y=0.92120.097Nx (X2=73.50 p,,0.001). This permits calculating the estimated %parous according to the number of wet days (x). If x=30, y=21.99, for example, parity can be obtained by inverting the logit equation as follows: parity~ ey 1zey =0.12, i.e. a parous rate of 12%. If the number of wet days is 0, parous rates of 72% would be obtained, while 15 wet days correspond to a 37% parous rate. Survival curves darlingi biting density (mosquitoes/ man/night) falciparum/vivax probability of surviving sporogony ratio Sporogonic cycle duration (days) Probability of surviving sporogony malariae/vivax probability of surviving sporogony ratio Aug 9.740 0.002 0.001 0.356 12.630 0.000 0.055 Nov 8.670 0.008 0.007 0.475 11.250 0.002 0.112 Jan 8.600 0.446 1.818 0.884 11.160 0.350 0.695 Mar 8.540 0.078 0.442 0.679 11.080 0.037 0.319 May 10.470 0.092 0.576 0.665 13.580 0.045 0.327 Jul 11.100 0.001 0.001 0.311 14.400 0.000 0.044 doi:10.1371/journal.pone.0022388.t005 Table 5. Sporogonic cycle durations for Plasmodium falciparum and P. malariae, probability of surviving sporogony, density of dangerously aged Anopheles darlingi and probability of surviving sporogony ratios between these species and P. vivax. Table 5. Sporogonic cycle durations for Plasmodium falciparum and P. malariae, probability of surviving sporogony, density of dangerously aged Anopheles darlingi and probability of surviving sporogony ratios between these species and P. vivax. Number of malaria cases in dry and wet seasons The number of malaria cases in the dry and wet seasons differed significantly by Mann-Whitney U test (U = 4.50; P,0.05). The number of malaria cases was higher during the dry season. Forward stepwise multiple regression for associating malaria cases with adult mosquito variables To explore the association of daily survival rates with meteorological parameters, survival rates in each month were used as the dependant variable in a forward multiple stepwise regression. Independent variables were the number of mosquitoes dissected, the number of wet days in the month of collection, adult densities, monthly rainfall, degree of wetness, temperature and humidity. The only significant variable to enter the model was the number of wet days (R2 = 0.80; F (1, 4) = 16.16; b = 20.89, SE b = 0.22; B = 20.01; P,0.05). A forward stepwise multiple regression was performed using the log(n+1) number of malaria cases as the dependent variable. Independent predictor variables were daily survival rate, parity ratio, adult densities, rainfall, number of mosquitoes surviving more than 10 days, temperature, and humidity. The only variable to be admitted in the model was daily survival rate (R2= 0.92; F (1, 4)= 23.36; b =0.92, SE b =0.19; B =1.65; P,0.01). Spearman’s rank correlation of daily survival rates with meteorological variables the Kaplan-Meier diagram (Figure 2). The observed number of mosquitoes is shown as the cumulative proportion surviving and a logarithmic % survival scale was used for obtaining linear regression curves. For January, R2 = 0.91, F(1, 142) = 755.31 (P,,0.001). For July, R2 = 0.97, F(1, 248)= 6985.55 (P,,0.001). The regression equation, determined in January, was y = 4.087Ne(xN-0.426), where x corresponds to the number of gonotrophic cycles that have already been completed. The regression equation, determined in July, was y = 5.177Ne(xN-1.148). Survival rates per gonotrophic cycle and per day were determined as shown in Table 6. Spearman’s rank correlation between daily survival rates and the number of wet days was significant with non-corrected P-levels (r = 20.88; P,0.05), but only marginally significant with Bonfer- roni adjusted significance (P = 0.07). Spearman’s rank correlation was not significant between daily survival rates and temperature (r = 0.02; Bonferroni adjusted P.1.0), humidity (r = 20.42; Bonferroni adjusted P.1.0) or degree of wetness (r = 20.25; Bonferroni adjusted P.1.0). Comparison of multiparous survival curves with daily survival rates With Davidson’s method we determined daily survival rates of 91% and 55% in January and July, respectively. The survival rate for completing one sporogonic cycle (pn) corresponds to 79.1% in January and 27.1% in July, while the survival rates determined by multiparous dissections for each sporogonic cycle were 65% in January and 30% in July (Table 6). Parity in wet and dry seasons A two-tailed Mann-Whitney U test of the percent parous in the dry and rainy seasons revealed significant differences (P,0.05), with higher parous rates in the dry season. Spearman’s rank correlation between adult densities and the log(n+1) of malaria cases in Sideroad 19 was not significant PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e22388 7 Mosquito Survivorship and Malaria in the Amazon Table 6. Number of dilatations in collections of Anopheles darlingi collected in January and July, 2004. No. of dilatations January 2004 No. (%) July 2004 No. (%) 0 53 (30.4) 185 (71.4) 1 40 (22.9) 38 (14.7) 2 30 (17.2) 18 (6.9) 3 17 (9.8) 8 (3.1) 4 3 (1.7) 1 (0.4) 5 1 (0.6) 0 (0) Uncountable 30 (17.2) 9 (3.5) Total 174 (100) 259 (100) Survival rate per cycle (per day*) 0.65 (0.83) 0.30 (0.58) = Presented sac-like dilatations and counting could not be performed because abortive ovarioles were not encountered. ** = using a 2.43 and 2.19 day long cycle in January and July, respectively. doi:10.1371/journal.pone.0022388.t006 per year (30 cases in three years) would be expected if parous rates were around 71%, but only 1 case per year if parous rates were around 16%. Defining the importance of adult density and daily survival rates: forward multiple regression (r = 0.70; Bonferroni adjusted P.0.72). But rank correlation was significant between daily survival rates and the log(n+1) of malaria cases in Sideroad 19 (r = 0.88; Bonferroni adjusted P,0.05). For differentiating adult densities from daily survival rates a forward multiple stepwise regression was performed using the log(n+1) malaria cases as the dependent variable and adult densities (human An. darlingi biting rate) and daily survival rates as predictor variables. A significant model (R2 = 0.81, F(1,4) = 16.98; P,0.05) was achieved only if daily survival rates was the only variable to be admitted. Also, higher b was found with daily survival rates (b = .072) than with adult densities (b = 0.21) in the two variable marginally significant model (R2 = 0.82, F(2,3) = 7.03, P = 0.07). This suggests that malaria incidence is better explained by daily survival rates than adult mosquito density. Simple regression of log malaria cases and biting density of dangerously aged mosquitoes A simple regression between the log(n+1) of malaria cases as the dependant variable and the density of dangerously aged mosquitoes as the grouping variable showed a significant association between the two parameters (R2 = 0.71, F(1, 4) = 9.97; P,0.05). The regression equation obtained, converted from log(n+1) cases to number of malaria cases, was the following: no:malaria~100:916zx:0:294{1, where x = density of dangerously aged mosquitoes. This means that a density of one dangerously aged An. darlingi per night would be associated with 5 cases of malaria per year in Sideroad 19 (15 in three years). For every increase in one dangerously aged mosquito per night the number of malaria cases doubles: two is associated with 10 cases per year, three with twenty etc. * = Presented sac-like dilatations and counting could not be performed because abortive ovarioles were not encountered. ** = using a 2.43 and 2.19 day long cycle in January and July, respectively. doi:10.1371/journal.pone.0022388.t006 Detinova’s classification There is still incomplete knowledge of the time needed for moving between each stage of sac-like dilations after oviposition occurs. Studies have traditionally considered sacular dilation to be present or absent. In this study we arbitrarily considered types A to C as representing recently oviposited females. Confusion could arise when classifying C and D dilations as recently oviposited or not, but these represented together less than 3% of dissected females. A, B and E types accounted for the majority of mosquitoes dissected. To our knowledge, this is the first attempt to relate Detinova’s sacular classification to gonotrophic cycle duration. We propose that the use of this classification may be advantageous, compared to the two category method, since it permits regression modeling and more accurate weighted means can be obtained. An accurate gonotrophic cycle determination is particularly important due to the sensibility of survival data to this estimate. On assuming constant gonotrophic cycles in longitudinal studies To our knowledge, this is the first study to document seasonal variation in gonotrophic cycle duration, for a Neotropical anopheline. We caution that using a constant gonotrophic cycle duration for fitting parous rate data must be performed with care. It may obscure changes in breeding site availability that may occur in accordance to rainfall. However, in our study, the gonotrophic cycle variations estimated in the dry and wet seasons was not important and controlling for cycle duration with a constant parameter would not change any of the observations, since very similar results would have been obtained if any constant gonotrophic cycle from 2.5 to 4.5 days was to be used. Shorter gonotrophic cycle durations in each season could reflect the increased abundance of larval habitats in the wet season, which appears to have been more determinant than the Methodological limitations: spatial distribution of larval habitats; possible subtle temporal variations in gonotrophic cycle duration; and sample size considerations For estimating adult survival rates, first the durations of the gonotrophic cycles must be accurately known. Limited informa- tion is available on the duration of the gonotrophic cycle of An. darlingi and methodological aspects have not been well defined. In Aripuana˜, Mato Grosso State, Central Brazil, Charlwood & Wilkes (1979) [38], reported that only a few parous females presented unstretched ovariolar terminals, and interpreted this as indicating that the gonotrophic cycle duration required at least three days. Charlwood’s minimum cycle method compared well with capture-recapture experiments in Jaru, Rondonia, Western Amazon, where Charlwood & Alecrim (1989) [20] determined that An. darlingi had an approximate 2.3 day oviposition cycle. The same authors also proposed a re-analysis of the data presented by Roberts et al. (1983) [17] arriving at a 2.6 day long cycle, but the data from the latter authors may have been slightly skewed because dissections were performed the following morning after collection. Our estimates of gonotrophic cycle durations for An. darling are within the range observed in the literature. We determined the gonotrophic cycle duration only twice during the year, by performing dissections soon after capture on a large number of mosquitoes. It is possible that short term temporal variations in cycle duration may have been overlooked. Also, minimal sample sizes for Charlwood’s minimum cycle method and the scale of temporal variations are not yet understood. More studies are necessary to better characterize the relative role of meteorological and geographic variables and to demonstrate how cycle durations vary in time. We suggest that capture-recapture experiments or laboratory studies focusing on the time needed for ovariolar stalk contraction may be particularly instructive. It is possible that the presence or proximity to dams may have biased the data. Dams may maintain constant recruitment because they form stable larval habitats, as compared to rivers. Adult density and survival rates near temporary rivers were not evaluated in this study. The proximity to fish-farming dams may also increase the proportion of parous to nulliparous mosquitoes, because ovipositing females tend to actively seek and concentrate at these areas [40]. The existence of spatial heterogeneity of parous rates was not evaluated in the presence study. Logistic regression of malaria cases and daily survival rates Logit regression was performed using parity status as the dependant variable, the log(n+1) number of malaria cases per month as the independent variable, and the percent parous or nulliparous mosquitoes as the count variable. The regression equation obtained was y = 23.344+2.851Nx (X2 = 73.75; P,,0.001). This means that approximately 10 cases of malaria Figure 2. Kaplan-Meier cumulative proportions plot with weighted least squares exponential regression fitting for Anopheles darlingi caught in January (unbroken line and circles) and July 2004 (dotted line and squares). doi:10.1371/journal.pone.0022388.g002 Figure 2. Kaplan-Meier cumulative proportions plot with weighted least squares exponential regression fitting for Anopheles darlingi caught in January (unbroken line and circles) and July 2004 (dotted line and squares). doi:10.1371/journal.pone.0022388.g002 August 2011 \| Volume 6 \| Issue 8 \| e22388 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 8 Mosquito Survivorship and Malaria in the Amazon relatively lower mean daily temperatures in the wet season (26uC, as compared to 29uC). Notes on the accuracy of Charlwood’s minimum cycle method Notes on the accuracy of Charlwood’s minimum cycle method Although Charlwood’s minimum cycle method appears to be of limited applicability because the possibility of the real cycle being longer than the estimate, the simplicity of the method is particularly useful for repeated measures studies. The method makes the following assumptions: 1- all females with well defined dilations (type E) have returned to bite no more than 24 hours after oviposition, i.e. have gonotrophic cycles of three days; 2- there is no delay between blood-feeding and oviposition; 3- the duration of egg development is constant. To account for the possibility of longer cycles, we performed the same statistical analyses assuming that these females had returned 2, 3, 4… up to 10 days after oviposition. Longer gonotrophic cycles increase the daily survival rates during the wet season disproportionally more than in the dry season, decreasing the overall magnitude of variance between seasons. However, non-significant statistical levels for the analyses performed (P.0.05) could only be obtained if one assumed that mosquitoes with well defined dilations had taken at least 4–5 days to find a host after oviposition, i.e. had gonotrophic cycles of 6–7 days. This is a highly unlikely scenario, since the variation in the literature has ranged from 2.3 to 4.4 days for this species, with different sampling methods [7] and the longest reported Nyssorhyncus cycle is 5 days [39]. Sideroad 19 differs from other sideroads in the area by the quantity of dams, and this may cause variation in gonotrophic cycle duration. Fish-farming dams are known important An. darlingi larval habitats [41]. While 10 dams were present in Sideroad 19, neighboring sideroads had a mean of only 4.25 dams (SD = 2.2). The mean distance between fish-farms and adult collecting stations used in the study was only 110 m (SD = 26.4 m). This distance decreased by 5–10 m during the wet season. More studies are necessary to demonstrate if gonotrophic cycle durations vary in different geographical areas. We caution that parity studies that make exclusive use of parous rates, instead of multiparous dissections, should attempt to control for recruitment fluctuations. To enable this, knowledge of larval binomics in the study area is highly recommended. Mosquito Survivorship and Malaria in the Amazon Mosquito Survivorship and Malaria in the Amazon predictor of daily survival rate was the number of wet days per month. We suggest that heavy rainstorms may cause increased adult mortality by direct impact of droplets on resting or flying mosquitoes. The frequency of raining may be more important than the amount of rain, but more studies are necessary to better evaluate this. Rainfall in Southern Roraima is of the convective type, with large drop size, high rain-rates and large amounts of water per downpour, typically reaching 20 to 40 mm/m2 in 2– 3 hours. Wind speed was not systematically measured in this study, but values from 12 to 18 km/h were common, as measured visually by the Beaufort scale, and could influence mosquito flight, dispersal [42] or even survival. significant with daily survival rates but not with density. This data suggests that malaria incidence in Sideroad 19 was better explained by daily survival rates than adult densities. To our knowledge, this paper is the first statistical study of variation of survival rates of Neotropical malaria vectors in relation to malaria incidence. Variations in malaria transmission have been related to mosquito survival rates per extrinsic incubation period, but not survivorship per feeding cycle [44]. In the Maroni River, French Guiana, we performed a reanalysis of the entomological data collected by Fouque et al. (2010) [7] with permission of the author, comprising almost 2,700 dissections during two years. The total number of mosquitoes, the number of parous mosquitoes and the number of malaria cases were significantly higher during the dry season, as compared to the wet season, by Mann Whitney U tests (P,0.001). Spearman rank correlation was significant (P,,0.001) between the number of malaria cases in Maripasoula and the number of parous mosquitoes per month. The simple linear regression equation between the number of malaria cases and the number of parous mosquitoes was also significant (P,,0.001), although neither survival rates alone nor mosquito density alone produced significant regressions with malaria incidence. We verified that recruitment fluctuations, secondary to decreased breeding rates, were likely to have occurred because decreases in adult density were higher than that explained by decreases in survival alone. These results suggest that, in the riverine Maroni region, where there are no dams, both survival rates and density variations caused by changes in larval breeding rates may be correlated to malaria incidence. Mathematical basis for comparisons of survival and density The greater influence of parous rates in determining transmis- sion can be explained by analyzing classic malaria modeling formulae [43]. Malaria incidence varies in a 1:1 proportion to the density of vectors and in a 1:1 proportion to the expectation of infective life ( pn {loge p). In this sense, a 16 fold increase in entomological inoculation rate could be brought about by an increase in 16 times in the man-biting rate or by an increase in 20% in survival rate [4]. Wet season-predominant alluvial malaria appears to differ epidemiologically from frontier zone or highland dry season malaria Parity studies may be particularly useful for explaining malaria incidence in areas where there is little seasonal change in anopheline biting rates. In this sense, malaria transmission in frontier zones appears to be epidemiologically distinct from alluvial malaria [45–46]. In frontier zones, the absence of large rivers and seasonal flooding, mean that larval habitats are relatively scarce and mosquito densities are lower. Vector breeding is dependent on the natural or artificially created water collections, being less dependent on the level of the water table. Malaria transmission occurs during the dry season [8,46–47] or the dry-wet and wet-dry transitions [9,21,48]. Meanwhile, near large rivers, the role of female mosquito density could be more important than survival because flooding occurs during the wet season, increasing the larval population and high adult densities are obtained [49– 51]. Longitudinal survival studies should keep this geographical Mosquito Survivorship and Malaria in the Amazon It is possible that in areas near year-round stable water collections only survival rates will be important for malaria incidence, while near small temporary rivers, where breeding may vary due to wet season flushing of larvae, both variations in survival rates and breeding will influence transmission. Capture-recapture experiments performed by Charlwood & Alecrim (1989) [20], at the beginning of the rainy season in that region of the Amazon, yielded daily survival rates of 83%. For the same period, we reported 81%, in May. A reanalysis of Charlwood’s (1980) [21] data from Mato Grosso, during the end of the rainy season in that region, was performed. Exponential regression models using the table provided by these authors, calculated daily survival rates of 63% with Davidson’s method, using the 2.3 day long cycle proposed by Charlwood & Alecrim (1989) [20]. We report 57% for the same season in Roraima. Fouque et al. (2010) [7] have also reported higher survival rates in the dry season than the wet season, in the Maroni area of French Guiana. Studies suggest that An. darlingi survival rates may vary as a function of seasonal influences. Variations may occur synchro- nously throughout areas of the Amazon with similar rainfall patterns, but more studies are necessary to verify this. Methodological limitations: possible subtle temporal variations in parous rates Parous rates were only determined once every two months. It is possible that transient variations in Davidson’s parous rates, in the order of weeks, could have been missed in the study. Fouque et al. 2010 [7], reported variations around 10–30% (SD = 12–24%) from one month to the next, but the possibility of recruitment fluctuation hindered better evaluation. More studies, with smaller sampling intervals and larger samples, are needed to verify more precisely how these rates vary in time. Fouque et al. (2010) [7] have proposed, as an alternative to the vectorial capacity for longitudinal studies, the use of a more simplified parameter describing the number of infected mosquitoes able to transmit malaria (IMT), calculated by IMT~HBR:pn:b, where b = proportion of infected mosquitoes. However, longitudi- nal studies frequently fail to encounter infected mosquitoes on many occasions and entolomological inoculation rates are determined with pooled data for the entire period of observation. We believe that b should be omitted from the equation, as was done in this study, to arrive at the simpler dangerously aged mosquitoes equation: q~HBR:pn. Variation in daily survival rates appears to be related to rainfall In our study, daily survival rates were lower during the wet season, as compared to the dry season. The best meteorological PLoS ONE \| www.plosone.org PLoS ONE \| www August 2011 \| Volume 6 \| Issue 8 \| e22388 PLoS ONE \| www.plosone.org 9 August 2011 \| Volume 6 \| Issue 8 \| e22388 Mosquito Survivorship and Malaria in the Amazon heterogeneity in mind. Parous rate studies using Davidson’s method may yield incorrect results near large rivers, where recruitment fluctuations are important. decreases in daily survival rates than P. vivax. This is compatible with the prevalence of Plasmodium spp. in Sideroad 19, where only two cases of P. falciparum malaria were detected, both in the dry season, and no cases of P. malariae, based on thick smears only. These results were not confirmed with Polymerase Chain Reaction (PCR) and asymptomatic subjects were not regularly sampled. Final comments Our results indicate that survival rates of An. darlingi may vary in accordance to rainfall and the wet season could be associated with lower adult survival rates, resulting in decreased malaria transmission. More studies are necessary to better evaluate the influence of meteorological parameters with survival rates. In small temporary rivers, An. darlingi breeding may be limited to the dry season [25–26]. Rainfall may cause larval mortality in small rivers by the following mechanisms: flushing out larvae with strong currents, direct impact of raindrops on the water, depending on the size of raindrops; ejection of immature stages onto muddy surroundings; and exhaustion of larvae by constantly moving away from the surface to avoid being struck by raindrops [58]. Malaria transmission in this setting would be limited by both mosquito survival and breeding rates. But the construction of small dams, blocking small waterways, may enable breeding throughout the year [25–26]. Dams permit constant recruitment of relatively small densities of An. darlingi and Barros et al (2011) [26] have proposed that the species prefers areas with obstructions to river flow, decreased luminance (shade) and proximity to human dwellings. Malaria transmission in this setting would be basically limited by mosquito survival alone. It appears that anthropic modification of the environment may remove the natural balance of limiting factors such as river currents caused by heavy raining [58]. Determining key transmission factors may help direct control efforts. If survival is the limiting factor, residual insecticide spraying would be particularly useful. If larval and/or adult Multiparous dissections can be used to validate Davidson’s daily survival rates and gonotrophic cycle durations Hypoendemic frontier malaria in the Amazon, the second colonization phase of frontier malaria [45], is composed mainly of P. vivax, followed by P. falciparum. Cases of P. malariae are considered to be relatively uncommon, when only thick smears are analyzed. We postulate that the more efficient transmission of P. vivax could be secondary to a vector survival-dependent limiting factor for disease transmission, compatible with sporogonic cycle durations of Plasmodium spp. It is possible that performing effective insecticide spraying increases the P. vivax to P. falciparum malaria ratios. The relative prevalence of Plasmodium spp. in the human population may also aid in identifying localities where mosquito survival is the limiting factor in disease transmission, but more studies are necessary to verify this. By comparing multiple methods of age determination in Anopheles farauti, Charlwood (1986) [52] suggested that rates based on multiparous dissections were more reliable than rates determined by parous dissections. This method does not require calculating gonotrophic cycle durations. For this reason, we dissected mosquitoes for obtaining survival curves. In our study, daily survival rates obtained by Davidson’s method compared well to the survival curves by multiparous dissections. The good correlation between the two data sets suggests that the gonotrophic cycle durations and daily survival rates that were determined with Davidson’s method were relatively accurate. Duration of the sporogonic cycle of Plasmodium spp Duration of the sporogonic cycle of Plasmodium spp The duration of the sporogonic cycle of P. vivax and other Plasmodium spp. in An. darlingi is poorly characterized. Although long experience with the Moshkovsky method in the former USSR has shown that it is useful for epidemiological analysis in temperate climates, we are unaware of any systematized validation with tropical malaria strains. For this reason, when we report the estimated percent of mosquitoes surviving enough to transmit malaria, both the percentage that would survive one or more cycle, as estimated by the Moshkovsky method, as well as the percentage that would survive 10 days is given, to show the results if temperature-dependant changes in sporogonic cycle duration were ignored. Relatively similar data were obtained with either method, with more dangerously aged mosquitoes in the dry season. However, the density of dangerously aged mosquitoes was better associated with malaria incidence than the 10 day old densities. For the latter parameter, the simple linear regression with log(n+1) malaria was not significant (P.0.1). Survival curves and constant versus age-dependant survival Many investigators have encountered problems while perform- ing the Polovoda technique for age-grading [53] and Hugo et al. [54] have found that it enabled correct classification of only 57.5% nulliparous and 1, 2 or 3-parous Ae. vigilax females. Hoc & Wilkes (1995) [55] have proposed that ovariole sacs contract to form basal distentions which are larger than the typical dilatations. These distensions would obscure signs of the typical dilatations, i.e. previous gonotrophic activity. The typical dilatations described by Polovodova would form only when follicles degenerate at an early stage during egg development. This means that in parous mosquitoes, multiple ovarioles must be checked to find those with one or more typical dilatations, as was performed in this study. The availability of the new molecular methods may help solve the problem of an adequate age-grading technique [54]. Fitting of the exponential model of survival curves initially suggest constant mortality rates in the different age groups of the mosquito population, rather than age dependant survival rates. However, studies with larger samples are needed to verify this and the existence of age-dependant survival rates is possible. Age- dependant survival would be suggested if the data fitted better a Gompertz [56] or a Weibull distribution rather than an exponential distribution. If mortality is, in fact, age-dependant, models will be more complex, p cannot be regarded as a constant, mathematical equations must be adapted and values recalculated. Age-independent models tend to overestimate the transmission potential of older mosquitoes, overestimating vectorial capacity [57]. Malaria incidence was better related to daily survival rates than adult densities Parous rates and adult densities were well correlated (0.82 correlation). This occurred because the changes observed in density were basically those expected by the decrease in survival rates, suggesting that survival, and not breeding, was the limiting factor determining density. 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Smith DL, McKenzie FE (2004) Statics and dynamics of malaria infection in Anopheles mosquitoes. Malar J 3: 13. Probability of surviving sporogony ratios and duration of the sporogonic cycles The duration of the extrinsic incubation periods of Plasmodium spp. are, in decreasing order: P. malariae.P. falciparum.P. vivax. Longer cycles decrease the probability that a mosquito will survive sporogony. For every day increase in extrinsic incubation cycles the probability of surviving sporogony decreases by pn+1. Plasmodium spp. with longer extrinsic incubation periods, such as P. falciparum and P. malariae, were more affected by seasonal PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e22388 11 Mosquito Survivorship and Malaria in the Amazon their field data for analysis and for the discussions that helped shape this paper. density is determinant, larvicidal methods or increasing the distance between larval habitats and humans should be favored. Acknowledgments Conceived and designed the experiments: FSB MA NH. Performed the experiments: FSB NH. Analyzed the data: FSB. Contributed reagents/ materials/analysis tools: FSB NH MA. Wrote the paper: FSB NH MA. Conceived and designed the experiments: FSB MA NH. Performed the experiments: FSB NH. Analyzed the data: FSB. Contributed reagents/ materials/analysis tools: FSB NH MA. Wrote the paper: FSB NH MA. To Jose´ Francisco Luitgards Moura, for the kind support and for helping our group with logistical problems. To Wanderli P. Tadei for the insightful comments that inspired this work. To Dr. Florence Fouque for providing References q 34. Hoc JQ, Charlwood JD (1990) Age determination of Aedes cantans using the ovarian oil injection technique. Med Vet Entomol 4: 227–33. 35. Kennedy AD, Gehan EA (1971) Computerized simple regression methods for survival time studies. Computer Programs in Biomedicine 1: 235–244. 8. 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(2007) Malaria vector incrimination in three rural riverine villages in the Brazilian Amazon, Am J Trop Med Hyg 76: 461–469. 39. dos Santos RLC, Forattini OP, Burattini MN (2002) Laboratory and field observations on duration of gonotrophic cycle of Anopheles albitarsis s.l. (Diptera: Culicidae) in southeastern Brazil. J Med Entomol 39: 926–930. 11. Rosa-Freitas MG, Tsouris P, Peterson AT, Hono´rio NA, Barros FSM, et al. (2007) An ecoregional classification for the State of Roraima, Brazil. The importance of landscape in malaria biology. Mem Inst Oswaldo Cruz 102: 349–356. 40. Menach AL, McKenzie FE, Flahault A, Smith DL (2005) The unexpected importance of mosquito oviposition behaviour for malaria: non-productive larval habitats can be sources for malaria transmission. Malaria J 4: 23. 12. Chaves SS, Rodrigues CL (2000) An initial examination of the epidemiology of malaria in the state of Roraima, in the Brazilian Amazon basin. Rev Inst de Med Trop de Sa˜o Paulo 42: 269–275. 41. Vittor AY, Pan W, Gilman RH, Tielsch J, Glass G, Shields T, et al. (2009) Linking Deforestation to Malaria in the Amazon: Characterization of the Breeding Habitat of the Principal Malaria Vector, Anopheles darlingi, Am J Trop Med Hyg 81: 5–12. 13. Furley PA The Forest Frontier, Routledge, London. 14. Consoli RAGB, Lourenc¸o-de-Oliveira R (1994) Princ importaˆncia sanita´ria no Brasil. Rio de Janeiro: Fiocruz. 14. p p q pp 57. Bellan SE (2010) The importance of age dependent mortality and the extrinsic incubation period in models of mosquito-borne disease transmission and control, PLoS ONE 5: Available: http://www.plosone.org/article/info:doi/10.1371/ journal.pone.0010165. Accessed 2011 Jun 4. 52. Charlwood JD (1986) Survival rate variation of Anopheles farauti (Diptera: Culicidae) between neighboring villages in coastal Papua New Guinea. J Med Entomol 23: 361–365. 53. Fox AS, Brust RA (1994) How do dilatations form in mosquito ovarioles? Parasitol Today 10: 19–23. 55. Hoc JQ, Wilkes TJ (1995) Age grading in the black fly Simulium woodi (Diptera: Simuliidae), a vector of onchocerciasis in Tanzania. Medical and Veterinary Entomology 9: 16–24. y 54. Hugo RL, Quick-Mile S, Kay BH, Ryan PA (2008) Evaluations of Mosquito Age Grading Techniques Based on Morphological Changes. Journal of Medical Entomology 453: 353–369. 56. Clements AN, Paterson GD (1981) The analysis of mortality and survival rates in wild populations of mosquitoes. J Appl Ecol 18: 373–399. 58. Paaijmans KP, Wandago MO, Githeko AK, Takken W (2007) Unexpected high losses of Anopheles gambiae larvae due to rainfall. Plos ONE 11: 1–7. Available: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001146. Ac- cessed 2011 Jun 4. References Souza-Santos R (2002) Distribuic¸a˜o sazonal de vetores da mala´ria em Machadinho d’Oeste, Rondoˆnia, Regia˜o Amazoˆnica, Brasil. Cad Sau´de Pu´b 18: 1813–1818. 22. Service MW (1993) Mosquito Ecology: Field Sampling Methods, second ed. London: Chapman and Hall. 23. Bliss CI (1967) Statistics in Biology. New York: McGraw-Hill. 49. Guarda JA, Sayag CR, Witzig R (1999) Malaria reemergence in the Peruvian Amazon region. Emerg Infect Dis 5: 209–215. 24. Hawley WA (1985) The effect of larval density on adult longevity of a mosquito, Aedes sirrensis: epidemiological consequences. J Animal Ecol 54: 955–964. 50. Klein TA, Lima JBP (1990) Seasonal distribution and biting patterns of Anopheles mosquitoes in Costa Marques, Rondonia, Brazil. J Amer Mosq Control Assoc 6: 700–707. 25. Barros FSM, Hono´rio NA, Arruda ME (2011) Temporal and spatial distribution of malaria within an agricultural settlement of the Brazilian Amazon. J Vect Ecol 36: 159–169. 51. Magris M, Rubio-Palis Y, Menares C, Villegas L (2007) Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela. Mem Inst Osw Cruz 102: 303–312. 26. Barros FSM, Arruda ME, Gurgel HC, Hono´rio NA (2011) Spatial clustering and longitudinal variation of Anopheles darlingi (Diptera: Culicidae) larvae in a river of the Amazon: the importance of the forest fringe and of obstructions to PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e22388 August 2011 \| Volume 6 \| Issue 8 \| e22388 12 56. Clements AN, Paterson GD (1981) The analysis of mortality and survival rates in wild populations of mosquitoes. J Appl Ecol 18: 373–399. 57. Bellan SE (2010) The importance of age dependent mortality and the extrinsic incubation period in models of mosquito-borne disease transmission and control, PLoS ONE 5: Available: http://www.plosone.org/article/info:doi/10.1371/ journal.pone.0010165. Accessed 2011 Jun 4. 58. Paaijmans KP, Wandago MO, Githeko AK, Takken W (2007) Unexpected high losses of Anopheles gambiae larvae due to rainfall. Plos ONE 11: 1–7. Available: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001146. Ac- cessed 2011 Jun 4. Mosquito Survivorship and Malaria in the Amazon Mosquito Survivorship and Malaria in the Amazon PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e22388 13
https://openalex.org/W4242121991	https://www.clim-past.net/11/1587/2015/cp-11-1587-2015.pdf	English	null	Glacier response to North Atlantic climate variability during the Holocene	null	2,015	cc-by	9,719	Glacier response to North Atlantic climate variability during the Holocene N. L. Balascio1,2, W. J. D’Andrea1, and R. S. Bradley3 1Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY 10964, USA 2Department of Geology, College of William & Mary, Williamsburg, VA 23187, USA 3Department of Geosciences, University of Massachusetts, Amherst, MA 01003, USA N. L. Balascio1,2, W. J. D’Andrea1, and R. S. Bradley3 1Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY 10964, USA 2Department of Geology, College of William & Mary, Williamsburg, VA 23187, USA 3Department of Geosciences, University of Massachusetts, Amherst, MA 01003, USA Correspondence to: N. L. Balascio (nbalascio@wm.edu) Correspondence to: N. L. Balascio (nbalascio@wm.edu) Received: 16 April 2015 – Published in Clim. Past Discuss.: 27 May 2015 Revised: 23 October 2015 – Accepted: 27 October 2015 – Published: 4 December 2015 Received: 16 April 2015 – Published in Clim. Past Discuss.: 27 May 2015 Revised: 23 October 2015 – Accepted: 27 October 2015 – Published: 4 December 2015 Abstract. Small glaciers and ice caps respond rapidly to climate variations, and records of their past extent provide information on the natural envelope of past climate vari- ability. Millennial-scale trends in Holocene glacier size are well documented and correspond with changes in North- ern Hemisphere summer insolation. However, there is only sparse and fragmentary evidence for higher-frequency varia- tions in glacier size because in many Northern Hemisphere regions glacier advances of the past few hundred years were the most extensive and destroyed the geomorphic evidence of ice growth and retreat during the past several thousand years. Thus, most glacier records have been of limited use for investigating centennial-scale climate forcing and feed- back mechanisms. Here we report a continuous record of glacier activity for the last 9.5 ka from southeast Greenland derived from high-resolution measurements on a proglacial lake sediment sequence. Physical and geochemical param- eters show that the glaciers responded to previously docu- mented Northern Hemisphere climatic excursions, including the “8.2 ka” cooling event, the Holocene Thermal Maximum, Neoglacial cooling, and 20th century warming. In addition, the sediments indicate centennial-scale oscillations in glacier size during the late Holocene. Beginning at 4.1 ka, a series of abrupt glacier advances occurred, each lasting ∼100 years and followed by a period of retreat, that were superimposed on a gradual trend toward larger glacier size. Glacier response to North Atlantic climate variability during the Holocene Thus, while declining summer insolation caused long-term cooling and glacier expansion during the late Holocene, climate system dynamics resulted in repeated episodes of glacier expan- sion and retreat on multi-decadal to centennial timescales. These episodes coincided with ice rafting events in the North Atlantic Ocean and periods of regional ice cap expansion, which conﬁrms their regional signiﬁcance and indicates that considerable glacier activity on these timescales is a normal feature of the cryosphere. The data provide a longer-term per- spective on the rate of 20th century glacier retreat and indi- cate that recent anthropogenic-driven warming has already impacted the regional cryosphere in a manner outside the nat- ural range of Holocene variability. Clim. Past, 11, 1587–1598, 2015 www.clim-past.net/11/1587/2015/ doi:10.5194/cp-11-1587-2015 © Author(s) 2015. CC Attribution 3.0 License. Clim. Past, 11, 1587–1598, 2015 www.clim-past.net/11/1587/2015/ doi:10.5194/cp-11-1587-2015 © Author(s) 2015. CC Attribution 3.0 License. 1 Introduction Glaciers and ice caps represent a small but important portion of the cryosphere (∼785 000 km2; Dyurgerov and Meier, 2005). Their mass wasting during the 20–21st century is responsible for 60 % of the sea-level rise unattributable to ocean warming (Meier, 2007) and they continue to retreat at an exceptional rate (Zemp et al., 2012). Moreover, be- cause small glaciers and ice caps respond rapidly to climate changes and there is a strong relationship between glacier mass balance and summer temperature (Oerlemans, 2005), past glacier extent can inform us about past climate variabil- ity. Holocene glacier activity in the Arctic is reasonably well documented at millennial timescales (Miller et al., 2010). Northern Hemisphere glaciers receded in the early Holocene and were smaller than present during the mid- Holocene. Centennial-scale variations, however, are not well constrained because there are few high-resolution and con- tinuous records, and because in many regions the most ex- tensive glacier advances since the early Holocene took place Published by Copernicus Publications on behalf of the European Geosciences Union. 1588 N. L. Balascio et al.: Glacier response to North Atlantic climate variability Figure 1. Location and catchment setting of Kulusuk Lake. The white dashed line marks the watershed boundary and the red dashed line deﬁnes the crest of moraines in front of both glaciers mapped in the ﬁeld. Cores were collected in the deepest basin (red circle). Image: Google, NASA. within the past few hundred years, destroying geomorphic evidence of intervening glacier positions. This is the case in Greenland, where historical (AD 1200–1940) advances of lo- cal glaciers were generally the most extensive since at least the early Holocene (Kelly and Lowell, 2009). y ( y , ) Evidence from the Greenland Ice Sheet (Kobashi et al, 2011), marine sediments (Bond et al., 1997; Thornalley et al., 2009; Moffa-Sánchez et al., 2014; Jiang et al., 2015), and terrestrial archives (D’Andrea et al., 2011; Larsen et al., 2012; Olsen et al., 2012) indicate that abrupt changes in atmospheric circulation and ocean dynamics, including abrupt cooling events, have punctuated the Holocene. These episodes have alternately been attributed to solar variabil- ity, freshwater forcing, volcanic activity, and/or changes in Atlantic Meridional Overturning Circulation (Wanner et al., 2011). How sensitive were glaciers to these abrupt episodes, and did glaciers throughout the North Atlantic respond uni- formly? 2 Study site Kulusuk Lake (0.8 km2, 69 m maximum depth) is located be- low a cirque with two small glaciers and is within a low arctic maritime region (MAT −1 ◦C, MAP 900 mm). Characteris- tic erosional features indicate that local glaciers have tem- perate thermal structures (Humlum and Christiansen, 2008). Distinct moraines deﬁned by sharp crests are located in front of both glaciers, and the recently glaciated area accounts for ∼50 % of the catchment, which is composed of Archaen gneisses (Bridgwater, 1976) (Fig. 1). Kulusuk Lake is ideally situated to capture and preserve a clear sedimentary record of glacier activity because (i) it only receives runoff from a very small catchment (catchment : lake area ratio of ∼2 : 1), mini- mizing the potential for long-term storage of sediments prior to deposition and limiting sediment input from non-glacial processes, (ii) the proximity of the glaciers to the lake results in minimal sediment transport distance, and (iii) the small size of the glaciers makes them sensitive to minor climate variations (Fig. 1). Therefore, bedrock erosion by the glaciers provides the primary source of minerogenic sediment to the lake and changes in glacier size should clearly be reﬂected in sediment properties. y g Lakes that receive meltwater from temperate glaciers can be used to develop continuous records of glacier activity. Bedrock erosion at the base of glaciers provides sediment supply for meltwater transport to proglacial lakes. In catch- ments where other sources of sediment are limited (such as from mass wasting, paraglacial effects, or the release of stored sediment) there is a strong relationship between sed- iment properties and glacier size (Nesje et al., 2000; Dahl et al., 2003; Jansson et al., 2005), which also follows from the assumption that large glaciers produce more minerogenic material and meltwater than small glaciers. Measurements of physical and geochemical properties of proglacial lake sediments can therefore be used to reconstruct records of past glacier size. Here we report a continuous 9.5 ka record of glacier activity on Kulusuk Island, southeast Greenland developed using sediment cores recovered from Kulusuk Lake (65.56◦N, 37.11◦W; 202 m) (Fig. 1). We character- ize changes in sedimentation using measurements of phys- ical sediment properties, including the following: bulk den- sity, organic matter content, magnetic susceptibility, and ac- cumulation rates. 2 Study site We also measured the relative elemental compositions of the sediment using scanning X-ray ﬂuores- cence (XRF) to characterize minerogenic changes at higher resolution and with greater sensitivity. These data provide de- tailed information on sedimentation in Kulusuk Lake related to glacier input. 1 Introduction During the period from AD 1250–1900, often re- ferred to as the “Little Ice Age”, well-resolved records from the North Atlantic region suggest coherence in ice cap activ- ity that was potentially driven by volcanic activity coupled with sea-ice/ocean feedbacks (Miller et al., 2012). However, prior to the last 1000 years there are sparse data for use in in- vestigating the synchrony of glacier response to climate vari- ability in the North Atlantic region. Figure 1. Location and catchment setting of Kulusuk Lake. The white dashed line marks the watershed boundary and the red dashed line deﬁnes the crest of moraines in front of both glaciers mapped in the ﬁeld. Cores were collected in the deepest basin (red circle). Image: Google, NASA. www.clim-past.net/11/1587/2015/ 3.2 Chronology An age–depth model was established based on 210Pb anal- ysis of the upper sediments and AMS radiocarbon dates on macrofossils. The 210Pb activity of samples taken every 1 cm from the upper 10 cm of the record was measured by Flett Research Ltd. (Winnipeg, Canada), and ages were modeled from these data using a constant rate of supply model. AMS radiocarbon measurements were made on plant/wood frag- ments and Daphnia ephippia that were wet sieved from core samples. All radiocarbon ages were calibrated to calendar years using CALIB v. 6.0 (Stuiver and Reimer, 1993) with the IntCal09 calibration data set (Reimer et al., 2009). Ages are presented in calendar years prior to AD 1950 (BP) unless otherwise indicated. Magnetic susceptibility, organic matter, and mass accu- mulation rate (MAR) proﬁles further deﬁne these lithologic changes with higher magnetic susceptibility values across in- tervals with coarser sediment and with lower organic con- tent (Fig. 3). Moderate organic matter, 5 %, and magnetic susceptibility, ∼400 SI 10−5, values characterize the inter- val from 2.5–1.8 m. From 1.8–1.6 m, magnetic susceptibility values decrease to zero and organic matter values increase to 19 % (with the exception of two brief intervals of de- creased values at 176 and 171 cm) and remain elevated to 1.4 m. From 1.4–1.2 m, organic content declines and mag- netic susceptibility values increase and then display more mi- nor ﬂuctuations across the upper 1.2 m. These intervals are clearly deﬁned by MARs, which incorporate sediment den- sity measurements that range from ∼0.8–1.8 g cm−3, but are primarily controlled by the large sedimentation rate changes (Fig. 2). 4.1 Sediment stratigraphy and chronology The 3.5 m composite sediment record from Kulusuk Lake contains distinct lithologic changes, deﬁned by visual stratig- raphy, magnetic susceptibility, organic matter content, and elemental data acquired by scanning XRF. The record can be divided into four lithologic units (Fig. 2). Unit I, 3.0–3.5 m, is a gravelly sand. Unit II, from 3.0 to 1.8 m, is a massive gray clayey silt. There is an abrupt transition to Unit III, a brown, organic-rich sediment that extends from 1.8–1.2 m. Unit IV, 1.2–0 m, is a laminated sequence with frequent sandy layers. Laminations consist of ﬁning-upward sequences and impart strong variability in all data sets. Chronologic data show that there are signiﬁcant changes in sedimentation rate that correspond to lithostratigraphic units (Fig. 2). An age–depth model was generated assuming that changes in sedimentation rate occurred at the boundaries of these units. In Unit IV, a third-order polynomial was applied to the radiocarbon ages, the core top date that represents when the cores were collected in AD 2010 (−60 cal yr BP), and the date of the base of the 210 Pb proﬁle at 10 cm (111 cal yr BP) (Table 1). This relationship was extrapolated to the base of Unit IV. Linear interpolation between the re- maining radiocarbon ages was used to generate the age– depth relationship for Units III and II. There is no chrono- logic control below 215 cm so we did not interpret sedimen- tation prior to 9.5 cal ka BP. 3.3 Scanning X-ray ﬂuorescence To characterize minerogenic changes at higher resolution and with greater sensitivity, an Itrax™XRF core scanner was used to produce proﬁles of relative elemental compositions (Croudace et al., 2006). The Itrax™continuously scans the surface of sediment cores at sub-mm resolution with a micro X-ray beam (20 mm × 100 µm), and the relative concentra- tions of a range of elements are determined based on the de- tection of dispersive energy spectra. Dispersive energy spec- tra are acquired across each measured interval, and peak area integrals are calculated for each element. Peak area integrals are related to elemental concentrations within the sediment, but can also be inﬂuenced by characteristics of the sedimen- tary matrix and therefore only indicate relative changes in elemental composition (Croudace et al., 2006; Rothwell et al., 2006). Our analysis focused on the elements K, Ca, Ti, Mn, Fe, Zn, Rb, Sr, which have detection limits that range 3.1 Sediment core collection and analysis Sediment cores were recovered from Kulusuk Lake in April 2010 when the lake was ice covered. Bathymetric mea- surements were made manually through holes drilled in the ice, and sediment cores were collected using Uwitec grav- Clim. Past, 11, 1587–1598, 2015 www.clim-past.net/11/1587/2015/ 1589 N. L. Balascio et al.: Glacier response to North Atlantic climate variability from 150 to 5 ppm (Croudace et al., 2006). All of the cores were scanned at 200 µm intervals with an exposure time of 10 s, voltage of 30 kV, and current of 55 nA. ity and percussion coring devices from the deepest location, which has a water depth of 69 m. A composite 3.5 m record was compiled by matching the physical stratigraphy and scanning XRF proﬁles from a 26 cm gravity core (Kul-10D- B) and multiple overlapping percussion cores (Kul-10G-A1, -B1, -A2). 4 Results The magnetic susceptibility of the cores was measured ev- ery 0.5 cm using a Bartington MS2E sensor. The organic- matter content of the cores was measured by loss-on-ignition (LOI) on contiguous 1-cm3 samples taken at 1 cm intervals. Organic-matter content was calculated as the difference be- tween the weight of dried 1-cm3 samples and their weight af- ter heating for 4 h at 550 ◦C (Dean, 1974). Bulk density mea- surements (g cm−3) and the calculated sedimentation rates (cm year−1) were used to determine mass accumulation rates (MAR; g cm−2 year−1). Grain size measurements were made at 10 cm increments. Samples were pre-treated with a 30 % hydrogen peroxide solution to digest organic material and analyzed using a Beckman Coulter LS200 particle-size an- alyzer. 4.2 Scanning XRF data analysis Elemental scans acquired by scanning XRF show a response similar to magnetic susceptibility with higher values across www.clim-past.net/11/1587/2015/ Clim. Past, 11, 1587–1598, 2015 N. L. Balascio et al.: Glacier response to North Atlantic climate variability 1590 N. L. Balascio et al.: Glacier response to North Atlantic climate variability p y Figure 2. Magnetic susceptibility proﬁle, percent sand, and mass accumulation rate (MAR) shown next to the age–depth model for the composite Kulusuk Lake record. The four lithostratigraphic units and the corresponding sedimentation rates are shown. A dash line marks the period below the last radiocarbon age (9.5 cal ka BP) where rates of sedimentation are extrapolated. Figure 2. Magnetic susceptibility proﬁle, percent sand, and mass accumulation rate (MAR) shown next to the age–depth model for the composite Kulusuk Lake record. The four lithostratigraphic units and the corresponding sedimentation rates are shown. A dash line marks the period below the last radiocarbon age (9.5 cal ka BP) where rates of sedimentation are extrapolated. Table 1. Geochronologic data for the Kulusuk Lake record. Table 1. Geochronologic data for the Kulusuk Lake record. Table 1. Geochronologic data for the Kulusuk Lake record. Statistical analysis of the scanning XRF data indicates that all of the elements are highly correlated and that there is a strong primary trend in the data. Correlation coefﬁcients show the strong signiﬁcant relationships among the majority of the elements (Table 2). Rather than relying on a single ele- ment (e.g., Ti), we used principal component analysis (PCA) to deﬁne the leading mode of variability (PC1) among the elemental data. PCA allows for a multidimensional exami- nation of the data set in order to identify the primary sig- nal(s). PCA results indicate that there is one strong primary trend in the elemental data with the ﬁrst eigenvector (PC1) accounting for 76 % of the total variance. The factor load- ings reveal the high correlations between individual element proﬁles and PC1 (Table 2). The trends in PC1 are similar to those in the lower-resolution magnetic susceptibility and or- ganic matter content records, justifying use of PC1 data to infer past minerogenic changes (Fig. 3). The choice to use PC1 rather than a single representative element (e.g., Ti) to represent changes in sedimentary minerogenic content has no impact on any of our conclusions. lakes. Glacier size, erosive ability, and meltwater produc- tion directly inﬂuence the amount of minerogenic sediment delivered to a proglacial lake. However, sedimentation in a proglacial lake can also be impacted by mass wasting processes in paraglacial environments (particularly in land- scapes with steep unstable slopes), and by the delayed re- lease of sediment stored along the transport pathway between the glacier and the lake (for example, sediment stored in ex- tensive meltwater stream channels). Relative to minerogenic material, organic sedimentation is typically a minor com- ponent in proglacial lakes and is related to the input of or- ganic matter from autochthonous and allochthonous primary productivity, and the preservation thereof. In proglacial en- vironments in the Arctic, low temperatures restrict vegeta- tion and soil cover, and minerogenic sediment input to lakes from glacial meltwater results in turbidity that impedes au- tochthonous productivity. Techniques for analyzing sediment from proglacial lakes therefore focus on investigating changes in the character of minerogenic sediment. The minerogenic content of lake sed- iments, used as a proxy for glacier size, is commonly eval- uated by measuring the magnetic susceptibility and organic matter content of the sediments. Table 1. Geochronologic data for the Kulusuk Lake record. Geochronologic data for the Kulusuk Lake record. Composite Description Laboratory 14C age Calibrated age range Median age depth (cm) IDa (yr BP) (1σ) (2σ) (cal yr BP) 0 Core Top – – – – −60 1 210Pb – – – – −53 2 210Pb – – – – −46 3 210Pb – – – – −36 4 210Pb – – – – −25 5 210Pb – – – – −7 6 210Pb – – – – 24 7 210Pb – – – – 44 8 210Pb – – – – 57 9 210Pb – – – – 83 10 210Pb – – – – 111 34 Daphnia ephippia OS-96479 335 ± 40 316–459 306–486 393 59.5 Plant/wood UCI-89386 940 ± 20 798–914 795–919 852 95 Daphnia ephippia OS-96454 1290 ± 25 1183–1276 1178–1283 1237 132 Plant/wood UCI-87240 3410 ± 60 3574–3814 3484–3832 3664 138.5 Plant/wood UCI-87241 3820 ± 60 4095-4378 4008-4415 4224 170.5 Daphnia ephippia OS-96461 7620 ± 50 8377–8452 8359–8539 8418 214.5 Daphnia ephippia OS-96746 8510 ± 130 9312–9659 9135–9887 9501 a UCI – University of California Irvine Keck-CCAMS Facility; OS – National Ocean Sciences AMS Facility Clim. Past, 11, 1587–1598, 2015 www.clim-past.net/11/1587/2015/ www.clim-past.net/11/1587/2015/ N. L. Balascio et al.: Glacier response to North Atlantic climate variability Table 2. XRF PC1 factor loadings and correlation matrix for scanning XRF elemental data. PC1 K Ca Ti Mn Fe Zn Rb Sr loadings K 0.950 1 Ca 0.868 0.891 1 Ti 0.969 0.938 0.797 1 Mn 0.815 0.672 0.594 0.783 1 Fe 0.945 0.876 0.748 0.946 0.814 1 Zn 0.861 0.759 0.609 0.842 0.727 0.836 1 Rb 0.831 0.743 0.601 0.791 0.637 0.790 0.727 1 Sr 0.686 0.689 0.803 0.583 0.412 0.488 0.436 0.455 1 N. L. Balascio et al.: Glacier response to North Atlantic climate variability 1591 Table 2. XRF PC1 factor loadings and correlation matrix for scanning XRF elemental data. coarser, clastic intervals. However, the XRF data have a greater sensitivity to minerogenic changes and were mea- sured at higher resolution (0.2 mm) (Fig. 3). We focused our analysis on the following elements: K, Ca, Ti, Mn, Fe, Zn, Rb, and Sr, which are common in siliciclastic sedi- ments. Changes in the concentrations of these elements re- ﬂect changes in the contribution of minerogenic material eroded from catchment bedrock and delivered to the lake. Table 1. Geochronologic data for the Kulusuk Lake record. The abundances of the ma- jor elements from bedrock material (measured by XRF) sim- ilarly serve as a proxy for the relative contribution of minero- genic material, versus organic matter, to the lake. Mag- netic susceptibility reﬂects the amount of magnetic minerals eroded and input to a lake, the abundance of major elements from bedrock (measured by XRF) also reﬂects the relative contribution of minerogenic material input to the lake, and organic matter content is a function of dilution by minero- genic input, changes in primary productivity, and preserva- tion. www.clim-past.net/11/1587/2015/ 5.1 Sedimentation in proglacial lakes At Kulusuk Lake, processes that can complicate the mech- anistic link between minerogenic input and glacier size are fundamentally limited. Input of sediment from non-glacial processes is restricted due to the small catchment and small catchment to lake-area ratio (∼2 : 1), and the proximity of the glaciers to the lake. These factors also limit the potential for sediment storage between the glaciers and the lake. Fur- thermore, the landscape surrounding the lake is composed Sedimentation in proglacial lakes can be the result of a complex set of physical processes associated with the ero- sion, storage, and transport of sediment within glacial and proglacial systems (Dahl et al., 2003; Jansson et al., 2005). It is important to consider these complicating factors when selecting sites for glacier reconstructions and when interpret- ing sedimentary records. Glaciers fundamentally impact the amount and character of minerogenic sediment in proglacial www.clim-past.net/11/1587/2015/ Clim. Past, 11, 1587–1598, 2015 1592 N. L. Balascio et al.: Glacier response to North Atlantic climate variability N. L. Balascio et al.: Glacier response to North Atlantic climate variability Figure 3. Kulusuk Lake record. (Top) First principal component of the scanning XRF data (PC1). (Middle) Magnetic susceptibility presented on a log scale. A dotted line deﬁnes the interval from 165–140 cm where some zero values were measured. (Bottom) Organic-matter content. Black bars indicate the location and age of chronologic control points (Table 1). The yellow shaded region on the PC1 plot shows where we have interpreted little to no glacier ice in the catchment during the Holocene Thermal Maximum (HTM). Blue shading deﬁnes the Neoglacial period when ice was reformed during the late Holocene (4.1 ka–present). Figure 3. Kulusuk Lake record. (Top) First principal component of the scanning XRF data (PC1). (Middle) Magnetic susceptibility presented on a log scale. A dotted line deﬁnes the interval from 165–140 cm where some zero values were measured. (Bottom) Organic-matter content. Black bars indicate the location and age of chronologic control points (Table 1). The yellow shaded region on the PC1 plot shows where we have interpreted little to no glacier ice in the catchment during the Holocene Thermal Maximum (HTM). Blue shading deﬁnes the Neoglacial period when ice was reformed during the late Holocene (4.1 ka–present). 5.3 Evidence for synchronous regional glacier response during the late Holocene The Kulusuk glacier reconstruction documents centennial- scale episodes of glacier advance during the Neoglacial (4.1 to 1.3 ka) coeval with other records of glacier growth in the North Atlantic region. After 4.1 ka, six major advances of the Kulusuk glaciers occurred (4.1, 3.9, 3.2, 2.8, 2.1, and 1.3 ka) and each successive advance resulted in greater glacier extent (Fig. 4). The progressive increase in glacier size is consis- tent with declining NH summer insolation, which is likely the mechanism driving millennial-scale changes in glacier size. However, each episode of glacier advance was followed by a period of retreat (or at least stabilization), suggesting that the glaciers repeatedly grew out of equilibrium with external insolation forcing and then retreated back toward an equilibrium state, indicating centennial-scale variability likely driven by internal climate dynamics. The episodic ad- vances of the Kulusuk glaciers during the past 4.1 ka are similar in timing to the cooling episodes in the North At- lantic Ocean inferred from ice-rafted debris (IRD) identiﬁed in marine sediment cores (Bond et al., 1997, 2001) (Fig. 4). Cooling events at these times have also been documented on the East Greenland and Icelandic shelves and attributed to in- creased strength of the East Greenland Current (Giraudeau et al., 2000; Jennings et al., 2002; Ran et al., 2008). Moreover, the Langjökull ice cap in Iceland advanced along with the Kulusuk glaciers and the North Atlantic IRD events (Larsen et al., 2012) (Fig. 4), and advances of the Bregne ice cap in east Greenland at ca. 2.6 and 1.9 ka (Levy et al., 2014), within chronological uncertainty of the Kulusuk glacier ad- vances ca. 2.8 and 2.1 ka, have also been documented. We propose that continuous records of glacier activity around the North Atlantic during the Neoglacial are beginning to show evidence for synchronous glacier response to abrupt episodes of climate change. At 4.1 ka, a sharp increase in XRF- and MS-inferred minerogenic content and decrease in organic matter content indicate the glaciers once again grew large enough to con- tribute minerogenic material to the lake. The regrowth of the Kulusuk glaciers represents the lowering of the regional snowline, and the precise timing could be considered unique to this catchment. However, the timing is contemporaneous with hydrologic changes at nearby Flower Valley Lake, likely related to an increase in the duration of lake ice cover (Bal- ascio et al., 2013). 5.2 Holocene glacier ﬂuctuations in southeast Greenland The timing of glacier size variations between radiocarbon-dated inter- vals since 4.1 ka are interpolated, and we estimate the ac- curacy to be better than ±100 years, the average 2-σ uncer- tainty of the ages. also marked by the advance of Jakobshavn Isbræ, an out- let glacier of the Greenland Ice Sheet in western Greenland (Young et al., 2011, 2013). The temporal resolution and age control of this section of our record cannot provide new con- straints on the exact timing of these events, however it clearly demonstrates the sensitivity of the Kulusuk glaciers to rapid, regional climate events. Between 7.8–4.1 ka, the Kulusuk glaciers were at their minimum Holocene extent, inferred from low minerogenic content, low MAR, and high organic matter content in the lake sediments (Fig. 3). We interpret this as an interval with little to no glacier ice in the catchment, primarily based on the XRF and magnetic susceptibility data, which are lowest and show reduced variability at this time, relative to the last 4.1 ka. This interval is also marked by extremely high organic matter content that is greater than 12 % (with a maximum of 19 %), suggesting that this period was accompanied by an in- crease in primary productivity due to a lack of input of glacial ﬂour. If the catchment was completely deglaciated, this indi- cates that the regional equilibrium-line altitude would have been greater than ∼676 m, which is the elevation of the mountain peak above the lake. Magnetic susceptibility re- mains close to zero throughout the mid-Holocene section of the core and appears insensitive to the minor minero- genic changes inferred from the XRF data, which could be attributed to paraglacial processes or seasonal runoff con- tributing very minor amounts of clastic sediment. There are two excursions in the PC1 record during this interval (ca. 7.2 and 6.2 ka), which we interpret as sediment inﬂuxes from paraglacial activity rather than as glacier advances be- cause they are short-lived and do not match the amplitude of variation observed elsewhere. Thus, this record provides well-dated constraints on the Holocene Thermal Maximum (HTM) in this area, which reﬁne previous estimates extrapo- lated for this region (Kaufman et al., 2004) and is similar to the interval when the Greenland Ice Sheet margin was behind its present limit, broadly constrained to ca. 7–4 ka (Larsen et al., 2015). 5.2 Holocene glacier ﬂuctuations in southeast Greenland of shallow, low-elevation slopes that minimize the likelihood of mass wasting events. Therefore, at Kulusuk Lake, it is reasonable to interpret changes in minerogenic content as a function of glacier size. Dramatic changes in minerogenic input to Kulusuk Lake over the last 9.5 ka reveal that the size of the Kulusuk glaciers has varied signiﬁcantly throughout the Holocene (Fig. 3). Begin- ning ca. 8.7 ka, increasing organic matter content and de- creasing minerogenic content, inferred from magnetic sus- ceptibility and XRF data, document signiﬁcant retreat of the Kulusuk glaciers, corresponding closely in time with the deglaciation of a nearby inland catchment ca. 8.4 ka (Balas- cio et al., 2013), following deglaciation of local coastal ar- eas ca. 11.1–9.5 ka (Long et al., 2008; Roberts et al., 2008). A brief interval of increased minerogenic input shows that this early Holocene retreat was interrupted by an episode of advance at 8.5 ka, coeval with reductions in sea surface temperatures and bottom water circulation in the subpolar North Atlantic, as seen in high-resolution marine records (El- lison et al., 2006; Kissel et al., 2013). Another abrupt episode of minerogenic input ca. 8.2 ka signiﬁes another glacier ad- vance. This advance occurred contemporaneously with the largest abrupt Holocene climate cooling event inferred from Greenland ice core records (Thomas et al., 2007), which is Variations in magnetic susceptibility, organic matter con- tent, and scanning XRF data (PC1) represent changes in the relative amount and grain size of minerogenic sediment de- livered to Kulusuk over the last 9.5 ka (Fig. 3). Magnetic sus- ceptibility and PC1 are directly related to, and organic con- tent is inversely proportional to, minerogenic content. The highest magnetic susceptibility and PC1 values correspond to intervals with coarser grain size. We therefore interpret the sedimentological system in Kulusuk Lake as follows: during periods of increased glacier size, more coarse minerogenic sediment was eroded from the bedrock and delivered to the lake by meltwater; during periods of smaller glacier size, less minerogenic sediment was deposited and a greater relative proportion of organic matter content accumulated. Clim. Past, 11, 1587–1598, 2015 www.clim-past.net/11/1587/2015/ N. L. Balascio et al.: Glacier response to North Atlantic climate variability 1593 tury retreat (Fig. 3). Importantly, the major sedimentologi- cal transitions in the record are all located near radiocarbon dates, thereby maximizing the certainty of their timing and the calculations of sediment accumulation rates. N. L. Balascio et al.: Glacier response to North Atlantic climate variability Figure 4. Regional response of glaciers to Holocene climate changes. (a) Kulusuk glaciers interpreted from PC1 data with July insolation anomalies at 65◦N (Berger and Loutre, 1991). (b) Hematite-stained grains (HSG) identiﬁed in core MC52-VM29-191 interpreted to indicate ice-rafting events (Bond et al., 1997). (c) Ratio of total organic carbon to total nitrogen (C / N) and (d) changes in sedimentation rate from Hvítárvatn, interpreted to reﬂect changes in the size of the Langjökull ice cap, Iceland, and catchment instability in response to climate cooling (Larsen et al., 2012). Yellow shading marks the timing of the Holocene Thermal Maximum (HTM), as interpreted at Kulusuk, and the dashed line on the PC1 plot shows where we have interpreted the absence of ice from the catchment during the HTM. Blue bars highlight intervals of glacier advance and increased ice rafting that deﬁne Neoglacial cooling events comparable among the records. Figure 4. Regional response of glaciers to Holocene climate changes. (a) Kulusuk glaciers interpreted from PC1 data with July insolation anomalies at 65◦N (Berger and Loutre, 1991). (b) Hematite-stained grains (HSG) identiﬁed in core MC52-VM29-191 interpreted to indicate ice-rafting events (Bond et al., 1997). (c) Ratio of total organic carbon to total nitrogen (C / N) and (d) changes in sedimentation rate from Hvítárvatn, interpreted to reﬂect changes in the size of the Langjökull ice cap, Iceland, and catchment instability in response to climate cooling (Larsen et al., 2012). Yellow shading marks the timing of the Holocene Thermal Maximum (HTM), as interpreted at Kulusuk, and the dashed line on the PC1 plot shows where we have interpreted the absence of ice from the catchment during the HTM. Blue bars highlight intervals of glacier advance and increased ice rafting that deﬁne Neoglacial cooling events comparable among the records. The amplitude of variability in the proxy measurements during the past 1.3 ka is lower than earlier in the Holocene, due to the greater size and stability of the Kulusuk glaciers; however, it is worthwhile to examine the changes in the sed- iment properties where advances are interpreted as sustained above average PC1 values. The very high sediment accu- mulation rates during this interval (0.8 mm yr−1) allow sub- annual XRF measurements and, if interpreted in the same manner as periods with smaller glacier size, can afford a de- tailed examination of changes in glacier size using the XRF PC1 data (Fig. 5). 5.3 Evidence for synchronous regional glacier response during the late Holocene We propose that this represents signiﬁcant cooling and the onset of the regional Neoglacial period. The oscillatory and stepwise increase in minerogenic input (de- crease in organic matter content) after 4.1 ka suggests that rather than advancing steadily toward their historical extent, the Kulusuk glaciers episodically advanced and retreated at centennial timescales until ca. 1.3 ka. After advancing at ca. 1.3 ka, they stabilized after 0.7 ka until their rapid 20th cen- It is also worth noting that Winsor et al. (2014) found ev- idence for an advance of an outlet glacier of the Greenland Ice Sheet in southern Greenland ending at ca. 1.5 ka, the tim- ing of which is supported by minimum-limiting radiocarbon ages from the same region dating to ca. 1.2 ka (Bennike and Sparrenbom, 2007). We acknowledge that this is the only lo- cation on the ice sheet margin where such a late Holocene advance has been documented, but nonetheless it highlights that changes in the ice margin position are beginning to be constrained more accurately. www.clim-past.net/11/1587/2015/ Clim. Past, 11, 1587–1598, 2015 1594 N. L. Balascio et al.: Glacier response to North Atlantic climate variability (b) Big Round Lake, Bafﬁn Island, varve thickness and magnetic susceptibility (Thomas and Briner, 2009; Thomas et al., 2010). (c) Bafﬁn Island ice cap activity reconstructed using vegetation kill dates with text showing original interpretations (Miller et al., 2012). (d) Langjökull ice cap, Iceland-based on varve thickness from Lake Hvítárvatn (Larsen et al., 2011). Blue shading marks periods of increased glacier size (sustained above average PC1 values). Figure 5. Change in the size of the Kulusuk glaciers since AD 700 compared with other high-resolution glacier and ice caps records from the region. (a) The Kulusuk PC1 record. Black horizontal line shows average value over this period. (b) Big Round Lake, Bafﬁn Island, varve thickness and magnetic susceptibility (Thomas and Briner, 2009; Thomas et al., 2010). (c) Bafﬁn Island ice cap activity reconstructed using vegetation kill dates with text showing original interpretations (Miller et al., 2012). (d) Langjökull ice cap, Iceland-based on varve thickness from Lake Hvítárvatn (Larsen et al., 2011). Blue shading marks periods of increased glacier size (sustained above average PC1 values). Lowell et al., 2013), unless the data are reinterpreted as sug- gested by Miller et al. (2013). similar distinct glacier advances at these times (Fig. 5) as well as an earlier advance ca. AD 1250–1300, which is also observed in the Kulusuk record (Thomas et al., 2010). How- ever, varve thickness data from Big Round Lake, which has previously been interpreted to represent summer tempera- ture, resemble trends in magnetic susceptibility (Thomas and Briner, 2009). This discrepancy can possibly be attributed to how the two proxies track different sedimentary processes operating over different timescales (annual vs. centennial), but without further analysis of those records we cannot ac- count for this apparent contradiction. We argue that the mag- netic susceptibility data from Big Round Lake are consistent with other data from around Greenland, indicating that the most extensive glacier advances since the early Holocene oc- curred between AD 1250 and 1900, and provide evidence for regionally coherent cooling phases during the Little Ice Age (Grove, 2001). We note that this timing contrasts with evidence from east Greenland that suggests the Istorvet Ice Cap advanced approximately 100 years earlier (ca. N. L. Balascio et al.: Glacier response to North Atlantic climate variability The overall trend reveals a small and very gradual glacier expansion after 0.7 ka followed by 20th cen- tury retreat, which resembles the overall trend in Arctic tem- peratures over the last 2 ka (Kaufman et al., 2009). Multi-decadal variations in inferred glacier size during the past 1.3 ka also appear to be synchronous with those of other glaciers in the region after ca. AD 1250 (Fig. 5). Kulusuk glaciers increased in size ca. AD 1250–1300 and again ca. AD 1450, similar to when ice caps on Bafﬁn Island (Miller et al., 2012) and Iceland (Larsen et al., 2011) were expand- ing (Fig. 4). After AD 1450 Kulusuk glaciers continued to expand, as did Langjökull on Iceland, while evidence from the Bafﬁn ice caps indicates continuous ice cover (Miller et al., 2012). Both Kulusuk and Langjökull glaciers appear to have ad- vanced in at least two phases, at ca. AD 1450–1630 and ca. AD 1700–1930. Magnetic susceptibility trends, linked to glacier size changes, from another high-resolution proglacial lake record on Bafﬁn Island (Big Round Lake) reveal two Clim. Past, 11, 1587–1598, 2015 www.clim-past.net/11/1587/2015/ N. L. Balascio et al.: Glacier response to North Atlantic climate variability 1595 N. L. Balascio et al.: Glacier response to North Atlantic climate variability Figure 5. Change in the size of the Kulusuk glaciers since AD 700 compared with other high-resolution glacier and ice caps records from the region. (a) The Kulusuk PC1 record. Black horizontal line shows average value over this period. (b) Big Round Lake, Bafﬁn Island, varve Figure 5. Change in the size of the Kulusuk glaciers since AD 700 compared with other high-resolution glacier and ice caps records from the region. (a) The Kulusuk PC1 record. Black horizontal line shows average value over this period. (b) Big Round Lake, Bafﬁn Island, varve thickness and magnetic susceptibility (Thomas and Briner, 2009; Thomas et al., 2010). (c) Bafﬁn Island ice cap activity reconstructed using vegetation kill dates with text showing original interpretations (Miller et al., 2012). (d) Langjökull ice cap, Iceland-based on varve thickness from Lake Hvítárvatn (Larsen et al., 2011). Blue shading marks periods of increased glacier size (sustained above average PC1 values). Figure 5. Change in the size of the Kulusuk glaciers since AD 700 compared with other high-resolution glacier and ice caps records from the region. (a) The Kulusuk PC1 record. Black horizontal line shows average value over this period. www.clim-past.net/11/1587/2015/ N. L. Balascio et al.: Glacier response to North Atlantic climate variability AD 1150; Therefore, there seems to have been regional coherence in glacier activity not only during the past 1.3 ka, as previously suggested (Miller et al., 2012), but also during the past 4.1 ka, and glacier growth in response to episodic climate change has been a common feature in the North Atlantic region through- out, at least, the last 4.1 ka. Cold events are an important feature of centennial-scale climate of the Holocene (Wanner et al., 2011). Cooling events in the North Atlantic region are possibly associated with changes in Atlantic Meridional Overturning Circulation (AMOC) (Denton and Broecker, 2008). IRD records suggest that periodic circulation changes of the North Atlantic Ocean resulted in an advection of cold, fresh surface water south and east during ice-rafting events throughout the Holocene (Bond et al., 1997). Ocean circulation and sea-surface tem- perature changes associated with IRD events have been at- tributed to solar forcing (Bond et al., 2001; Moffa-Sánchez et al., 2014; Jiang et al., 2015), and some modeling studies have Clim. Past, 11, 1587–1598, 2015 N. L. Balascio et al.: Glacier response to North Atlantic climate variability 1596 Figure 6. Relative rates of change in the size of the Kulusuk glaciers interpreted from scanning XRF PC1 data. Red bars show 105-year intervals when the average rate was positive indicating glacier retreat, and blue bars show intervals when the average rate was negative indicating glacier advance. Values not calculated during the mid-Holocene when we interpret glaciers to be absent. Figure 6. Relative rates of change in the size of the Kulusuk glaciers interpreted from scanning XRF PC1 data. Red bars show 105-year intervals when the average rate was positive indicating glacier retreat, and blue bars show intervals when the average rate was negative indicating glacier advance. Values not calculated during the mid-Holocene when we interpret glaciers to be absent. that the glaciers were sensitive to a number of previously documented regional climate ﬂuctuations and improves our understanding of Holocene climate dynamics in this sector of the Arctic. In particular, the record clearly constrains the Holocene Thermal Maximum at this site to between 7.8 and 4.1 ka, when the glaciers likely completely melted away. The regrowth of the Kulusuk glaciers at 4.1 ka corresponds with regional hydrologic changes and reﬂects the onset of the Neoglacial period. N. L. Balascio et al.: Glacier response to North Atlantic climate variability The last 4.1 ka is marked by a series of abrupt glacier advances as the size of the Kulusuk glaciers increased. These episodes of glacier growth correspond with ice rafting events in the North Atlantic Ocean, as well as re- gional ice cap expansion, and demonstrate that glaciers in this sector of the Arctic were very active during the late Holocene in response to abrupt cooling events that punctu- ated millennial-scale insolation-driven cooling. The recon- struction of Kulusuk glacier activity provides a new and re- ﬁned perspective on late Holocene cold events, which are im- portant features of centennial-scale climate variability. conﬁrmed that AMOC can switch between distinct modes in response to a small external forcing, such as solar variabil- ity (Jongma et al., 2007). However, modeling results are in- consistent and it is also possible that cooling events might have simply resulted from internal ocean dynamics (Schulz and Paul, 2002). Regardless of the mechanism, our results demonstrate that glaciers responded quite actively to natural climate variations of the Holocene. 5.4 Rates of glacier change during the Holocene This well-dated, high-resolution record of changes in the size of the Kulusuk glaciers also allows comparison among the rates of past glacier size variations. We present relative rates of change inferred from the ﬁrst derivative of the XRF PC1 data in 105-year binned averages, an interval chosen using the interval with the lowest resolution (Fig. 6). We acknowl- edge the caveat that they are based on the assumption that the relationship between minerogenic input and glacier size has remained constant. The analysis indicates that the rate of 20th century retreat of the Kulusuk glaciers was greater than during any other century of the past 1.3 ka, including during the Medieval Climate Anomaly. Furthermore, the 20th cen- tury retreat rate was 2–3 times the rate of any other period of retreat during the past 4.1 ka, and almost twice as rapid as the early Holocene retreat that marked the transition into the re- gional HTM (Fig. 6). This comparison helps to place the rate of 20th century glacier loss in the context of natural episodes of past glacier activity. Acknowledgements. This research was supported by a LDEO Postdoctoral Fellowship to NLB, NSF grant ARC-0851642 to WJD, NOAA grant NA09OAR4600215 and NSF grant ARC- 0909354 to RSB. We thank Lucien von Gunten, Sam Davin, and Greg de Wet for assistance with ﬁeld work, as well as Jason Briner, Anders Carlson, and three anonymous reviewers for comments on earlier drafts. Acknowledgements. This research was supported by a LDEO Postdoctoral Fellowship to NLB, NSF grant ARC-0851642 to WJD, NOAA grant NA09OAR4600215 and NSF grant ARC- 0909354 to RSB. We thank Lucien von Gunten, Sam Davin, and Greg de Wet for assistance with ﬁeld work, as well as Jason Briner, Anders Carlson, and three anonymous reviewers for comments on earlier drafts. Edited by: V. Rath Edited by: V. Rath References The Kulusuk Lake sediment record was used to generate a high-resolution record of changes in the size of the Kulusuk glaciers over the last 9.5 ka. Characteristics of the lake and catchment limit the potential for sedimentation from non- glacial processes making it ideally situated to clearly cap- ture changes related to glacier activity. The record shows Balascio, N. L., D’Andrea, W. J., Bradley, R. S., and Perren, B. B.: Biogeochemial evidence for hydrologic changes during the Holocene in a lake sediment record from southeast Greenland, The Holocene, 23, 1428–1439, 2013. Bennike, O. and Sparrenbom, C. J.: Dating of the Narssarssuaq stade in southern Greenland, The Holocene, 17, 279–282, 2007. Bennike, O. and Sparrenbom, C. 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ULUSAL BİYOKİMYA KONGRESİ 27-30 Ekim 2021, Gaziantep, Türkiye TBS INTERNATIONAL BIOCHEMISTRY CONGRESS 2021 32ND NATIONAL BIOCHEMISTRY CONGRESS 27-30 October 2021, Gaziantep, Turkey 2021 Cilt [Volume] 46 Ek Sayı [Supplement] 2 Türk Biyokimya Derneği’nin yayın organıdır. 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Hamdi Öğüş, Department of Medical Biochemistry, Faculty of İ. Hamdi Öğüş, Department of Medical Biochemistry, Faculty of Medicine, Near East University, Nicosia, Emeritus, TRNC Erdal Karaöz, Department of Histology and Embryology, Faculty of Medicine, İstinye University, İstanbul, TURKEY Erdal Karaöz, Department of Histology and Embryology, Faculty of Medicine, İstinye University, İstanbul, TURKEY İ. Hamdi Öğüş, Department of Medical Biochemistry, Faculty of Medicine, Near East University, Nicosia, Emeritus, TRNC Medicine, Near East University, Nicosia, Emeritus, TRNC , y y, , Reşat Apak, Department of Chemistry, Faculty of Engineering, İstanbul University Cerrahpaşa Research Information System, İstanbul, TURKEY Khosrow Adeli, Molecular Medicine, Research Institute, Th e Hospital for Sick Children, University of Toronto, Toronto, Ontario, CANADA Diler Aslan, Department of Biochemistry, Faculty of Medicine, y Yeşim Özarda, Department of Medical Biochemistry, Faculty of y y Reşat Apak, Department of Chemistry, Faculty of Engineering, İstanbul University Cerrahpaşa Research Information System, İstanbul, TURKEY ş p y y Medicine, Uludağ University, Bursa, TURKEY Medicine, Uludağ University, Bursa, TURKEY ğ y Tomris Özben, Department of Medical Biochemistry, Faculty of ğ y Tomris Özben, Department of Medical Biochem Medicine, Akdeniz University, Antalya, TURKEY Nazmi Özer, Faculty of Pharmacy, Girne American University, Nicosia TRNC Pamukkale University, Denizli, TURKEY Pamukkale University, Denizli, TURKEY İsrael Pecht, Department of Immunology, Weizmann Institute of Cumhur Bilgi, Department of Medical Biochemistry, University of Yüksek İhtisas, Ankara, TURKEY İsrael Pecht, Department of Immunology, Weizmann Institute of Science, Rehovot, ISRAEL Science, Rehovot, ISRAEL Mario Plebani, Department of Medical Sciences, University of Padova, Padova, ITALY Center “Mother Teresa”, Tirana, ALBANIA Demetrios Rizos, Hormonal and Biochemical Laboratory, Are Medicine, Karadeniz Technical University, Trabzon, TURKEY Hospital, University of Athens, Athens, GREECE Elif Demirkan, Department of Biology, Faculty of Arts & Sciences, George Russev, Bulgarian Academy of Sciences, Institute of Molecular Biology, Sofi a, BULGARIA Uludağ University, Bursa, TURKEY Z. Günnur Dikmen, Department of Bio Z. Günnur Dikmen, Department of Biochemistry, Facult i Fahri Saatçioğlu, Department of Biosciences, University of Oslo, Oslo, NORWAY Z. e-ISSN 1303-829X All information regarding notes for contributors, subscriptions, open access, back volumes and orders is available online at www.degruyter.com/view/j/tjb. RESPONSIBLE EDITOR Doğan Yücel PhD, Assoc Professor of Biochemistry, Department of Medical Biochemistry, Ankara Training and Research Hospital, University of Health Sciences, 06340, Ankara, Turkey, Tel: +90312 595 321, Email: doyucel@yahoo.com JOURNAL COORDINATOR Heike Jahnke, De Gruyter, Genthiner Straße 13, 10785 Berlin, Germany. Tel.: +49 (0) 30 260 05–220, Email: heike.jahnke@degruyter.com JOURNAL COORDINATOR Heike Jahnke, De Gruyter, Genthiner Straße 13, 10785 Berlin, Germany. Tel.: +49 (0) 30 260 05–220, Email: heike.jahnke@degruyter.com RESPONSIBLE FOR ADVERTISEMENTS Markus Kügel, De Gruyter, Rosenheimer Str. 143, 81671 Munich, Germany, Tel.: +49 89 76 902-424, e-mail: anzeigen@degruyter.com RESPONSIBLE FOR ADVERTISEMENTS Markus Kügel, De Gruyter, Rosenheimer Str. 143, 81671 Munich, Germany, Tel.: +49 89 76 902-424, e-mail: anzeigen@degruyter.com EDITORIAL BOARD [EDİTÖRLER KURULU] Günnur Dikmen, Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, TURKEY Miral Dizdaroğlu, National Institute of Standards and Technology, h b Ferhan Girgin Sağın, Department of Medical Biochemistry, Faculty of Miral Dizdaroğlu, National Institute of Standards and Technology, Ferhan Girgin Sağın, Department of Medical Biochemistry, Faculty of M di i E U i it İ i TURKEY Gaithersburg, MD, USA Medicine, Ege University, İzmir, TURKEY Aziz Sancar, Department of Biochemistry and Biophysics, U North Carolina School of Medicine, Chapel Hill, NC, USA Natural Sciences, Imperial College, London, UNITED KINGDOM Natural Sciences, Imperial College, London, UNITED KINGDOM Figen Erkoç, Department of Biology Education, Faculty of Gazi Praveen Sharma, All India Institute of Medical Sciences, Jodhpur, INDIA i Figen Erkoç, Department of Biology Education, Faculty of Gazi Education, Gazi University, Emeritus, Ankara TURKEY Education, Gazi University, Emeritus, Ankara TURKEY y Gökhan Hotamişlıgil, Department of Genetics and Complex Diseases, Harvard School of Public Healt, Boston, USA y Gökhan Hotamişlıgil, Department of Genetics and Complex Diseases, Emin Sofi c, Department of Chemistry, Faculty of Science, Sa Gökhan Hotamişlıgil, Department of Genetics and Complex Diseases, Harvard School of Public Healt, Boston, USA i University, Sarajevo, BOSNIA AND HERZEGOVIA Mehmet Kesimer, Department of Pathology and Laboratory Medicine, Mehmet Kesimer, Department of Pathology and Laboratory Medicine, Eser Sözmen, Department of Medical Biochemistry, Faculty of Medicine, University of Ege, İzmir, TURKEY Marsico Lung Institute, University of North Carolina at Chapel Hill, NC, USA Marsico Lung Institute, University of North Carolina at Chapel Hill, NC, USA Abdullah Tuli, Department of Medical Biochemistry, Fac Medicine, Çukurova University, Adana, TURKEY İrfan Küfrevioğlu, Department of Chemistry, Faculty of Art & Sciences, İrfan Küfrevioğlu, Department of Chemistry, Faculty of Art & Sciences, Ali Ünlü, Department of Biochemistry, Faculty of M Ali Ünlü, Department of Biochemistry, Faculty of Medicine, Selçuk Atatürk University, Erzurum, TURKEY y Nada Majkic-Singh, Institute of Medical Biochemistry, Pharmaceutical Nada Majkic-Singh, Institute of Medical Biochemistry, Pharmaceutical RESPONSIBLE FOR ADVERTISEMENTS Markus Kügel, De Gruyter, Rosenheimer Str. 143, 81671 Munich, Germany, Tel.: +49 89 76 902-424, e-mail: anzeigen@degruyter.com TYPESETTING TNQ Technologies, Chennai, India © 2021 Walter de Gruyter GmbH, Berlin / Boston © 2021 Walter de Gruyter GmbH, Berlin / Boston
https://openalex.org/W2099044642	https://hal.science/hal-02929129/file/acp-11-5655-2011.pdf	English	null	Variability and budget of CO&lt;sub&gt;2&lt;/sub&gt; in Europe: analysis of the CAATER airborne campaigns – Part 1: Observed variability	Atmospheric chemistry and physics	2,011	cc-by	17,031	To cite this version: Irène Xueref-Remy, C. Messager, D. Filippi, Maud Pastel, P. Nedelec, et al.. Variability and budget of CO2 in Europe: analysis of the CAATER airborne campaigns – Part 1: Observed variability. Atmospheric Chemistry and Physics, 2011, 11 (12), pp.5655-5672. 10.5194/acp-11-5655-2011. hal- 02929129 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02929129 https://hal.science/hal-02929129v1 Submitted on 4 Sep 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ doi:10.5194/acp-11-5655-2011 © Author(s) 2011. CC Attribution 3.0 License. Atmospheric Chemistry and Physics 4Laboratoire d’A´erologie (LA), 14 avenue Edouard Belin, 31400 Toulouse, France Received: 23 September 2009 – Published in Atmos. Chem. Phys. Discuss.: 26 February 2010 Revised: 24 November 2010 – Accepted: 12 January 2011 – Published: 20 June 2011 Received: 23 September 2009 – Published in Atmos. Chem. Phys. Discuss.: 26 February 2010 Revised: 24 November 2010 – Accepted: 12 January 2011 – Published: 20 June 2011 Abstract. Atmospheric airborne measurements of CO2 are very well suited for estimating the time-varying distribution of carbon sources and sinks at the regional scale due to the large geographical area covered over a short time. We present here an analysis of two cross-European airborne campaigns carried out on 23–26 May 2001 (CAATER-1) and 2–3 Oc- tober 2002 (CAATER-2) over Western Europe. The area covered during CAATER-1 and CAATER-2 was 4◦W to 14◦E long; 44◦N to 52◦N lat and 1◦E to 17◦E long; 46◦N to 52◦N lat respectively. High precision in situ CO2, CO and Radon 222 measurements were recorded. Flask sam- ples were collected during both campaigns to cross-validate the in situ data. During CAATER-1 and CAATER-2, the mean CO2 concentration was 370.1 ± 4.0 (1-σ standard devi- ation) ppm and 371.7 ± 5.0 (1-σ) ppm respectively. A HYS- PLIT back-trajectories analysis shows that during CAATER 1, northwesterly winds prevailed. In the planetary boundary layer (PBL) air masses became contaminated over Benelux and Western Germany by emissions from these highly urban- ized areas, reaching about 380 ppm. Air masses passing over rural areas were depleted in CO2 because of the photosyn- thesis activity of the vegetation, with observations as low as 355 ppm. During CAATER-2, the back-trajectory analysis showed that air masses were distributed among the 4 sectors. HAL Id: hal-02929129 https://hal.science/hal-02929129v1 Submitted on 4 Sep 2020 Air masses were enriched in CO2 and CO over anthropogenic emission spots in Germany but also in Poland, as these coun- tries have part of the most CO2-emitting coal-based plants in Europe. Simultaneous measurements of in situ CO2 and CO combined with back-trajectories helped us to distin- guish between fossil fuel emissions and other CO2 sources. The 1CO/1CO2 ratios (R2 = 0.33 to 0.88, slopes = 2.42 to 10.37), calculated for anthropogenic-inﬂuenced air masses over different countries/regions matched national inventories quite well, showing that airborne measurements can help to identify the origin of fossil fuel emissions in the PBL even when distanced by several days/hundreds of kms from their sources. We have compared airborne CO2 observa- tions to nearby ground station measurements and thereby, conﬁrmed that measurements taken in the lower few me- ters of the PBL (low-level ground stations) are representa- tive of the local scale, while those located in the free tro- posphere (FT) (moutain stations) are representative of atmo- spheric CO2 regionally on a scale of a few hundred kilome- ters. Stations located several 100 km away from each other differ from a few ppm in their measurements indicating the existence of a gradient within the free troposphere. Obser- vations at stations located on top of small mountains may match the airborne data if the sampled air comes from the FT rather than coming up from the valley. Finally, the anal- ysis of the CO2 vertical variability conducted on the 14 pro- ﬁles recorded in each campaign shows a variability at least 5 to 8 times higher in the PBL (the 1-σ standard deviation associated to the CO2 mean of all proﬁles within the PBL is 4.0 ppm and 5.7 ppm for CAATER-1 and CAATER-2, re- spectively) than in the FT (within the FT, 1-σ is 0.5 ppm and 1.1 ppm for CAATER-1 and CAATER-2, respectively). The CO2 jump between the PBL and the FT equals 3.7 ppm for the ﬁrst campaign and −0.3 ppm for the second campaign. Correspondence to: I. Xueref-Remy (irene.xueref@lsce.ipsl.fr) Correspondence to: I. Xueref-Remy (irene.xueref@lsce.ipsl.fr) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 5656 I. 1 Introduction Atmospheric greenhouse gas (GHG) concentrations have been increasing since the pre-industrial era, due to human ac- tivities such as the combustion of fossil fuel compounds and deforestation. The most signiﬁcant growth has been in the presence of CO2, its atmospheric concentration having in- creased by more than 30% during the last 150 yr. Political ef- forts have been made through the United Nations Framework Convention on Climate Change to stabilize atmospheric CO2 concentration levels. Such efforts require independent ver- iﬁcation of anthropogenic and natural ﬂuxes on a regional scale, a task which represent a huge scientiﬁc and politi- cal challenge (Intergovernmental Panel on Climate Change, 2007). The aims of these campaigns were: (1) to validate a new airborne in situ CO2 analyzer; (2) to characterize CO2 vari- ability in the low troposphere above Western Europe; (3) to evaluate the contributions of anthropogenic and biospheric ﬂuxes to this variability; (4) to assess the representativeness of ground stations; and (5) to better characterize the gradients of CO2 between the PBL and the FT. The atmosphere is a strong integrator of CO2 surface sources and sinks. Observations can thus be used to quan- tify surface ﬂuxes over relatively large scales by matching them with modelled ﬁeld simulations from transport models. This method, known as inverse modelling, is the approach most used to quantify CO2 ﬂuxes at regional or global scales. However, the ﬂux partition, especially at the regional scale (100–1000 km) is still poorly understood. Indeed for Europe (based on data from 1992–1996), Gurney et al. (2003) have compared 16 inverse models: all indicate that Europe is a sink for CO2 but they show very large differences, with a mean annual ﬂux of 0.6 GtC yr−1 and a standard deviation of ±0.4 GtC yr−1 i.e. 66% of the mean. In this paper, we show the results of these campaigns con- ducted in the low troposphere (<4000 m) aboard the Falcon 20 of the Deutsches Zentrum f¨ur Luft und Raumfahrt (Oberp- faffenhofen Germany). During this experiment the Falcon was equipped with a continuous CO2 analyser (CONDOR), a continuous CO analyser (MOZAIC CO analyser, Labora- toire d’A´erologie, France) which was used as a combustion tracer and a sequential Radon 222 analyser (AVIRAD) which was used as a tracer of continental air masses. The conditions of the campaigns and the instrumentation deployed are presented in Sect. 2. In Sect. HAL Id: hal-02929129 https://hal.science/hal-02929129v1 Submitted on 4 Sep 2020 In a companion pa- per (Xueref-Remy et al., 2011, Part 2), a comparison of ver- tical proﬁles from observations and several modeling frame- works was conducted for both campaigns. HAL Id: hal-02929129 https://hal.science/hal-02929129v1 Submitted on 4 Sep 2020 Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 Globally at this level, reducing inverse modelling uncer- tainties requires a better characterization of atmospheric CO2 vertical and horizontal variability through in situ observa- tions. Over the past 10 yr, the atmospheric CO2 global mon- itoring network has been largely used to retrieve large scale distribution of sources and sinks at the surface. Recent stud- ies have demonstrated the need for more data over the con- tinental region in order to make feasible robust estimations of the biospheric contribution to the regional carbon bud- get. Stephens et al. (2007) have highlighted the need for in situ vertical observations to better constrain CO2 ﬂuxes. In- deed, because of the large geographical area they can span within a short time, airborne measurements are very well suited to studying atmospheric CO2 variability on a regional scale, vertically and horizontally, and, in particular, can give crucial information on the gradients between the PBL and the free troposphere (FT). Over Europe, airborne campaigns conducted in the boundary layer are very limited as resumed in Geels et al. (2007). The Co-ordinated Access to Aircraft for Transnational Environmental Research (CAATER-1 and CAATER-2), an European initiative, has given us the op- portunity to perform two intensive airborne campaigns over Western Europe in May 2001 and October 2002. A very striking zonal CO2 gradient of about 11 ppm was ob- served in the mid-PBL during CAATER-2, with higher con- centrations in the west than in the east. This gradient may originate from differences in atmospheric mixing, ground emission rates or Autumn’s earlier start in the west. More airborne campaigns are currently under analysis in the frame- work of the CARBOEUROPE-IP project to better assess the likelihood of these different hypotheses. In a companion pa- per (Xueref-Remy et al., 2011, Part 2), a comparison of ver- tical proﬁles from observations and several modeling frame- works was conducted for both campaigns. A very striking zonal CO2 gradient of about 11 ppm was ob- served in the mid-PBL during CAATER-2, with higher con- centrations in the west than in the east. This gradient may originate from differences in atmospheric mixing, ground emission rates or Autumn’s earlier start in the west. More airborne campaigns are currently under analysis in the frame- work of the CARBOEUROPE-IP project to better assess the likelihood of these different hypotheses. 2 Description of the campaigns and of instrumentation Two airborne campaigns were carried out in May 2001 and in October 2002, here called CAATER-1 and CAATER- 2. The two ﬂight routes are different, but they both extend across a domain of roughly 20◦in longitude between West- ern France and West Hungary. The Falcon-20 ﬂights for each campaign are shown in Fig. 1. The CAATER-1 campaign consisted of 5 ﬂights for a total of 14 h during 23–26 May 2001, and the CAATER-2 campaign consisted of 3 ﬂights for a total of 8 h during 2–3 October 2002 (Tables 1 and 2). Vertical CO2 proﬁles and in the case of CAATER-2 also CO and 222-Rn, were collected between the ground and 4000 m alt during each campaign. We have 14 proﬁles in total for each campaign. 1 Introduction 3, we conduct an analysis of air mass origins using back-trajectories. In Sect. 4, we analyse the contribution of anthropogenic emis- sions to CO2 variability using CO data. In Sect. 5, we as- sess the representativeness of ground station measurements using aircraft observations. Finally Sect. 6 focuses on CO2 vertical variability, especially on characterizing CO2 gradi- ents between the PBL and the FT in function of the air mass origins. In a companion paper (Xueref-Remy et al., 2011, Part 2), we conduct a comparison between observa- tions and models: ﬁrstly to assess the capability of a global model versus a mesoscale model to reproduce the observed gradients, incorporating also several biospheric ﬂuxes, and secondly, to present a comparison between model-based ﬂuxes obtained using a new method coupling backplumes Brieﬂy, these large differences originate for several rea- sons. Firstly, most of the studies are based on measurements done on remote and marine sites, far from where terrestrial ﬂuxes are taking place: there is a lack of measurements over the continents to constrain ﬂux calculations by inverse mod- elling (Geels et al., 2007). Secondly, models have difﬁcul- ties in representing atmospheric transport in the continental planetary boundary layer (PBL) (Gerbig et al., 2003). Sev- eral intercomparison studies have been undertaken to make progress in the modeling of the ﬂuxes and especially on a seasonal scale (e.g. Gurney et al., 2004; Baker et al., 2006; Law et al., 2008; Patra et al., 2008; Carouge et al., 2010a, b). Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ 5657 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 Fig. 1. Averaged ﬂux maps (for the days of the campaigns) and ﬂight patterns (red: 1st day, green: 2nd day, pink: 3rd day, blue: 4th day of the campaigns) according to latitude, longitude and altitude (in km). Net Ecosystem Exchange (NEE) ﬂuxes are from ORCHIDEE and fossil fuel (FF) ﬂuxes from Andres et al. (1996) (left: CAATER-1, right: CAATER-2). Oceanic ﬂuxes are from Takahashi et al. (1999) and are almost zero therefore do not appear in the ﬁgure. Fig. 1. Averaged ﬂux maps (for the days of the campaigns) and ﬂight patterns (red: 1st day, green: 2nd day, pink: 3rd day, blue: 4th day of the campaigns) according to latitude, longitude and altitude (in km). Net Ecosystem Exchange (NEE) ﬂuxes are from ORCHIDEE and fossil fuel (FF) ﬂuxes from Andres et al. (1996) (left: CAATER-1, right: CAATER-2). Oceanic ﬂuxes are from Takahashi et al. (1999) and are almost zero therefore do not appear in the ﬁgure. and a priori ﬂuxes, and observation-based ﬂuxes calculated with the Radon method (Schmidt et al., 2003). dard meteological parameter sensors (i.e., pressure, temper- ature, horizontal and vertical windspeeds, dewpoint temper- ature, absolute and relative humidities, virtual temperature and virtual potential temperature). I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5658 Table 1. Airport acronyms and geographical information. Table 1. Airport acronyms and geographical information. Code Name Country Latitude Longitude Altitude (m a.s.l.) BZH Brest France 48◦26′ N 04◦25′ W 99 OBP Oberpfaffenhofen Germany 48◦05′ N 11◦17′ E 593 ORL Orleans France 47◦53′ N 02◦10′ E 120 PDB Paderborn Germany 51◦36′ N 08◦37′ E 213 SOB Sarmellek Hungary 46◦42′ N 17◦06′ E 408 m a.s.l. = meters above sea level. Table 2. Flight information for the CAATER 1 and CAATER 2 campaigns. Date Time range (hh:mm UTC) Flight pattern Flight number 23 May 2001 12:12–14:43 OBP – BZH 1 24 May 2001 15:03–17:50 Atlantic, south of BZH 2 25 May 2001 11:26–13:43 BZH – PDB 3 26 May 2001 08:56–15:46 PDB – OBP 4 2 October 2002 08:48–11:10 OBP – ORL 5 2 October 2002 11:10–13:16 ORL – PDB 6 3 October 2002 10:02–12:20 PDB* – SOB 7 3 October 2002 14:19–15:52 SOB – OBP 8 * No data available between PDB and THU. Table 2. Flight information for the CAATER 1 and CAATER 2 campaigns. * No data available between PDB and THU. Table 3. Ground station acronyms and geographical information. Table 3. Ground station acronyms and geographical information. Ground altitude Type of station Site Name Country Latitude Longitude (m a.s.l.a) (m.a.g.l)b CBW Cabauw Holland 51◦58′ N 04◦55′ E −0.7 Tower (213 m) CMN Monte Cimone Italy 44◦11′ N 10◦42′ E 2165 Surface (mountain top) HUN Hegyhatsal Hungary 46◦57′ N 16◦39′ E 248 Tower (115 m) MHD Mace Head Ireland 53◦19′ N 09◦53′ W 26 Surface PRS Plateau Rosa Italy 45◦56′ N 07◦42′ E 3480 Surface (mountain top) PUY Puy-de-Dome France 45◦45′ N 03◦00′ E 1465 Surface (mountain top) SCH Schauinsland Germany 47◦55′ N 07◦55′ E 1205 Surface (mountain top) WES Westerland Germany 54◦56′ N 08◦19′ E 12 Surface a m a.s.l. = meters above sea level; b m a.g.l. = meters above ground level. 2.1 The CAATER campaigns The CAATER aircraft measurement (Co-ordinated Ac- cess to Aircraft for Transnational Environmental Research) programme is coordinated by the German DLR, French INSU/CNRS and M´et´eo-France, and by the UK Met Of- ﬁce. The programme was funded between 2000 and 2003 by the European Commission to provide research aircraft fa- cilities for pilot projects and new studies. The objective of the CAATER Carbon Dioxide pilot project detailed in this study is to measure the vertical and horizontal variability of CO2 over Western Europe during two contrasted seasons. We used the DLR-Falcon 20 jet aircraft (http://www.dlr.de/fb/en/ desktopdefault.aspx/tabid-3714/5789 read-8405/) equipped with (1) a CO continuous infra-red analyzer, (2) a CO2 non dispersive infra-red gas analyzer, (3) a 222Rn sequential sampler instrument, (4) a ﬂask sampling unit and (5) stan- The paper also uses a dataset from different in situ sam- pling sites described in Table 3. These data will be used in Sect. 5 to assess the stations footprint by comparing them to the CAATER measurements. Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ 2.2 CO2 surface ﬂuxes conditions higher photosynthetic activity in May than in October (see Appendix A), but vegetation index is not easy to translate into a CO2 source/sink map. We show in Fig. 1, overlaid with the campaign ﬂights, the three CO2 continental ﬂux components, i.e.: the Net Ecosystem Exchange (NEE) ﬂuxes given by the process-based ecosystem model ORCHIDEE (Krinner et al., 2005) also described in the companion paper (Xueref-Remy et al., 2011, Part 2) with a spatial resolution of 0.35◦×0.35◦forced by synoptic weather data with a 3 h resolution averaged on the days of the campaign (the diurnal cycle thus being smoothed); annual fossil fuel (FF) emission maps with a spatial resolution of 1◦×1◦from Because the focus of this study is to analyze the variability of CO2 across Western Europe, it is important to understand the underlying ﬂuxes, which we provide in this section. Figure 1 shows the average CO2 ﬂux maps over Western Europe during the sampling interval of each campaign. The net CO2 ﬂux is the sum of fossil fuel CO2 emissions and of the Net Ecosystem Exchange ﬂux, which can be positive (source) or negative (sink) depending upon the vegetation status. The Normalized Difference Vegetation Index (NDVI) provided by the SPOT VGT-4 satellite system reveals a Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. 2.2 CO2 surface ﬂuxes conditions Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5659 2 October 2002 3 October 2002 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 10 10 10 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 20 10 10 10 10 10 10 23 May 2001 24 May 2001 25 May 2001 26 May 2001 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 10 10 10 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 20 10 10 10 10 10 10 23 May 2001 24 May 2001 25 May 2001 26 May 2001 1029 1026 1023 1020 1017 1014 1011 1008 1005 1002 1026 1023 1020 1017 1014 1011 1008 1005 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 20 Oct 2nd, 2002 Oct 3rd, 2002 1029 1026 1023 1020 1017 1014 1011 1008 1005 1002 1026 1023 1020 1017 1014 1011 1008 1005 1029 1026 1023 1020 1017 1014 1011 1008 1005 1002 1026 1023 1020 1017 1014 1011 1008 1005 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 20 20 20 Oct 2nd, 2002 Oct 3rd, 2002 Fig. 2.3 Synoptic weather conditions 2 October 2002 1029 1026 102 1020 1017 1014 101 100 1005 1002 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 Oct 2nd, 2002 1029 1026 102 1020 1017 1014 101 100 1005 1002 1029 1026 102 1020 1017 1014 101 100 1005 1002 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 20 Oct 2nd, 2002 3 October 2002 1026 1023 1020 1017 1014 1011 1008 1005 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 Oct 3rd, 2002 1026 1023 1020 1017 1014 1011 1008 1005 1026 1023 1020 1017 1014 1011 1008 1005 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 20 Oct 3rd, 2002 3 October 2002 Oct 3rd, 2002 Oct 3rd, 2002 Synoptic weather conditions encountered during both cam- paigns are shown in Fig. 2 (http://weather.ou.edu/∼cgodfrey/ reanalysis/). During CAATER 1, the synoptic situation was mainly anti-cyclonic, as shown by mean sea level pressure and 850 hPa wind maps. Note that 850 hPa represents the mean aircraft altitude during the campaign (1457 m above sea level). No major cloud system was present over Europe during the campaign. During 23–24 May 2001 winds from the north-east prevailed over the campaign domain in North- ern France, established around a high-pressure system lo- cated over the British Isles. On 25 May 2001, this high- pressure moved towards southern Scandinavia and north- easterly winds continued to prevail. Fig. 2. Meteorological maps showing mean sea-level pressure (in hPa) and wind speed (in m s−1) at 850 hPa for each day of the CAATER campaigns at 12:00:00 h UTC (http://weather.ou.edu/ ∼cgodfrey/reanalysis/). Latitude (vertical scale) is given in ◦N and longitude (horizontal scale) in ◦E. During CAATER 2, dry conditions were encountered, apart from 3 October 2002 when a few showers occured over Th¨uringen, in Eastern Germany. By 2 October 2002, a small high-pressure system over Norway induced airﬂow from the north into the aircraft route over Southern Germany. But an- other high-pressure placed over the Balkans region creates a second ﬂow from Southern Europe into the aircraft route over France. We thus expect a very signiﬁcant change in air mass origins at the boundary between these two different ﬂow regimes. 2.2 CO2 surface ﬂuxes conditions -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 10 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 20 10 10 23 May 2001 25 May 2001 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 10 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 20 20 10 10 23 May 2001 25 May 2001 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 10 10 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 10 10 10 10 24 May 2001 26 May 2001 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 10 10 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 -10 -5 0 5 10 15 20 25 30 70 65 60 55 50 45 40 35 30 10 10 10 10 24 May 2001 26 May 2001 Andres et al. 2.2 CO2 surface ﬂuxes conditions (1996) updated to the year of each campaign; and oceanic ﬂuxes from Takahashi et al. (1999, 2002). As oceanic ﬂuxes are negligible compared to NEE and fossil fuel ﬂuxes, they do not appear in the maps. These maps show that for this period in May 2001, Europe was mainly acting as a CO2 sink (reaching about −1 gC m−2 day−1) while in early October 2002 it was acting more like a source of CO2 north of 47◦N (about 1 gC m−2 day−1) and a tiny sink south of 45◦N (about 0.4 gC m−2 day−1). Largest fossil fuel emissions occur from the London megacity area, Benelux, Ruhr, the Berlin metropolitan area and the Warsaw city regions. The most emitting coal-fed power plants (the so-called “dirty thirty” identiﬁed by the WWF: http://www.panda.org/what we do/knowledge centres/ climate change/problems/cause/coal/dirty 30/) are located here and represent 10% of European CO2 emissions climate change/problems/cause/coal/dirty 30/) are located here and represent 10% of European CO2 emissions. 2.2 CO2 surface ﬂuxes conditions 2. Meteorological maps showing mean sea-level pressure (in hPa) and wind speed (in m s−1) at 850 hPa for each day of the CAATER campaigns at 12:00:00 h UTC (http://weather.ou.edu/ ∼cgodfrey/reanalysis/). Latitude (vertical scale) is given in ◦N and longitude (horizontal scale) in ◦E. 2.3 Synoptic weather conditions On 3 October 2002, two high-pressure sys- tems were observed, west and east of the aircraft route, over France and Greece. This situation gives rise to complex wind patterns, as further evidenced from the back-trajectory anal- ysis in Sect. 3. uous NDIR airborne analyzer developed at LSCE (Fig. 3). This instrument is based on a commercial sensor Li-COR 6262 with a fast response detector (1 Hz acquisition), with temperature, pressure and ﬂow rates of air being regulated at constant values (Table 4). Outside air was pumped and dried by a magnesium perchlorate cartridge before being analyzed. More details can be found in Filippi et al. (2002). Airborne CO2 measurements are useful for carbon cy- cle studies (inversions) if they have a precision better than 0.5 ppm (Gloor et al., 2000). Frequent calibrations (ev- ery 30 min) give an instantaneous precision of 0.1 ppm, but slow instrument drifts due to changes in surrounding physi- cal parameters such as pressure and temperature require fre- quent calibrations during ﬂights. We used two calibration gases in high-pressure cylinders of 2 l, with concentrations in the NOAA X-93 scale of 365.92 ± 0.05 ppm (Low) and 401.29 ± 0.05 ppm (High). Each standard is passed through the analyzer for 3 min, and only the last minute of acqui- sition is retained to compute the calibration curve and cal- culate outside air CO2. Given the limited volume of each 2.4.3 In situ continuous CO measurements The CO analyser is the same as the one developed for routine measurements onboard passenger aircrafts for the MOZAIC program (http://mozaic.aero.obs-mip.fr/web/). CO was only measured during CAATER-2 and its characteristics are sum- marized in Table 4. This analyser described in Nedelec et al. (2003) is a fully automated instrument designed to reach an accuracy of 5%. It is based on the commercial IR cor- relation gas analyser Model48C produced by Thermo En- vironment Instruments [TEI, USA]. It is a Gas Filter Cor- relation instrument based on the principle of infra-red ab- sorption by the 4.67 µm fundamental vibration-rotation band of CO. Radiation from an infrared source is chopped and passed through a gas ﬁlter which alternates between CO and N2. The radiation then passes through a narrow band pass ﬁlter and a multiple optical pass sample cell where absorp- tion by the sample gas occurs. The IR radiation exits the sample cell and falls on a lead-selenium solid state IR de- tector. Other gases do not cause modulation of the detector signal since they absorb the reference and measure beams equally. Thus, the Gas Filter Correlation System responds speciﬁcally to CO. The Model 48CTL is also qualiﬁed by US EPA designated Method (EQSA-0486-060). The speciﬁ- cation of the commercial instrument is 10 ppbv CO for 300 s integration time. Major improvements have been brought by N´ed´elec et al. (2003): periodic accurate zero measurements, new IR detector with better cooling and temperature regula- tion, pressure increase and regulation in the absorption cell, increased ﬂow rate to 4 l min−1, water vapor trap, and ozone ﬁlter. The speciﬁcations achieved for 30 s integration time (response time of the instrument) are a precision of ±5 ppbv CO with a minimum detectable of 10 ppbv of CO. The in- strument was calibrated before and after the campaign with Fig. 3. Schematic of the CONDOR analyzer. cylinder, the time taken for calibration and the sought pre- cision, an optimal compromise had to be found. We carried out ≈30 calibrations during each campaign. The average sta- bility (1-σ std. deviation on 1-Hz data) was 0.03 ppm dur- ing the last minute of each calibration passage. To compute the instrument accuracy, each calibration gas was treated as an unknown target. The difference between the true and the measured target value is 0.10 ppm for the high standard and 0.08 ppm for the low one. 2.4.1 In situ continuous CO2 measurements In addition to standard meteorological parameters (wind, rel- ative humidity, temperature, pressure) with a 1-Hz acquisi- tion, the CO2 concentrations were measured with a contin- Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5660 Table 4. Characteristics of the CO2 and CO analyzers. Table 4. Characteristics of the CO2 and CO analyzers. Parameter CO2 analyzer (CONDOR) CO analyzer Precision ≤0.20 ppm for 1 s ±5 ppbv for 30 s Sampling frequency 1 Hz 0.03 Hz Power supply 18-32VDC/15A max 24V/10A max Cells pressure 1080 hPa 2532 hPa Flow rates 50 sccm (Reference cell) 400 sccm (Sample cell) 4000 sccm Temperature 35 ◦C 30 ◦C Calibrations 1 calibration (6 mn) every 30 mn Before and after each campaign Volume 95×55×40 cm3 60×31×43 cm3 Weight 80 kg 45 kg Fig. 3. Schematic of the CONDOR analyzer. centration was systematically rejected if the two measure- ments differed by more than 0.10 ppm (3.2% of the samples). The mean difference between the in situ NDIR and the ﬂasks was less than 0.20 ppm for both campaigns (see Appendix B, Fig. B1). 2.4.3 In situ continuous CO measurements Also, we found that, even when using one single calibration result before each ﬂight, the in- strument accuracy is still greater than 0.20 ppm. 3.2 Air masses sampled in May 2001 during CAATER-1 pp In order to investigate the origin of the sampled air masses, we computed for each ﬂight, 5-days backtrajectories at dif- ferent measurement points of the ﬂight path. The HYSPLIT- 4 (Hybrid Single-Particle Lagrangian Integrated Trajectory) was used to compute these backtrajectories (Draxler and Hess, 1998). Four-dimensional (x,y,z,t) gridded meteoro- logical ﬁelds from NOAA/NCEP (National Centers for En- vironmental Predictions, http://www.ncep.noaa.gov/) global reanalysis data were used to drive HYSPLIT-4 (analysis ev- ery 6 h, 1◦×1◦horizontal resolution, 14 vertical levels). Each trajectory was calculated as the time integrated advection of a single particle. The integration time-step can vary during the simulation, as it is computed from the requirement that the advection distance per time-step should be less than 75% of the meteorological grid spacing. This linear integration method is common (e.g. Kreyszig, 1968) and has been used for trajectory analysis (Petterssen, 1940) for quite some time. Advection is computed from the average of the 3-D-velocity vector for the position at time t and the position at time t −1. The accuracy of the model has been quantiﬁed by testing the model trajectories against balloon data: the difference was about 10 to 20% (Draxler, 1998). Back-trajectories calculated for each ﬂight of CAATER 1 are shown in Fig. 5 (a projection of Fig. 4 for CAATER 1 in the horizontal longitude-latitude plan). About 58% of the air masses sampled came from the north-west, 37% from the north-east and only 5% from the south-east. y On 23 May 2001 (Fig. 5a), between 4◦E and 2◦W, the back-trajectories indicate a continental origin from the north- east, with air masses being advected in the boundary layer (<2000 m) and carrying low CO2 values of ≈360 ppm. As the aircraft moved west of 2◦W, the sampled air mass reached values in the range of 372–375 ppm. Indeed dur- ing that day, from ECMWF reanalysis, we know that there was no special feature in the vertical structure of the atmo- sphere such as convection that can explain this difference in concentration. Backtrajectories show that between 2◦E and 2◦W, air masses were advected from the east-south-east in the boundary layer, while they were advected from the north- east passing above the ocean between 2◦W and 4◦W before landing in Brest. 3.1 Mean CO2, CO concentrations and back-trajectories The CO2 distribution observed over the European continent results from the mixing of oceanic background air with con- tinental signals from fossil fuel emissions and biospheric sources and sinks. The lower tropospheric CO2 mean con- centrations between 0–4000 m alt were 370.1 ± 4.0 ppm dur- ing CAATER-1 (May) and 371.7 ± 5.0 ppm during CAATER 2 (October). Thus, despite a stronger biogenic (biospheric) uptake in Spring, reducing background CO2 over the whole Northern Hemisphere and CO2 over the European continent, the lower troposphere mean level in May was roughly similar to October. For CAATER-2, the mean CO concentration was 177 ± 61 ppb. As a reference for our analysis, we computed marine back- ground concentrations of CO2 and CO in the boundary layer, called MBL values. These MBL values were computed us- ing records from the Mace Head station that is part of the RAMCES network (https://ramces.lsce.ipsl.fr/). Mace Head is located on the west coast of Ireland, thus, marine back- ground concentrations during CAATER could be inferred by selecting the marine wind sector data averaged over each campaign’s duration. For CO2, the MBL values obtained are 374.5 ± 0.3 ppm during CAATER 1 and 367.9 ± 0.2 ppm during CAATER 2; for CO the MBL value computed for CAATER 2 is 131.1 ± 0.2 ppb. Fig. 4. CO2 concentration 3-D distribution according to longitude, latitude and altitude. The ﬂight patterns are projected on the longi- tude/latitude plan. (top: CAATER-1, bottom: CAATER-2). 2.4.2 Flasks analysis To independently assess the instruments accuracy, about 50 1-l glass ﬂasks were sampled during each campaign. Af- ter 5 min of ﬂushing, the sampled air was compressed at 1 bar above atmospheric pressure to avoid any contamination due to leakage. The CO2 concentration was measured by gas chromatography at LSCE with a precision <0.10 ppm (P´epin et al., 2001), each ﬂask being measured twice. A ﬂask con- Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5661 Fig. 4. CO2 concentration 3-D distribution according to longitude, latitude and altitude. The ﬂight patterns are projected on the longi- tude/latitude plan. (top: CAATER-1, bottom: CAATER-2). a traceable CO cylinder from the National Institute of Stan- dards and Technology in Boulder, Colorado, USA. a traceable CO cylinder from the National Institute of Stan- dards and Technology in Boulder, Colorado, USA. I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. c- Flight 3 (25 May 2001) : BZH - PDB d- Flight 4 (26 May 2001) : PDB-OBP 11:30 12:00 12:30 13:00 13:30 Time (hh:mm UTC) 10:00 12:00 14:00 16:00 Time (hh:mm UTC) b- Flight 2 (24 May 2001) : Atlantic survey south of BZH a- Flight 1 (23 May 2001) : OBP-BZH 12:30 13:00 13:30 14:00 14:30 Time (hh:mm UTC) 15:00 15:30 16:00 16:30 17:00 17:30 18:00 Time (hh:mm UTC) Fig. 5. (a, b) Back-trajectories computed over 96 h for ﬂight 1 and ﬂight 2. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back- trajectories. Right panels show the altitude of the ﬂight, and back- trajectories colored according to air mass altitude. Black circles rep- resent ground stations named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. c- Flight 3 (25 May 2001) : BZH - PDB d Flight 4 (26 May 2001) : PDB OBP 11:30 12:00 12:30 13:00 13:30 Time (hh:mm UTC) a- Flight 1 (23 May 2001) : OBP-BZH 12:30 13:00 13:30 14:00 14:30 Time (hh:mm UTC) c- Flight 3 (25 May 2001) : BZH - PDB a- Flight 1 (23 May 2001) : OBP-BZH Time (hh:mm UTC) d- Flight 4 (26 May 2001) : PDB-OBP d- Flight 4 (26 May 2001) : PDB-OBP b- Flight 2 (24 May 2001) : Atlantic survey south of BZH b- Flight 2 (24 May 2001) : Atlantic survey south of BZH 15:00 15:30 16:00 16:30 17:00 17:30 18:00 Time (hh:mm UTC) Fig. 5. (c, d) Back-trajectories computed over 96 h for ﬂight 3 and ﬂight 4. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back- trajectories. Right panels show the altitude of the ﬂight, and back- trajectories colored according to air mass altitude. Black circles rep- resent ground stations named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5662 b- Flight 2 (24 May 2001) : Atlantic survey south of BZH a- Flight 1 (23 May 2001) : OBP-BZH 12:30 13:00 13:30 14:00 14:30 Time (hh:mm UTC) 15:00 15:30 16:00 16:30 17:00 17:30 18:00 Time (hh:mm UTC) Fig. 5. (a, b) Back-trajectories computed over 96 h for ﬂight 1 and ﬂight 2. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back- trajectories. Right panels show the altitude of the ﬂight, and back- trajectories colored according to air mass altitude. Black circles rep- resent ground stations named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. c- Flight 3 (25 May 2001) : BZH - PDB d- Flight 4 (26 May 2001) : PDB-OBP 11:30 12:00 12:30 13:00 13:30 Time (hh:mm UTC) 10:00 12:00 14:00 16:00 Time (hh:mm UTC) Fig. 5. (c, d) Back-trajectories computed over 96 h for ﬂight 3 and ﬂight 4. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back- trajectories. Right panels show the altitude of the ﬂight, and back- trajectories colored according to air mass altitude. Black circles rep- resent ground stations named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. c- Flight 3 (25 May 2001) : BZH - PDB d- Flight 4 (26 May 2001) : PDB-OBP 11:30 12:00 12:30 13:00 13:30 Time (hh:mm UTC) 10:00 12:00 14:00 16:00 Time (hh:mm UTC) Fig. 5. (c, d) Back-trajectories computed over 96 h for ﬂight 3 and ﬂight 4. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back- trajectories. Right panels show the altitude of the ﬂight, and back- trajectories colored according to air mass altitude. Black circles rep- resent ground stations named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). 3.2 Air masses sampled in May 2001 during CAATER-1 It is thus very likely that the depletion of CO2 observed between 2◦E and 2◦W on 23 May 2001 was due to advection of air formerly exposed to terrestrial CO2 uptake. Indeed two days later (see Fig. 5c) we encountered www.atmos-chem-phys.net/11/5655/2011/ Atmos. Chem. Phys., 11, 5655–5672, 2011 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 a- Flight 5 (2 October 2002) : OBP - ORL b- Flight 6 (2 October 2002) : ORL - PDB (Germany) 09:00 09:30 10:00 10:30 11:00 Time (hh:mm UTC) 11:00 11:30 12:00 12:30 13:00 Time (hh:mm UTC) Fig. 6. (a, b) Back-trajectories computed over 96 h for ﬂights 5 and 6. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back-trajectories. Right panels show the altitude of the ﬂight, and back-trajectories colored according to air mass altitude. Black circles represent ground sta- tions named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. a- Flight 5 (2 October 2002) : OBP - ORL 09:00 09:30 10:00 10:30 11:00 Time (hh:mm UTC) Europe when compared to the “biospheric” minimum further east is of the order of 20 ppm in the whole boundary layer. a- Flight 5 (2 October 2002) : OBP - ORL pp y y On 26 May 2001 (Fig. 5d), we sampled oceanic, bio- spheric and anthropogenic air masses. Around 12◦E in the mid-afternoon, we measured low concentrations (around 365 ppm). The easterly ﬂow associated with these mini- mum CO2 values corresponds to air masses advected into the boundary layer, and thus directly exposed to continu- ous biospheric uptake. In contrast around 14◦E, an air mass coming from the west and north-west was sampled. Back trajectories indicated that this air mass is exposed to anthro- pogenic emissions, and contains CO2 values 25 ppm above these low concentrations. Note that all these measurements were recorded in the PBL at altitudes lower than 400 m a.s.l.; backtrajectories show that the high-CO2 air mass sampled at 14◦E was advected from the north-west at altitudes below 500 m, with an origin that can be approximately traced to anthropogenic emissions over Northern Germany and Den- mark. 3.3 Air masses sampled in October 2002 during CAATER-2 We also analyze the relationship between CO and CO2 ob- served during the CAATER 2 campaign (unfortunately, no CO data is available for CAATER 1). In air masses inﬂu- enced by combustion processes, we expect a positive corre- lation between the residual concentrations of CO and CO2 computed to background concentrations. This should enable the identiﬁcation of fossil fuel CO2 in total CO2 in sampled air masses (e.g. Levin and Karstens, 2007). This (too) simple theory is however complicated by (1) different ratios of CO to fossil fuel CO2 emissions in the various types of combus- tion, (2) mixing of air rich in fossil fuel CO2 with oceanic or vegetation ﬂuxes, a process that changes CO2 without chang- ing CO, and (3) chemical production of secondary CO from biogenic volatile organic compounds, which in summer can contribute as much CO as anthropogenic combustions (Riv- ier et al., 2006); this process tends to increase CO but not CO2. During the CAATER-2 campaign, these three pro- cesses (different mix of combustion, mix of oceanic, vege- tation and fossil fuel CO2, secondary CO production) are un- fortunately at work simultaneously. This, in principle, makes the inferrence of fossil fuel CO2 very difﬁcult, as already noted when using ground-based station records (Karstens et al., 2006; Levin et al., 2007). In this context, it is interest- ing to check the relationship between CO and CO2 in the diverse sampled air masses during the CAATER-2 campaign (see Sect. 3). Fig. 6. (a, b) Back-trajectories computed over 96 h for ﬂights 5 and 6. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back-trajectories. Right panels show the altitude of the ﬂight, and back-trajectories colored according to air mass altitude. Black circles represent ground sta- tions named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. we computed the linear regression correlation (R2 determi- nation factor) and the slope (S) of 1CO vs. 1CO2 (delta standing for the difference of the observed concentration to the marine background concentration) for each air mass. The results are presented in Fig. 8. I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 On the other hand, air masses coming from altitudes higher than 1700 m from the West, with tropospheric air de- coupled from surface ﬂuxes, are associated to CO2 concen- trations (∼374 ppm) close to the marine background value deﬁned in Sect. 3.1 (374.5 ppm). b- Flight 6 (2 October 2002) : ORL - PDB (Germany) b- Flight 6 (2 October 2002) : ORL - PDB (Germany) b- Flight 6 (2 October 2002) : ORL - PDB (Germany) 11:00 11:30 12:00 12:30 13:00 Time (hh:mm UTC) Time (hh:mm UTC) I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. Fig. 5. (a, b) Back-trajectories computed over 96 h for ﬂight 1 and ﬂight 2. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back- trajectories. Right panels show the altitude of the ﬂight, and back- trajectories colored according to air mass altitude. Black circles rep- resent ground stations named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. over the Bay of Biscay ﬁrst sampled air advected from the English Channel at ≈4000 m alt with oceanic CO2 concen- trations of 372–374 ppm, and then continental air from north- eastern Europe, with CO2 depleted by plant uptake down to 360 ppm. a similar CO2 depletion between 2◦E and 2◦W, also below 2000 m, around midday and with similar pressure and wind conditions than on 23 May 2001. For this ﬂight, simulta- neous in-situ CO2 and 222Rn measurements allowed us to identify the role of terrestrial CO2 uptake over France at that period (Xueref-Remy et al., 2011, Part 2). Between 2◦W and 4◦W, we can conclude from the HYSPLIT backtrajecto- ries that an oceanic air mass was sampled, whose CO2 con- centration was close to the MBL value deﬁned in Sect. 3.1 (374.5 ppm). On 25 May 2001 (Fig. 5c), the air mass was ﬁrst oceanic (374 ppm). Moving eastwards between 3◦W and 1◦E, sam- pled continental air masses gave CO2 concentrations of ap- proximately 360 ppm (biospheric-inﬂuenced values, that are similar to those sampled during 23 May on roughly the same route westwards: see above), then oceanic air, followed again by anthropogenic emission plumes over the Ruhr area with CO2∼380 ppm. The signal of this high emission region of On 24 May 2001 (Fig. 5b) over the Bay of Biscay, air masses were inﬂuenced by local anthropogenic emissions near the surface close to BZH (∼378 ppm). The aircraft ﬂight Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ 5663 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. 3.3 Air masses sampled in October 2002 during CAATER-2 c- Flight 7 (3 October 2002) : THU - SOB (Hungary) 10:00 10:30 11:00 11:30 12:00 Time (hh:mm UTC) c- Flight 7 (3 October 2002) : THU - SOB (Hungary) Time (hh:mm UTC) d- Flight 8 (3 October 2002) : SOB - OBP 14:30 15:00 15:30 Time (hh:mm UTC) d- Flight 8 (3 October 2002) : SOB - OBP Fig. 7. CO, CO2 and altitude time series during the CAATER 2 ﬂights (top: 2 October 2002; bottom: 3 October 2002). Striking events have been selected with colorbars (pink is for C: high CO and CO2 correlation level events, green is for M: mixed determination factor events, blue is for B: tropospheric background events). 15:00 Time (hh:mm UTC) On 3 October 2002, during the (return) ﬂight from SOB to OBP (Fig. 6d), the aircraft climbed above 4000 m (event B6). Background values of CO, CO2 at 4000 m were checked and conﬁrmed to be unchanged from the ﬂight above the Or- leans forest (event B2). Then, an horizontal route was fol- lowed in the boundary layer (mean altitude 300 m; event C5 in Fig. 7). Near-surface anthropogenic emission plume sam- pled west of Hegyhatsal with a high 1CO vs. 1CO2 cor- relation (R2 = 0.88, slope = 10.37 ppb CO ppm−1 CO2) was most likely of local origin, from Graz (second largest city in Austria with 300 000 people and an international airport). Finally, the airplane crossed Eastern Germany westwards to OBP at 2200m altitude. Surprinsingly low CO2 values for the month of October (360 ppm) were observed during that last ﬂight, as well as low CO values (110 ppb). The back- trajectories of this ﬂight show a complex pattern, with some air coming from the Alps before reaching the aircraft posi- tion. Fig. 6. (c, d) Back-trajectories computed over 96 h for ﬂights 7 and 8. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back-trajectories. Right panels show the altitude of the ﬂight, and back-trajectories colored according to air mass altitude. Black circles represent ground sta- tions named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. 3.3 Air masses sampled in October 2002 during CAATER-2 The R2 and slope values showed a wide range of variation when all air masses are considered without distinction (Table 5). To all R2, we com- puted the associated p-value (Table 5): in all cases the corre- lation signiﬁcance is higher than 99%. On 3 October 2002, during the ﬂight from OBP to SOB (Table 2; Fig. 6c) a vertical proﬁle was sampled over a rural location in the Th¨uringen area (event B4 in Fig. 7). This proﬁle shows CO2 and CO values close to background. The same tropospheric values at 4000 m as those measured First, we identiﬁed the different air masses in the CAATER-2 dataset classiﬁed by their origins (Fig. 6). Then Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5664 d- Flight 8 (3 October 2002) : SOB - OBP c- Flight 7 (3 October 2002) : THU - SOB (Hungary) 10:00 10:30 11:00 11:30 12:00 Time (hh:mm UTC) 14:30 15:00 15:30 Time (hh:mm UTC) Fig. 6. (c, d) Back-trajectories computed over 96 h for ﬂights 7 and 8. The distance between 2 triangles is 24 h. Left panels show CO2 concentrations along the ﬂight path and back-trajectories. Right panels show the altitude of the ﬂight, and back-trajectories colored according to air mass altitude. Black circles represent ground sta- tions named in Fig. 1. Altitude is given in meters above the sea level (m a.s.l.). Note that some of the back-trajectories are outside the latitude and longitude chosen borders, but these are so diluted that they do not give any relevant information. 09:00 10:00 11:00 12:00 13:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 Time (hh:mm UTC) Fig. 7. CO, CO2 and altitude time series during the CAATER 2 ﬂights (top: 2 October 2002; bottom: 3 October 2002). Striking events have been selected with colorbars (pink is for C: high CO and CO2 correlation level events, green is for M: mixed determination factor events, blue is for B: tropospheric background events). 3.3 Air masses sampled in October 2002 during CAATER-2 one day earlier over ORL shows that the signal of sources and sinks is mainly conﬁned to the boundary layer during the CAATER-2 campaign, because of anticyclonic condi- tions and reduced mixing. An anthropogenic emission plume (event C3 in Fig. 7) was sampled between 12◦and 13.5◦E, with a small positive 1CO vs. 1CO2 correlation (R2 = 0.26, slope = 1.78 ppb CO ppm−1 CO2). In this C3 event, the ori- gin of the air masses was traced to the high emission Ruhr industrial region. At longitude 17◦E (event C4 in Fig. 7) we encountered a better-deﬁned anthropogenic emission plume (R2 = 0.47, slope = 3.2 ppb CO ppm−1 CO2) during the ver- tical proﬁle over the tall tower of Hegyatsall. 4 Comparison of CAATER observations to CO:CO2 emission ratios from inventories for anthropogenic- inﬂuenced air masses 4 Comparison of CAATER observations to CO:CO2 emission ratios from inventories for anthropogenic- inﬂuenced air masses Colors represent correlated events C1 to C5 as deﬁned in the text. See Sect. 3 for marine background concentration deﬁnition. see in Fig. 6a that C1 is not only inﬂuenced by Eastern Ger- many, but also by industrial regions (Malm¨o) in Southern Sweden and in Western Poland, where the CO:CO2 emission ratio are higher regionally (range 11.1 to 12 kt CO/Mt CO2). C1 C2 C3 C4 & C5 Delta [CO2] (ppm) Delta [CO] (ppb) C1 C2 C3 C4 & C5 Delta [CO2] (ppm) Delta [CO] (ppb) Air mass C2 was exposed to Rhone valley emissions as well as to CO2 uptake by vegetation in that region (Sect. 3; see also Fig. 1 (NEE) and the high NDVI values in Fig. A1). This air mass shows a weak 1CO2−1CO correlation (R2 = 0.65) and a lower slope (3.52 ppb CO ppm−1 CO2) than would be expected from fossil fuel addition alone (France CO:CO2 emission inventory ratio ρ = 14 kt CO/Mt CO2), in- dicating a strong biospheric contribution reducing CO2 while leaving CO unaffected. Air mass C3, sampled at ∼900 m, was exposed to emis- sions in Western Germany from the Ruhr region (Fig. 6c). The R2 factor is not very high (0.26) and the slope, S = 1.78 ppb CO ppm−1 CO2, is quite low compared to the slope of 5.1 kt CO/Mt CO2 from the emission inventory ratio for Germany, meaning that these emissions have probably been diluted. Fig. 8. 1CO (i.e. CO minus CO marine background concentration) versus 1CO2 (i.e. CO2 minus CO2 marine background concentra- tion) for the CAATER 2 ﬂights. Colors represent correlated events C1 to C5 as deﬁned in the text. See Sect. 3 for marine background concentration deﬁnition. Air mass C4, sampled at low altitude (∼400 m), showed exposure to fossil fuel ﬂuxes in Austria and Czech Republic (Fig. 6c). The mean observed slope is 3.2 ppb CO ppm−1 CO2, close to the emission inventory ratio value for Czech Republic (4.4 kt CO/Mt CO2), but less close to the one for Austria (10.4 kt CO/Mt CO2). Thus, fossil fuel CO2 from Czech Republic must have had a dominant inﬂuence on these air masses. However, the large scattering around the linear regression line (R2 = 0.47) indicates interplay of combustion sources with different ratios. Air mass C1 was exposed to fossil fuel emission ﬂuxes in Eastern Germany/Western Poland/Southern Sweden (see Sect. 3). 4 Comparison of CAATER observations to CO:CO2 emission ratios from inventories for anthropogenic- inﬂuenced air masses We selected here only the well-identiﬁed anthropogenic- inﬂuenced air masses diagnosed from back-trajectories (events C1 to C5). These air masses all show high R2 values (0.33 to 0.88), but their slope varies from 2.42 to 10.37 ppb CO ppm−1 CO2 (Table 5). These slope values are compared below to the CO:CO2 slope (ρ in kt CO Mt−1 CO2) given by emission inventories (EMEP, 2008) for the region of inﬂu- ence of each air mass. Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5665 Table 5. Evaluation of the correlation level between CO2 and CO for the different events selected from Fig. 7. Each event has been attributed to a class according to its back-trajectory location and direction (from Fig. 6) as explained in the text. Event Determination p-value Slope 1CO/1CO2 Day factor R2 (number of pairs) (in ppb CO ppm−1 CO2) (in October 2002) C1 0.82 3.60×10−42 (n = 60) 6.38 2 C2 0.65 3.14×10−39 (n = 66) 3.52 2 C3 0.26 0. (n = 1673) 1.78 3 C4 0.47 0. (n = 530) 3.2 3 C5 0.69 0. (n = 1461) 8.93 3 M1 0.13 0. (n = 141) 0.9 2 M2 0.35 0. (n = 2198) 0.76 2 M3 −0.42 0. (n = 2968) −2.88 2 M4 −0.06 9.11×10−3 (n = 1848) −0.36 2 B1 −0.67 1.75×10−5 (n = 100) −11.4 2 B2 0.54 0. (n = 2234) 12.1 2 B3 −0.41 0. (n = 886) −10.1 2 B4 0.15 6.67×10−6 (n = 922) 3.56 3 B5 −0.56 0. (n = 861) −7.15 3 B6 −0.43 0. (n = 556) −2.56 3 B7 −0.73 0. (n = 915) −2.36 3 Table 5. Evaluation of the correlation level between CO2 and CO for the different events selected from Fig. 7. Each event has been attributed to a class according to its back-trajectory location and direction (from Fig. 6) as explained in the text. C1 C2 C3 C4 & C5 Delta [CO2] (ppm) Delta [CO] (ppb) C1 C2 C3 C4 & C5 Delta [CO2] (ppm) Delta [CO] (ppb) Fig. 8. 1CO (i.e. CO minus CO marine background concentration) versus 1CO2 (i.e. CO2 minus CO2 marine background concentra- tion) for the CAATER 2 ﬂights. 4 Comparison of CAATER observations to CO:CO2 emission ratios from inventories for anthropogenic- inﬂuenced air masses It has the highest R2 (0.82), and a slope S = 6.38 ppb CO ppm−1 CO2 higher than the mean inventory de- rived slope for Germany (ρ = 5.1 kt CO/Mt CO2). One can Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5666 Longitude (°E) Longitude (°E) Longitude (°E) Longitude (°E) Fig. 9. CO2 concentrations along the ﬂight patterns represented in function of longitude and altitude during the CAATER 1 (left) and CAATER 2 (right) campaigns. Longitude (°E) Longitude (°E) Longitude (°E) Longitude (°E) Air mass C5 was sampled at quite low altitude (∼400 m) over rural west Hungary. It was inﬂuenced by ﬂuxes from West Hungary (local sources), as well from Austria, Switzer- land and Southern Germany (remote sources). Despite the rather complex spiraling back-trajectories shown in Fig. 6d for C5, this air mass keeps a tight positive correlation be- tween 1CO and 1CO2 (R2 = 0.69) and the measured slope (8.93 ppb CO ppm−1 CO2) is close to the regional inventory ratios (Hungary, Austria and Switzerland: ρ = 10.1, 10.4 and 8.3 kt CO/Mt CO2, respectively). In summary, this analysis shows that despite mixing of fossil fuel CO2 ﬂuxes with biospheric CO2 ﬂuxes (which can be >0 in the northern part of Europe and <0 in its southern part; Fig. 1), and spatial and temporal variability in the CO:CO2 fossil fuel combustion ratios in the differ- ent countries (reﬂecting different reliances on fossil fuel for energy production), it is still possible to use the observed 1CO:1CO2 slope in atmospheric measurements to quantify the contribution of fossil among other sources of CO2 a few days/hundreds of km distant from the original source. Here the integrative properties of synoptic atmospheric transport help to average the contrasted CO:CO2 ratios of local emis- sions. Fig. 9. CO2 concentrations along the ﬂight patterns represented in function of longitude and altitude during the CAATER 1 (left) and CAATER 2 (right) campaigns. -13 -8 -2 2 8 -13 -8 -2 2 8 -13 -8 -2 2 8 -13 -8 -2 2 8 Fig. 10. Interpolation of the CO2 concentrations for the whole cam- paign and for each leg according to longitude and altitude during the CAATER 1 (left) and CAATER 2 (right) campaigns. 4 Comparison of CAATER observations to CO:CO2 emission ratios from inventories for anthropogenic- inﬂuenced air masses The concen- tration scale refers to the MBL background concentration of each campaign, as deﬁned in the text. Flight paths are shown in black. Ground station concentrations, averaged in Sect. 1, are shown at the respective station coordinates. Note that for plot convenience, HUN and WES stations during CAATER 2 are shown as being at the max- imum of the chosen concentration scale, while indeed they show even higher concentrations (respectively: 12.7 ppm and 38.5 ppm above the marine background concentration, deﬁned in Sect. 3). 5 Comparison of aircraft measurements with surface station measurements Fig. 10. Interpolation of the CO2 concentrations for the whole cam- Fig. 10. Interpolation of the CO2 concentrations for the whole cam- paign and for each leg according to longitude and altitude during the CAATER 1 (left) and CAATER 2 (right) campaigns. The concen- tration scale refers to the MBL background concentration of each campaign, as deﬁned in the text. Flight paths are shown in black. Ground station concentrations, averaged in Sect. 1, are shown at the respective station coordinates. Note that for plot convenience, HUN and WES stations during CAATER 2 are shown as being at the max- imum of the chosen concentration scale, while indeed they show even higher concentrations (respectively: 12.7 ppm and 38.5 ppm above the marine background concentration, deﬁned in Sect. 3). Here we compare the aircraft-measured CO2 distribution with ground-based station records. The goal is to produce a consistent 3-D picture of the CO2 ﬁeld, and to analyze the reasons for concentration differences between altitude and ground level. We used available CO2 data from sev- eral ground-based observatories taken at the time of the cam- paigns (CBW, PUY, CMN and PRS for CAATER-1; WES, HUN, SCH, PUY, CMN and PRS for CAATER-2; described in Table 3). Hourly data from the stations were selected from 12:00:00 to 18:00:00 h UTC to ﬁlter out night-time and morning data, not representative of regional-scale con- ditions. Indeed in the morning, most of the time, the PBL is growing and encroaches air from aloft while loosing CO2 accumulated by respiration the former night (Gibert et al., 2007), making CO2 concentrations more variable. The PBL gets well-mixed and reaches to a relatively stable height only around midday. The footprint of the stations is thus better de- ﬁned with afternoon values, and more adapted to do a com- parison with aircraft data. 200 km horizontally and 100 m vertically. In Fig. 10, the ma- rine background value (reminder: 374.5 ppm for CAATER- 1; 367.9 ppm for CAATER-2) was removed from all the data. Figure 11 shows a comparison between ground sta- tion values and the mean proﬁle recorded during each cam- paign. The CO2 vertical distributions and cross-sections (Figs. 6 Analysis of the vertical variability CMN agrees better with the aircraft observations than the other stations do, even if it is located outside the cam- paign domain. CO2 at CMN (2165 m) compares within 0.7 ppm (0.3 ppm) to the CAATER-1 (CAATER-2) inter- polated data for the station’s location. During CAATER- 1 (May 2001), the high amplitude of the diurnal cycle of CO2 at CMN (http://gaw.kishou.go.jp/wdcgg/products/ cd-rom/cd 14/A/metadata/co2/data/200612120073.html) re- vealed that this station lay within the PBL. However, as CMN is located at a relatively high altitude and far from local anthropogenic sources, during CAATER-2 it was now lo- cated in the free troposphere. It was measuring similar air masses from the West as those recorded by the aircraft dur- ing the closest proﬁle (around 12◦E), on which the interpo- lation at CMN mainly relies. Interestingly, at PRS (4000 m) which was located in the free troposphere during both cam- paigns, interpolated airborne data and station observations are different by about 3 ppm and 0.5 ppm for CAATER-1 and CAATER-2, respectively. Indeed, the CAATER-1 interpola- tion lies on one proﬁle done above northern Germany with air masses coming from the north (Fig. 5) while PRS is lo- cated further south, sampling air masses from the south, east and west as it can be seen on Fig. 5. This reveals a gradient of a few ppm in the free troposphere over Western Europe. The CO2 gradient between the boundary layer and the free troposphere is a key parameter to optimize ﬂuxes using in- verse methods. For each proﬁle, the PBL height was deter- mined as the altitude at which the vertical gradient of the po- tential temperature begins to decrease, and where CO2 and H2O present step changes (Gerbig et al., 2003; Ramonet et al., 2002). To illustrate the CO2 vertical variability, the altitude of each single proﬁle was then normalized versus the PBL height; the normalized proﬁles are shown on Fig. 12 for CAATER-1 and Fig. 13 for CAATER-2. To improve read- ability, data have been averaged over 50 m layers. As already noticed in the previous section, CO2 values show a strong variability in the PBL compared to the FT. The mean and standard deviation (std) computed on all data within the PBL are, respectively, 369.9 ppm and 4.0 ppm dur- ing CAATER-1; 371.5 ppm and 5.7 ppm during CAATER- 2. I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5667 Fig. 11. Variability observed in the PBL and the FT during each campaigns (left: CAATER 1, right: CAATER 2) using proﬁles only. The altitude of each individual proﬁle has been normalized to the relevant PBL height. The plot shows the mean and standard devi- ation (1-σ) of CO2 concentration over layer of 1/10th of the PBL height. The global mean and variability (±1-σ standard deviation) in the PBL (upper grey bar) and FT (lower grey bar) are shown ac- cording to the CO2 concentration scale of the plot. receives air advected by up-slope winds, bringing anthro- pogenic emissions from valleys that have accumulated dur- ing the night; and in the late afternoon, air is cleared of this local inﬂuence on windy days (Schmidt et al., 2001). Therefore, CO2 concentrations at SCH averaged between 12:00 UTC and 18:00 UTC and are less contaminated, but not exempt from urban emissions from the Ruhr region cities. Station data match CAATER-2 campaign aircraft data very well (no data was available during CAATER-1). At PUY, lo- cal mesoscale circulations can either bring emission plumes from Clermont-Ferrand, or air from rural areas, depending on wind direction. Both at SCH and PUY, the mixing of surface air with tropospheric air masses is always present, explaining why CO2 values at these stations are between the PBL and the FT (Fig. 10). Fig. 11. Variability observed in the PBL and the FT during each campaigns (left: CAATER 1, right: CAATER 2) using proﬁles only. The altitude of each individual proﬁle has been normalized to the relevant PBL height. The plot shows the mean and standard devi- ation (1-σ) of CO2 concentration over layer of 1/10th of the PBL height. The global mean and variability (±1-σ standard deviation) in the PBL (upper grey bar) and FT (lower grey bar) are shown ac- cording to the CO2 concentration scale of the plot. 5 Comparison of aircraft measurements with surface station measurements 9 and 10) show a large range in the PBL (354 ppm to 378 ppm for CAATER-1; 358 ppm to 378 ppm for CAATER- 2) and more homogeneous values in the FT (372 ppm to 374 ppm for CAATER-1; 367 ppm to 373 ppm for CAATER- 2). This is true for both campaigns but even more so for CAATER-1 than CAATER-2 (Fig. 11). Figure 10 shows that CO2 in the CAATER-1 domain is globally below the MBL value (374.5 ppm), whereas it is above the MBL back- ground curve during CAATER-2 (367.9 ppm). One can also observe that CO2 measured at the CARBOEUROPE stations 200 km horizontally and 100 m vertically. In Fig. 10, the ma- rine background value (reminder: 374.5 ppm for CAATER- 1; 367.9 ppm for CAATER-2) was removed from all the data. Figure 11 shows a comparison between ground sta- tion values and the mean proﬁle recorded during each cam- paign. The CO2 vertical distributions and cross-sections (Figs. 9 and 10) show a large range in the PBL (354 ppm to 378 ppm for CAATER-1; 358 ppm to 378 ppm for CAATER- 2) and more homogeneous values in the FT (372 ppm to 374 ppm for CAATER-1; 367 ppm to 373 ppm for CAATER- 2). This is true for both campaigns but even more so for CAATER-1 than CAATER-2 (Fig. 11). Figure 10 shows that CO2 in the CAATER-1 domain is globally below the MBL value (374.5 ppm), whereas it is above the MBL back- ground curve during CAATER-2 (367.9 ppm). One can also observe that CO2 measured at the CARBOEUROPE stations Figure 9 provides a projection of Fig. 4 on the vertical lon- gitude/altitude plan. It has been interpolated to produce ver- tical cross-sections in Fig. 10. For interpolation, the 1 Hz data are ﬁrst averaged into bins of 100 m altitude. Then a Delaunay triangulation in the altitude, longitude plane was applied (function TRIANGULATE of IDL package). Af- ter triangulation, the values were interpolated onto a regular grid (function TRIGRID of IDL package) at resolutions of Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ 6 Analysis of the vertical variability The impact of anthropogenic emissions from the R¨uhr region is visible in proﬁles recorded near the German-French border. Proﬁles recorded in East Germany, close to Berlin, also show some local emission plumes. In the mid PBL, no depletion linked to the biospheric activity was observed, a difference with Spring 2001. The signal is quite homogeneous between the mid PBL and the FT. However, there is a striking zonal gradient in the CO2 mean mid-PBL concentration of about 11 ppm, revealing: (1) a weaker bio- spheric activity i.e. Autumn starting earlier in Western Eu- rope than in Eastern Europe – however, this hypothesis does not match with the ﬂux maps given in Fig. 1, that indicates on averaged higher ﬂuxes in the East than in the West of Europe. Also, it does not ﬁt with the NDVI maps as well, given in An- nexe 1 – ; (2) a greater mixing of continental and oceanic air in the West of Europe; and/or (3) more CO2 emissions in the west of Europe such as in France than in the eastern part. Inventories from UNFCCC (2002) give 403.15 Mt CO2/yr emitted by France against 863.9 Mt CO2/yr emitted by Ger- many which mitigates against this last supposition. On the other hand, inventories for Austria (71.0 Mt CO2/yr), Hun- gary (57.7 Mt CO2/yr), Slovenia (16.3 Mt CO2/yr), and Croa- tia (20.9 MtCO2/yr) as well as likely emissions of the Fed- eral Republic of Yugoslavia and Bosnia-Herzegovinia (no inventories available) make it probable that the total of the emissions from these countries (covered by the red region on Fig. 13) are less than those of France and so support sup- position 3. However, ﬂuxes shown on Fig. 1 do not match with this hypothesis. Indeed, from ECMWF reanalysis data, we inferred that some convection activity occured in France on 2 October along the ﬂight path to Orleans: this can ex- plain that the dark-blue proﬁles are straight, since the air was well-mixed likely containing a mixture of CO2 biospheric emissions from the ground and CO2 emissions advected from the Rhone valley (blue footprint on Fig. 13), thus charged in anthropogenic CO2 (CO in the range of 130–160 ppm). Also, the green proﬁle, recorded above Th¨uringen is straight. 6 Analysis of the vertical variability In the FT, these values are 373.6 ppm and 0.5 ppm for CAATER-1; 371.2 ppm and 1.1 ppm for CAATER-2. In other terms, the atmospheric CO2 variability over Europe is at least 5 to 8 ppm higher in the PBL than in the FT. The PBL variability was higher during Spring 2001 than during Fall 2002. Interestingly, the mean CO2 jump is 3.7 ppm during CAATER-1 and −0.3 ppm during CAATER- 2, with a similar FT averaged CO2 concentration. Also, the mean proﬁle during CAATER-1 shows a minimum in the mid PBL which is not present in in the CAATER-2 mean proﬁle. These 2 points are the result of a higher photosynthetic activ- ity in Spring than in Autumn. CO2 measurements at surface stations CBW and WES are inﬂuenced by large nearby urban emissions from the Ams- terdam urban area and Northern German cities, which diffuse with altitude and so differ signiﬁcantly from aircraft observa- tions. CO2 at CBW and WES (respectively WES and HUN) was more than 4 ppm higher (respectively 11 ppm) than the PBL value sampled by the aircraft during CAATER-1 (re- spectively during CAATER-2). To go deeper into the variability analysis, all individual proﬁles have been plotted and colored in Figures 12 and 13 according to the location they were recorded in and the ori- gin of the air masses, deduced from backplumes obtained with the LMDZ model and described in the companion pa- per (Xueref-Remy et al., 2011). Both PUY and SCH stations are located on the top of mid-elevation mountains (1205 m alt and 1465 m alt respec- tively for SCH and PUY) over urbanized valleys. During the morning and early afternoon, in spring and summer, SCH Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5668 For CAATER 1 (Fig. 12), the CO2 concentration range is very large in the bottom half of the PBL (355 to 378 ppm), with highest CO2 values in the Brittany region due to local anthropogenic emissions from the Brest city area, and lowest values in Northern Germany with air depleted by photosyn- thetic activity. In Southern and Eastern Germany, vegeta- tion seems less active but the signal does not contain urban emissions. 6 Analysis of the vertical variability In the mid-troposphere, proﬁles recorded in Brit- tany come from air masses, travelling between the ocean, the English Channel and rural regions in North-Western France. CO2 concentrations range between 362 and 368 ppm, de- pending on the relative importance of continental air masses depleted by photosynthetic activity compared to oceanic air masses. In the FT, the atmosphere does not interact as much with ground sources and sinks, and the signal is therefore constant. CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 -12 -8 -4 0 4 8 12 16 20 60 56 52 48 44 Longitude (°E) Latitude (°N) Altitude / PBL height CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 -12 -8 -4 0 4 8 12 16 20 60 56 52 48 44 Longitude (°E) Latitude (°N) Altitude / PBL height CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 -12 -8 -4 0 4 8 12 16 20 60 56 52 48 44 Longitude (°E) Latitude (°N) Altitude / PBL height Fig. 12. CAATER 1 vertical variability analysis. Left panel: all CO2 proﬁles binned over 50 m layers and standardized to their re- spective PBL height. Right panel: schema of the regions covered by the back-trajectories of the proﬁles (proﬁle locations are indicated by circles). Colors of right and left panels correspond. CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 Altitude / PBL height CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 Altitude / PBL height CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 Altitude / PBL height -12 -8 -4 0 4 8 12 16 20 60 56 52 48 44 Longitude (°E) Latitude (°N) -12 -8 -4 0 4 8 12 16 20 60 56 52 48 44 Longitude (°E) Latitude (°N) -12 -8 -4 0 4 8 12 16 20 60 56 52 48 44 Longitude (°E) Latitude (°N) Fig. 12. CAATER 1 vertical variability analysis. Left panel: all CO2 proﬁles binned over 50 m layers and standardized to their re- spective PBL height. Right panel: schema of the regions covered by the back-trajectories of the proﬁles (proﬁle locations are indicated by circles). Colors of right and left panels correspond. For CAATER 2 (Fig. 13), the CO2 concentration range in the bottom half of the PBL is lower than for CAATER 1 (364 to 381 ppm). 6 Analysis of the vertical variability There was some convection developing in this region, mean- ing that likely the air was well-mixed and inﬂuenced by an- thropogenic CO2 emissions (CO again in the range of 130– 160 ppm) as well as a priori less-concentrated CO2 signals from the biosphere activity advected from Germany (green footprint on Fig.13). The shape of the turquoise proﬁles and even more of the red proﬁles is very different, with a marked depletion in the mid-PBL. No convection activity could be identiﬁed at these locations and times. The CO signal reaches values as low as 100 ppb, thus, excluding anthropogenic con- tribution. The air mass footprints are continental (east Ger- many for the turquoise proﬁles, Central Europe for the red proﬁles), excluding as well oceanic signal contribution. It is thus likely, that there was some biospheric sinks still acting in east Germany and even in a stronger manner in Central Europe at that time. Once again, model ﬂuxes do not relate this point, but biospheric ﬂuxes are not always properly mod- elled as we discuss in the companion paper (Xueref-Remy et al., 2011). It would be interesting in the future to conduct new airborne campaigns to assess if the observed gradient is always present at the fall season, or if it was only punctual. Using tracers such as CO, Radon 222 and carbon isotopes would also help to discriminate the role of anthropogenic, biospheric and oceanic sources and sinks. 7 Conclusions During CAATER-1, the action of the biospheric sink over rural areas produced atmospheric CO2 values as low as 355 ppm, while the emissions from anthropogenic- inﬂuenced regions such as Benelux and the Ruhr valley gave atmospheric CO2 concentration levels of about 380 ppm at approx. 1000 m a.s.l. above these regions. gradient of about 11 ppm in the CO2 mean mid-PBL con- centration with higher concentrations in the west during the CAATER 2 campaign is striking. This gradient could origi- nate from a transport effect (a better mixing in Western Eu- rope), an earlier Autumn in the west compared to the east, or higher emissions from Western Europe than Eastern Europe. To better understand this gradient and discriminate correctly beween these hypotheses, more airborne vertical proﬁles are needed. Such a program has been undertaken in the frame- work of the CARBOEUROPE-IP project in 5 sites (Grifﬁth, Scotland; Orleans, France; Hegyhatsal, Hungary; Bialystok, Poland; and La Muela, Spain), with one ﬂight every 5 days. The data are currently being analyzed and future publications are planned. In the companion paper (Xueref-Remy et al., 2011, Part 2) we conduct a comparison of vertical proﬁles between models and observations. We also attempt to calcu- late CO2 ﬂuxes during the CAATER 1 campaign using the so-called “Radon method” based on simultaneous CO2 and Radon-222 observations (Schmidt et al., 2003) and modeling tools. pp g During CAATER-2, CO2 and CO, were recorded simul- taneously which helped us to trace fossil fuel emissions. After a classiﬁcation of air masses according to their ori- gin,we calculated the 1CO to 1CO2 ratio (1 standing for the difference with the marine boundary layer con- centration) and selected only well-identiﬁed anthropogenic- inﬂuenced air masses diagnosed from back-trajectories. The anthropogenic-inﬂuenced air masses show high R2 values ranging from 0.33 to 0.88 and a large range of slopes (2.42 to 10.37 ppb CO ppm−1 CO2). Comparing these slopes to the ones from EMEP inventories for Western Europe countries, we have observed that it is possible to distinguish the contri- bution of fossil fuel combustion from other sources of CO2 even at a distance of a few days or hundreds of kms from the source. Aircraft data were compared to surface station observa- tions (CBW, PUY, CMN and PRS for CAATER 1; WES, HUN, SCH, PUY, CMN and PRS for CAATER 2). 7 Conclusions This paper focuses on atmospheric CO2 variability observed during the CAATER campaigns that occured above western Europe on 23–26 May 2001 (CAATER 1) and 2–3 Octo- ber 2002 (CAATER 2) between the ground and 4000 m a.s.l. Although the aircraft paths were slightly different during CAATER-1 and CAATER-2, the campaigns give a good picture of CO2 concentration variability over Europe dur- ing one Spring and one Fall. The instrumentation provided measurements of in situ CO2 (precision of 0.2 ppm), CO2 ﬂasks samples (precision of 0.1 ppm), Radon-222 (precision of 30%) during CAATER-1, and in-situ CO (precision of Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 5669 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 [CO2] (ppm) 360 365 370 375 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 [CO2] (ppm) 360 365 370 375 CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 Longitude (°E) Latitude (°N) Altitude / PBL height -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 [CO2] (ppm) 360 365 370 375 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 [CO2] (ppm) 360 365 370 375 CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 Longitude (°E) Latitude (°N) Altitude / PBL height -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 [CO2] (ppm) 360 365 370 375 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 [CO2] (ppm) 360 365 370 375 CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 Longitude (°E) Latitude (°N) Altitude / PBL height Fig. 13. CAATER 2 vertical variability analysis. Left panel: all CO2 proﬁles binned over 50 m layers and standardized to their re- spective PBL height. 7 Conclusions Right panel: schema of the regions covered by the backplumes associated with the proﬁles (proﬁle locations are indicated by circles). Colors of right and left panels correspond. [CO2] (ppm) 360 365 370 375 [CO2] (ppm) 360 365 370 375 CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 Altitude / PBL height [CO2] (ppm) 360 365 370 375 [CO2] (ppm) 360 365 370 375 CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 Altitude / PBL height [CO2] (ppm) 360 365 370 375 [CO2] (ppm) 360 365 370 375 CO2 (ppm) 360 365 370 375 2.0 1.5 1.0 0.5 0.0 Altitude / PBL height 5 ppb) during CAATER-2. Despite a stronger biogenic up- take in Spring, the lower tropospheric mean level and the mean variability in May (370.1 ± 4.0 ppm) are roughly simi- lar in October (371.7 ± 5.0 ppm). However, this mean value is lower than the marine boundary layer concentration for CAATER-1 (374.5 ppm) and higher for CAATER-2 (the ma- rine background, 367.9 ppm, being more depleted). A back- trajectory analysis shows that during CAATER-1, dominant winds were coming from the north-west, while they were more balanced between the 4 sectors during CAATER-2. -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 Longitude (°E) Latitude (°N) -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 Longitude (°E) Latitude (°N) -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 -8 -4 0 4 8 12 16 20 24 58 54 50 46 42 38 Longitude (°E) Latitude (°N) Fig. 13. CAATER 2 vertical variability analysis. Left panel: all CO2 proﬁles binned over 50 m layers and standardized to their re- spective PBL height. Right panel: schema of the regions covered by the backplumes associated with the proﬁles (proﬁle locations are indicated by circles). Colors of right and left panels correspond. 7 Conclusions Only af- ternoon values were selected so as to get data representative of the regional scale. Urban stations such as CBW and WES are strongly inﬂuenced by local emissions and were decou- pled from the airborne observations. Depending on the time of the day and the meteorological situation, stations on top of small mountains such as SCH and PUY may be located in the boundary layer (PBL) or in the free troposphere (FT); they match the airborne observations if in the FT and not con- taminated by air emanating from the valley. Stations when in the FT such as CMN match airborne measurements quite well, as the atmosphere is well mixed at that altitude. How- ever, PRS which is in the FT but a few 100 km further from the aircraft path differs by 3 ppm from the airborne measure- ments, which highlights the existence of a CO2 gradient of a few ppm above Europe in the FT. The publication of this article is ﬁnanced by CNRS-INSU. The publication of this article is ﬁnanced by CNRS-INSU. Appendix B Andres, R.J., Marland, G., Fung, I., and Matthews, E.: A 1◦×1◦ distribution of carbon dioxide emissions from fossil fuel con- sumption and cement manufacture 1950–1990, Global Bio- geochem. Cy., 10, 419–429, 1996. Edited by: J. Rinne The publication of this article is ﬁnanced by CNRS-INSU. Fig. B1. Coordinated ﬂights over Thringen on 26 May 2001 at 14:30:00 h UTC (in situ: plain line, LSCE ﬂasks: plain circles; Jena ﬂasks: open triangles). NDVI maps for Western Europe during the CAATER campaigns These NDVI maps provided by the SPOT VGT-4 satellite system give us information on the photosynthetic activity. They clearly show a higher photosynthetic activity during Spring 2001 than during Fall 2002 in Western Europe. As the gradient between the PBL and the FT is a key pa- rameter for inverse modeling we have also analyzed CO2 vertical variability. The mean jump between the PBL and the FT is of the order of 3.7 ppm during CAATER 1 and −0.3 ppm during CAATER 2, and the variability is at least 5 to 8 times higher in the PBL than in the FT. A strong zonal Atmos. Chem. Phys., 11, 5655–5672, 2011 www.atmos-chem-phys.net/11/5655/2011/ 5670 I. Xueref-Remy et al.: Variability and budget of CO2 in Europe – Part 1 Fig. A1. NDVI maps on 21 May 2001 for CAATER 1 (left) and on 1 October 2002 for CAATER 2 (right). Fig. A1. NDVI maps on 21 May 2001 for CAATER 1 (left) and on 1 October 2002 for CAATER 2 (right). 3000 2500 2000 1500 1000 500 364 366 368 370 372 374 CO2 concentration (ppm) Altitude a.s.l. (m) 3000 2500 2000 1500 1000 500 364 366 368 370 372 374 CO2 concentration (ppm) Altitude a.s.l. (m) Fig. B1. Coordinated ﬂights over Thringen on 26 May 2001 at 14:30:00 h UTC (in situ: plain line, LSCE ﬂasks: plain circles; Jena ﬂasks: open triangles). Acknowledgements. This work has been funded by the Institut National des Sciences de l’Univers, France, and by the European Commission in the framework of the AEROCARB project. We thank Ingeborg Levin and Martina Schmidt for helpful discussions on the Radon calculations. We thank David Picard for his partic- ipation in the campaign. NECP meteorological maps are thanks to Christopher Godfrey, the University of Oklahoma School of Meteorology, and the National Centers for Environmental Predic- tion/National Center for Atmospheric Research 40-Year Reanalysis Project. We thank Cyril Moulin and Fabienne Maignan from LSCE for providing NDVI maps. We are grateful to Nicolas Viovy for providing ORCHIDEE ﬂuxes. Many thanks to Mary Minnock for improving the English of the text. 3000 2500 2000 1500 1000 500 364 366 368 370 372 374 CO2 concentration (ppm) Altitude a.s.l. (m) 3000 2500 2000 1500 1000 500 364 366 368 370 372 374 CO2 concentration (ppm) Altitude a.s.l. (m) Edited by: J. Rinne The publication of this article is ﬁnanced by CNRS-INSU. Edited by: J. Rinne Validation of the CO2 in situ analyser As explained in Sect. 2, ﬂask measurements were compared to CONDOR measurements. We show here (Fig. B1) a com- parison for 26 May 2001 over Th¨uringen at 14:30:00 h UTC, during which the Falcon ﬂight was co-ordinated with those of a small aircraft equipped with a ﬂask sampler from the MPI-BGC Jena group. LSCE ﬂasks (circles), Jena ﬂasks (tri- angles) and CONDOR (plain line) data agree within 0.2 ppm. Baker, D. F., Doney, S. C., and Schimel, D. S.: Variational data as- similation for atmospheric CO2, Tellus B, 58(5), 359–365, 2006. Carouge, C., Bousquet, P., Peylin, P., Rayner, P. 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https://openalex.org/W4226320464	https://www.researchsquare.com/article/rs-635786/latest.pdf	English	null	Effect of Volume and Renewal of the Storage Media on the Release of Monomer from Dental Composites	International journal of dentistry	2,021	cc-by	4,898	Effect of Volume and Renewal of the Storage Media on the Release of Monomer from Dental Composites Sima Shahabi Tehran University of Medical Sciences Maryam Sayyari Tehran University of Medical Sciences Sima Sadrai Tehran University of Medical Sciences Sara Valizadeh (  valizadeh_s@sina.tums.ac.ir ) Tehran University of Medical Sciences Hamidreza Hajizamani Tehran University of Medical Sciences Alireza Sadr University of Washington Research Article Version of Record: A version of this preprint was published at International Journal of Dentistry on December 28th, 2021. See the published version at https://doi.org/10.1155/2021/9769947. Page 1/13 Page 1/13 Page 1/13 Abstract Background: released monomers to the oral environment from dental composite, cause systemic or local side effects on the tissues and cells and also affect mechanical properties of the resins. Methods: This study evaluated the effect of the volume and renewing of storage media on monomer leachability from dental composite. Samples of two dental composites (BEAUTIFIL II Gingiva (BG) and Filtek bulkfill flowable (FBF)) were stored after polymerization in 1 and 3 milt storage media (ethanol/water 75%) for seven days. Refreshing of storage media was done in half of the samples of each group. The amount of releasing monomers (UDMA, BisGMA, TEGDMA) in storage media were measured by high performance liquid chromatography. (HPLC) Data was analyzed using Two-way ANOVA and T-test. (α=0.05). Methods: This study evaluated the effect of the volume and renewing of storage media on monomer leachability from dental composite. Samples of two dental composites (BEAUTIFIL II Gingiva (BG) and Filtek bulkfill flowable (FBF)) were stored after polymerization in 1 and 3 milt storage media (ethanol/water 75%) for seven days. Refreshing of storage media was done in half of the samples of each group. The amount of releasing monomers (UDMA, BisGMA, TEGDMA) in storage media were measured by high performance liquid chromatography. (HPLC) Data was analyzed using Two-way ANOVA and T-test. (α=0.05). Results: Elution of TEGDMA and UDMA from both composites was significantly higher in 3ml storage media. In groups with refreshing of storage media, BisGMA had higher amounts of release. Saturation makes the storage media volume important factor in monomer elution. Conclusion: Refreshing of storage media had significant effect on monomer release before the elution of 50% of total released monomer. Conclusion: Refreshing of storage media had significant effect on monomer release before the elution of 50% of total released monomer. Background Considering the ever-increasing popularity of composites, there are concerns about the release of composite compounds and their toxicity. In other words, there is a probability that resin materials would release substances such as unpolymerized monomers, additives and filler component when placed in the oral cavity [1]. In clinical conditions, with a short-time curing time (usually less than 40 seconds) and the temperature of 37°C, composites will not be fully polymerized, due to the crosslinking reactions that can considerably reduce the movements of the monomers. The degradation process occurring in the oral cavity can also increase the release of certain substances from resin materials, as mechanical, enzymal and hydraulic processes can break the polymeric chains and release the products of this breakage as monomer or polymer molecules [1, 2]. Resin matrix usually consists of two oligomers: UDMA and BisGMA [3]. Bis-GMA was synthesized from bisphenol A (BPA) and glycidyl methacrylate (GMA) [4]. The release of monomers and additives can be harmful and might have adverse local or systemic effects. Other than the allergenic properties of monomers, some of the released substances can be cytotoxic, genotoxic, and carcinogenic, and might be toxic to the reproduction system [1]. BPA is one kind of endocrine disrupting compound that can cause several health problems [3]. Recognition and evaluation of such risks require knowledge about the exact quantity of the released substances [5]. The vast heterogeneity in the evaluation methods in different studies regarding the measurement of the released compounds from dental composites has had a considerable effect on the results of such studies. While a great number of analytic studies have been carried out, not using the standard means of measurement and varieties in presenting the results, prevent an accurate analysis of the quantity of the released monomers [1, 4]. In other words, the amount of monomers released in the oral cavity cannot be precisely determined, rendering it more difficult to evaluate the possible risks of resin composites. The amount of released monomers can differ due to approximately 100,000 factors in different studies. Page 2/13 Page 2/13 Although such significant variation can be explained with the fact that different composite materials have been used in each study, many factors play an important role in this matter, such as the storage media (pure ethanol, a mixture of water ethanol, artificial saliva, etc.) incubation conditions (temperature and duration) and the methods used for analysis. [1, 3, 5]. Background The most frequently used protocol to measure monomer elution, has been described by ISO 10993-12 2010 [6] which suggests that the ratio of the mass of the test sample to the volume of the test storage media should be at least 1gr:10ml [7]. The word “at least” and also the statement of ISO which mentions interference of degradation product with the progress of degradation process should be considered as two major factors in validity of results. However, there is a weak yet significant correlation between the amount of the storage media and the quantity of the released monomers. In other words, the greater the storage media volume is, the higher the monomer concentrations are [1, 5]. This finding can be justified with the saturation of the storage media by the released monomers. Considering the storage media reaching a state of equilibrium and a consequent diminished monomer release, it can be interpreted that the recorded values in the past in-vitro studies were speculated with less than the actual values. Nevertheless, in in-vivo conditions, reaching the saturation state does not seem possible, as saliva is continuously being refreshed. One way to rectify this problem in in-vitro conditions is to renew the solvent medium within equal intervals, rendering it impossible to reach saturation state [6, 7]. In this study, other than the effect of the volume of storage media on the release of monomers from composites, the effect of renewal of storage media within equal intervals was evaluated. These results are essential for improving experimental protocols regarding monomer release, which will be carried out in future analytic studies with the aim of evaluating the long-term effects of released components from composites in near future. The null hypothesis is that volume and renewing of storage media do not affect monomer leachability. Keeping samples in storage media in different methods The storage media in groups 1,2,5 and 6 were incubated for 7 days in 37°C temperature. The storage media in groups 3,4,7 and 8 were incubated for 24 hours in 37°C temperature, and After each 24 hours, the whole storage media was taken up for analysis following which the samples were immersed in 1mL or 3 ml of fresh ethanol/water solution. To be more precise, each composite disc was taken from the solvent, rinsed and, dried by low power air and immersed in fresh solvent. The renewed storage media was again incubated in 37°C temperature. The renewal and incubation of the storage media were repeated every 24 hours for the next 6 days. Schematic illustration of the research method used in this study is available in Figure 1. Preparation of the specimens Two types of composites (BEAUTIFUL II gingiva (BG) & Filtek bulk-fill (FBF)) were selected (Table 1). Table 1 Two types of composites (BEAUTIFUL II gingiva (BG) & Filtek bulk-fill (FBF)) were selected (Table 1). Table 1 the properties of the used composites Composite Brand Manufacturer Resin Matrix Filler Type Filler Amount Filtek Bulk Fill Flowable (1) 3M ESPE GmbH, Germany Bis-GMA, BisEMA, Procry-lat, UDMA Zirconia or Silica, Ytterbium Trifluoride Loading Percentage by Weight: 64.5% BEAUTIFUL II Gingiva (2) Shofu Inc., Japan Bis-GMA, TEGDMA S-PRG filler based on Fluoroboroaluminosilicate glass, polymerization initiator, pigments and others Loading Percentage by Weight: 81% 64 Disk-shaped composite samples (height: 2mm, width: 6mm) were prepared in a Teflon mold. Before polymerization, the disks were covered with a glass plate to prevent the formation of an oxygen-inhibited layer. The specimens were cured with the 3W light curing device High power Blue light LED (guilin woodpecker, china), with the light output of 500 mw/cm2 for 40 seconds according to the manufacturer's instruction. 64 Disk-shaped composite samples (height: 2mm, width: 6mm) were prepared in a Teflon mold. Before polymerization, the disks were covered with a glass plate to prevent the formation of an oxygen-inhibited layer. The specimens were cured with the 3W light curing device High power Blue light LED (guilin woodpecker, china), with the light output of 500 mw/cm2 for 40 seconds according to the manufacturer's instruction. Storage media preparation The storage media was prepared using pure ethanol (99.99%, Merck, Germany) and water, with the ethanol/water ratio of 75% in two volumes of 1mlit and 3mlit. The composite disks were placed in the storage media according to table 2 (8 groups and 8 composite discs in each group). Table 2 Test groups Group Composite Type Solvent Volume Daily Renewal 1 Filtek Bulk Fill Flowable 1 ml No 2 BEAUTIFUL II Gingiva 1 ml No 3 Filtek Bulk Fill Flowable 1 ml Yes 4 BEAUTIFUL II Gingiva 1 ml Yes 5 Filtek Bulk Fill Flowable 3 ml No 6 BEAUTIFUL II Gingiva 3 ml No 7 Filtek Bulk Fill Flowable 3 ml Yes 8 BEAUTIFUL II Gingiva 3 ml Yes Table 2 Test groups Measurement of Monomer Release The amount of released monomer (UDMA, BisGMA, TEGDMA) was evaluated by the HPLC 600 E waters System Controller (Waters, MA, USA) method, through The Perfect target ODS-3 column (125 mm height, 4mm width, and silica particle size of 5 µm), with a UV detector at 230 nm wave length. The mobile phase was 70% acetonitrile and Page 4/13 Page 4/13 30% distilled water, at 0.8 mm flow rate, and 20µlit injection volume at room temperature. Figure 2 shows the chemical structure of these monomers. At first, different concentrations of each monomer (0.5–50 µg/lit) were injected into the system, and a standard curve was obtained which was used to analyze the produced curve of samples. It is noticeable that in refreshing samples, the sample of each day was injected in the system in order to gain the daily releasing pattern of monomers. Statistical Analysis The obtained data were analyzed using the SPSS statistical software 25th version. Continuous variables were presented as mean and standard deviation. Three-way ANOVA and T-test were used. The significant level was considered at 0.05. Results Three tracked monomers were detected in all groups of storage media. Volume of solvent did not affect BisGMA release while TEGDMA and UDMA from had significantly higher elution in 3ml extraction solvent. To be more precise, in samples without refreshing, UDMA from both composites and TEGDMA from FBF eluted in higher amount in 3ml solvents. To define the effect of composite type in monomer elution, it should be considered that TEGDMA eluted higher from BG composite while UDMA eluted more from FBF. Refreshing of extraction solvent caused more elution of BisGMA in all samples. Besides, UDMA from FBF in 3 ml eluted more in samples with refreshing in comparison with the samples without refreshing. This result was also true in TEGDMA elution in both solvent 's volume from FBF composite. However, for TEGDMA elution from BG in 3ml solvents the reverse is true. (Table 3) Table 3 monomers elution in tested groups. (µg) monomer TEGDMA UDMA BisGMA volume 1 mL 3 mL 1 mL 3 mL 1 mL 3 mL refreshing Yes No Yes No Yes No Yes No Yes No Yes No FBF mean 0.19 0.01 0.31 0.13 66.99 47.28 76.12 75.39 0.25 0.04 0.18 0.06 sd 0.16 0.01 0.07 0.25 8.36 6.92 4.79 25.09 0.45 0.00 0.00 0.01 BG mean 0.42 0.36 0.19 0.67 7.83 3.36 6.09 9.64 0.09 0.07 0.19 0.09 sd 0.11 0.06 0.05 0.11 1.34 0.65 1.11 12.02 0.01 0.02 0.01 0.01 Daily elution patterns of monomers show that except UDMA monomer eluted from BG composite in 3ml solvent, all monomers in all volume had their maximum elution in day one. While TEGDMA and UDMA released more than half of their total elution in first day. Composite BG in 1 ml storage media released 83% of its total released TEGDMA in the first day. (Tables 4 and 5) Page 5/13 Page 5/13 Table 4 Daily release of monomers from FBF. Results (µg) Monomer TEGDMA UDMA BisGMA volume 1ml 3ml 1ml 3ml 1ml 3ml Day 1 mean 0.19 0.31 44.89 47.57 0.03 0.04 Sd 0.16 0.07 8.73 4.13 0.00 0.00 Day 2 Mean 0.00 0.00 7.60 9.59 0.01 0.03 Sd 0.00 0.00 0.74 0.18 0.00 0.00 Day 3 Mean 0.00 0.00 5.73 6.31 0.01 0.02 Sd 0.00 0.00 0.36 0.45 0.00 0.00 Day 4 Mean 0.00 0.00 1.40 2.46 0.01 0.02 sd 0.00 0.00 0.03 0.31 0.00 0.00 Day 5 Mean 0.00 0.00 1.29 1.69 0.01 0.02 sd 0.00 0.00 0.00 0.51 0.00 0.00 Day 6 Mean 0.00 0.00 3.80 3.39 0.01 0.02 sd 0.00 0.00 0.40 0.19 0.00 0.00 Day 7 Mean 0.00 0.00 2.28 4.59 0.01 0.02 sd 0.00 0.00 0.82 0.50 0.00 0.00 Table 4 Page 6/13 Page 6/13 Page 6/13 Table 5 daily release of monomers from BG. (µg) Monomer TEGDMA UDMA BisGMA volume 1ml 3ml 1ml 3ml 1ml 3ml Day 1 mean 0.35 0.15 4.85 0.21 0.04 0.05 Sd 0.11 0.04 1.28 0.56 0.01 0.01 Day 2 Mean 0.04 0.03 0.63 0.97 0.01 0.02 Sd 0.01 0.01 0.13 0.07 0.00 0.00 Day 3 Mean 0.00 0.00 0.62 0.80 0.01 0.02 Sd 0.00 0.00 0.15 0.07 0.00 0.00 Day 4 Mean 0.00 0.00 0.00 1.77 0.01 0.02 sd 0.00 0.00 0.00 0.56 0.00 0.00 Day 5 Mean 0.00 0.00 0.66 0.69 0.01 0.02 sd 0.00 0.00 0.13 0.28 0.00 0.00 Day 6 Mean 0.01 0.01 0.65 0.69 0.01 0.02 sd 0.00 0.00 0.08 0.13 0.00 0.00 Day 7 Mean 0.01 0.01 0.42 0.96 0.01 0.02 sd 0.00 0.00 0.10 0.13 0.00 0.01 Table 5 daily release of monomers from BG. (µg) Discussion Higher amount of storage media reaches saturation point later, so monomer elution is higher. According to Van Landuyt KL et al’s [1] meta-analysis, release of monomers is a chemical equilibrium reaction and in smaller amounts of storage media equilibrium was achieved faster and prevented more monomer release. Constant refreshing of saliva and pulpal fluid in in vivo situation prevent saturation status. High volume of storage media and refreshing of it could be used to overcome this condition. measured monomer elution in 1, 7 and 28 days and one year and storage media were refreshed in 28 days. Monomer elution was equal in 28 days and one year which indicates saturation of storage media. In fact, saturation of storage media prevents further elution of monomers. Higher amount of storage media reaches saturation point later, so monomer elution is higher. According to Van Landuyt KL et al’s [1] meta-analysis, release of monomers is a chemical equilibrium reaction and in smaller amounts of storage media equilibrium was achieved faster and prevented more monomer release. Constant refreshing of saliva and pulpal fluid in in vivo situation prevent saturation status. High volume of storage media and refreshing of it could be used to overcome this condition. In this study 2 volumes of storage media were used to evaluate effect of storage media volume on monomer elution. In order to evaluate the effect of storage media refreshing on monomer elution, in half of the samples, storage media was refreshed daily. Thus, daily pattern of monomer elution was obtained. Alshali et al [15] measured monomer elution after one day, one month and three months. In their study, 70% of total three month- elution of TEGDMA and 50% of the total BisGMA elution occurred on the first day. Nalcaci et al [16] declared that TEGDMA eluted faster than BisGMA in methanol as in one sample group, 92% of TEGDMA eluted in first 6 hours while at the same time BisGMA reached 57% elution. Sideridou ID et al [17] measured monomer elution in 3,6 and 24 hours and also 3,6 and 30 days. Similar to the current study, the highest amount of release of TEGDMA and UDMA was observed in first day, unlike BisGMA. The first mechanism of monomer elution, is elution from the composite surface that occurs in the first 24 hours. Discussion In this study two different composite materials were used to measure the effect of storage media volume and refreshing of it on monomer elution. In this study two different composite materials were used to measure the effect of storage media volume and refreshing of it on monomer elution. Bulkfill composites like FBF are in high demand these days because of their increased depth of cure and reduced clinical time of operation [8]. BG can provide esthetics in conservative restorations with gingival recession [9]. Despite the manufacturer’s information, elution of TEGDMA from FBF- as seen in Pongprueksa P et al’s [10] study, and also UDMA from BG was observed. According to Cokic SM et al [6] manufacturers do not release the exact component of composites because of trade reasons. Bulkfill composites like FBF are in high demand these days because of their increased depth of cure and reduced clinical time of operation [8]. BG can provide esthetics in conservative restorations with gingival recession [9]. Despite the manufacturer’s information, elution of TEGDMA from FBF- as seen in Pongprueksa P et al’s [10] study and also UDMA from BG was observed. According to Cokic SM et al [6] manufacturers do not release the exact component of composites because of trade reasons. Ethanol was used as storage media to intensify monomer elution. In aqueous media such as artificial saliva, monomers are released in smaller amounts [11, 12]. Also, FDA declared that ethanol can simulate exposure to nutrition materials such as light drinks and chocolate [13, 14]. According to VanLanduyt et al [1] UDMA wasn’t detectable during first day of elution. According to Cokic SM et al [6] storage media volume had major effect on monomer elution. The larger the volume of the storage media, the higher the amounts of monomer elution that happens. Also, incubation time between 7 and 30 days had no significant effect in monomer release. These two observations lead to considering the limitation of monomer solubility in storage media, as a major factor in monomer elution. Polydorou O et al [14] Page 7/13 Page 7/13 measured monomer elution in 1, 7 and 28 days and one year and storage media were refreshed in 28 days. Monomer elution was equal in 28 days and one year which indicates saturation of storage media. In fact, saturation of storage media prevents further elution of monomers. Discussion Gonzalez-Bonet A et al [18] described that TEGDMA has two hydrolysable ester groups and hydrolyzing these groups creates methacrylate acid (MA), 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl methacrylate (TEGMA) and TEG. Finer Y et al [19] detected TEGDMA products such as TEGMA, triethylene glycol and methacrylicacid. They suggested that total TEGDMA elution is equal to amount of the main TEGDMA plus its products. It can be obtained that FBF releasing products such as additives and filler components could have hydrolyzed TEGDMA to its products while refreshing the storage media released TEGDMA and prevented hydrolyzing of this monomer. TEGDMA elution from FBF is significantly higher in case of refreshing storage media despite its daily elution pattern and rate of elution. Gonzalez-Bonet A et al [18] described that TEGDMA has two hydrolysable ester groups and hydrolyzing these groups creates methacrylate acid (MA), 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl methacrylate (TEGMA) and TEG. Finer Y et al [19] detected TEGDMA products such as TEGMA, triethylene glycol and methacrylicacid. They suggested that total TEGDMA elution is equal to amount of the main TEGDMA plus its products. It can be obtained that FBF releasing products such as additives and filler components could have hydrolyzed TEGDMA to its products while refreshing the storage media released TEGDMA and prevented hydrolyzing of this monomer. 3ml storage media of BG composites at the first day, eluted monomers in small amounts; Which caused more monomer elution in 1ml storage media cases with refreshing and also more elution of TEGDMA in 3ml storage media in cases without refreshing. Conclusion It can be suggested that in studies related to monomer elution, especially in long term evaluations, refreshing the storage media should be conducted before first 24 hours and after that due to slower rate of elution it can be done in longer periods. Higher amount of storage media volume is preferable while balance between preventing saturation and limit of detection should be considered. Monomers hydrolyzing to their products and measuring their products should be noticed. Discussion Subsequently monomer elution continues with a slower rate, since increasing the volume of polymeric chains and release of unreacted monomers from composite, takes substantial time [11]. In the current study TEGDMA and UDMA from FBF and UDMA from BG eluted more in 3 ml storage media (when not refreshing the solvent) because of the faster saturation of 1ml storage media. In cases of refreshing storage media, two different volumes had no significant difference in reaching saturation status and thus monomer elution. According to Cokic SM et al there are two barriers for monomer release; decreasing the concentration gradient between the sample and the storage media [6] and saturation of storage media with monomers. Thus, rather than the storage media volume and concentration of monomer in the storage media, the concentration of unreacted monomers in the composite plays an important role in reaching equilibrium. Elution of TEGDMA from BG (in cases of not refreshing the solvent) had no significant difference between two volumes of storage media. It can be observed that high concentrations of unreacted TEGDMA in BG led to the continued elution of TEGDMA in 1ml storage media. Daily pattern of TEGDMA elution also confirms this result. Since elution of this monomer from BG, despite of its high rate in elution, continued till day 7. While FBF released TEGDMA only on the first day. Elution of BisGMA from FBF and BG was in small amounts, which justifies not reaching saturation in 1ml volume and not having a significant difference in elution of this monomer in two storage media volumes. Refreshing of storage media had significant effect on BisGMA elution. Considering that BisGMA couldn’t reach saturation during 7 days because of its small amounts, refreshing of storage media should cause another phenomenon rather than preventing saturation. Refreshing storage media will reduce concentration of monomers in storage media and increase the concentration gradient. BisGMA has a heavy aromatic core, a higher molecular weight [17] and a lower elution rate than TEGDMA and UDMA. BisGMA is the only monomer which eluted lower than 50% of its total elution in day one. Storage media refreshing and increasing the concentration gradient, affect more than 50% of BisGMA ‘s total elution while for TEGDMA and UDMA this effect is not significant. Page 8/13 TEGDMA elution from FBF is significantly higher in case of refreshing storage media despite its daily elution pattern and rate of elution. This study was conducted under the approval of the ethical committee of Tehran University of Medical Sciences. Page 9/13 Page 9/13 Page 9/13 Abbreviations BG: Beautifil II Gingiva FBF: Filtek Bulkfill Flowable BisGMA: Bisphenol A Diglycidyl Ether DiMethacrylate UDMA: Urethan DiMethacrylate TEGDMA: Tri Ethylene Glycol DiMethacrylate HPLC: High Performance Liquid Chromatography BPA: Bisphenol A GMA: Glycidyl Methacrylate LED: Light Emitting Diode ANOVA: Analysis of Variance Declarations Ethics Approval and Consent to Participate This study was conducted under the approval of the ethical committee of Tehran University of Medical Sciences. BG: Beautifil II Gingiva Authors' contributions SSh Study design and concept, MS and SS Experiment performing, SV Study design and concept, Writing the manuscript, HH Manuscript English editing, AS Reviewing the final manuscript. all authors have read and approved the manuscript Competing Interest The authors declare no conflict of interest, financial or otherwise. Funding This study was part of DDS,MSc thesis in Tehran University of Medical Sciences (Thesis Code : 6355) and was funded and supported by Dental Research Center of Dentistry, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran.(Grant No. :9111272030) Acknowledgements Authors are thankful for statistical analysis to Dr. MohamadJavad Kharazifard Authors are thankful for statistical analysis to Dr. MohamadJavad Kharazifard Authors are thankful for statistical analysis to Dr. MohamadJavad Kharazifard Page 9/13 It is an in Vitro Study, so Consort for participant is not applicable. Availability of Data and Materials The data supporting the findings of the article is available in request. References 1. Van Landuyt KL, Nawrot T, Geebelen B, De Munck J, Snauwaert J, Yoshihara K, Scheers H, Godderis L, Hoet P, Van Meerbeek B. How much do resin-based dental materials release? A meta-analytical approach. Dental Materials. 2011 Aug 1;27(8):723 – 47. 1. 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Alshali RZ, Salim NA, Sung R, Satterthwaite JD, Silikas N. Analysis of long-term monomer elution from bulk-fill and conventional resin-composites using high performance liquid chromatography. Dental Materials. 2015 Dec 1;31(12):1587-98. 16. Nalcaci A, Ulusoy N, Atakol O. Time-based elution of TEGDMA and BisGMA from resin composite cured with LED, QTH and high-intensity QTH lights. Operative Dentistry. 2006 Feb;31(2):197–203. 16. Nalcaci A, Ulusoy N, Atakol O. Time-based elution of TEGDMA and BisGMA from resin composite cured with LED, QTH and high-intensity QTH lights. Operative Dentistry. 2006 Feb;31(2):197–203. 17. Sideridou ID, Achilias DS. Page 10/13 Elution study of unreacted Bis-GMA, TEGDMA, UDMA, and Bis‐EMA from light‐cured dental resins and resin composites using HPLC. Journal of Biomedical Materials Research Part B: Applied Biomaterials: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials. 2005 Jul;74(1):617 – 26. 18. Gonzalez-Bonet A, Kaufman G, Yang Y, Wong C, Jackson A, Huyang G, Bowen R, Sun J. Preparation of dental resins resistant to enzymatic and hydrolytic degradation in oral environments. Biomacromolecules. 2015 Sep 23;16(10):3381-8. 18. Gonzalez-Bonet A, Kaufman G, Yang Y, Wong C, Jackson A, Huyang G, Bowen R, Sun J. Preparation of dental resins resistant to enzymatic and hydrolytic degradation in oral environments. Biomacromolecules. 2015 Sep 23;16(10):3381-8. 19. Finer Y, Santerre JP. Influence of silanated filler content on the biodegradation of bisGMA/TEGDMA dental composite resins. Journal of Biomedical Materials Research Part A. 2007 Apr;81(1):75–84. 19. Finer Y, Santerre JP. Influence of silanated filler content on the biodegradation of bisGMA/TEGDMA dental composite resins. Journal of Biomedical Materials Research Part A. 2007 Apr;81(1):75–84. 19. Finer Y, Santerre JP. Influence of silanated filler content on the biodegradation of bisGMA/TEGDMA dental composite resins. Journal of Biomedical Materials Research Part A. 2007 Apr;81(1):75–84. Page 11/13 Figures Page 12/13 Figure 1 Schematic illustration of the research method used in this study Figure 1 Schematic illustration of the research method used in this study Page 12/13 Page 12/13 Figure 2 Monomer chemical Structure measured in this study Page 13/13
https://openalex.org/W3033642252	http://cds.cern.ch/record/2746015/files/10.1088_1742-6596_1526_1_012004.pdf	English	null	First observation and study of the K <sup>±</sup> → π <sup>±</sup> π <sup>0</sup> e <sup>+</sup> e <sup>−</sup> decay with the NA48/2 experiment at CERN	Journal of physics. Conference series	2,020	cc-by	3,659	1526 (2020) 012004 IOP Publishing doi:10.1088/1742-6596/1526/1/012004 1526 (2020) 012004 IOP Publishing doi:10.1088/1742-6596/1526/1/012004 International Conference on Kaon Physics 2019 Journal of Physics: Conference Series 1526 (2020) 012004 doi:10.1088/1742-6596/1526/1/012004 2 for the NA48/2 Collaboration: G. Anzivino, R. Arcidiacono, W. Baldini, S. Balev, J.R. Batley, M. Behler, S. Bifani, C. Biino, A. Bizzeti, B. Bloch-Devaux, G. Bocquet, N. Cabibbo, M. Calvetti, N. Cartiglia, A. Ceccucci, P. Cenci, C. Cerri, C. Cheshkov, J.B. Ch`eze, M. Clemencic, G. Collazuol, F. Costantini, A. Cotta Ramusino, D. Coward, D. Cundy, A. Dabrowski, P. Dalpiaz, C. Damiani, M. De Beer, J. Derr´e, H. Dibon, L. DiLella, N. Doble, K. Eppard, V. Falaleev, R. Fantechi, M. Fidecaro, L. Fiorini, M. Fiorini, T. Fonseca Martin, P.L. Frabetti, L. Gatignon, E. Gersabeck, A. Gianoli, S. Giudici, A. Gonidec, E. Goudzovski, S. Goy Lopez, M. Holder, P. Hristov, E. Iacopini, E. Imbergamo, M. Jeitler, G. Kalmus, V. Kekelidze, K. Kleinknecht, V. Kozhuharov, W. Kubischta, G. Lamanna, C. Lazzeroni, M. Lenti, L. Litov, D. Madigozhin, A. Maier, I. Mannelli, F. Marchetto, G. Marel, M. Markytan, P. Marouelli, M. Martini, L. Masetti, E. Mazzucato, A. Michetti, I. Mikulec, M. Misheva, N. Molokanova, E. Monnier, U. Moosbrugger, C. Morales Morales, D.J. Munday, A. Nappi, G. Neuhofer, A. Norton, M. Patel, M. Pepe, A. Peters, F. Petrucci, M.C. Petrucci, B. Peyaud, M. Piccini, G. Pierazzini, I. Polenkevich, Yu. Potrebenikov, M. Raggi, B. Renk, P. Rubin, G. Ruggiero, M. Savri´e, M. Scarpa, M. Shieh, M.W. Slater, M. Sozzi, S. Stoynev, E. Swallow, M. Szleper, M. Valdata-Nappi, B. Vallage, M. Velasco, M. Veltri, S. Venditti, M. Wache, H. Wahl, A. Walker, R. Wanke, L. Widhalm, A. Winhart, R. Winston, M.D. Wood, S.A. Wotton, A. Zinchenko, M. Ziolkowski. First observation and study of the K± →π±π0e decay with the NA48/2 experiment at CERN B Bloch-Devaux1,2 1 Universit`a di Torino, Dipartimento di Fisica Sperimentale, Torino, Italy B Bloch-Devaux1,2 1 Universit`a di Torino, Dipartimento di Fisica Sperimentale, Torino, Italy E-mail: brigitte.bloch-devaux@cern.ch Abstract. The ﬁrst observation of the rare decay K± →π±π0e+e−is reported by the NA48/2 experiment at CERN, based on 4919 candidates with 4.9% background contamination. From the analysis of 1.7 × 1011 kaon decays collected in 2003–2004, the branching ratio in the full kinematic region is measured to be BR(K± →π±π0e+e−)= (4.24 ± 0.14) × 10−6. A detailed study of the kinematic space shows evidence for a structure dependent contribution. Both measured BR and structure dependent contribution are in perfect agreement with theoretical predictions based on Chiral Perturbation Theory. Several P- and CP-violating asymmetries are also evaluated. 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 1526 (2020) 012004 doi:10.1088/1742-6596/1526/1/012004 Journal of Physics: Conference Series decay rate consists of three terms: the dominant long-distance IB contribution (electric part E only), the DE component (both electric E and magnetic M parts), and their interference. The interference term collects the diﬀerent contributions, IB-E, IB-M and E-M. The P-violating IB-M and E-M terms cancel upon angular integration and do not contribute to the total rate. Few theoretical publications investigate the K± →π±π0e+e−mode [1][2][3] and no experimental observation has so far been reported. The authors of [2] were able to predict, on the basis of the NA48/2 measurement of the magnetic and electric terms in the K± →π±π0γ decay mode [4], the contributions of the single components relative to IB. Including more experimental measurements from radiative kaon decays [5], the authors of [3] were able to reﬁne their predictions. 3. Analysis strategy and measurements Events in the signal mode are selected simultaneously with events in the normalization mode (K± →π±π0 D, π0 D →γe+e−) chosen to have a ﬁnal state topology (π±γe+e−) diﬀering from the signal (π±γγe+e−) by only one photon while satisfying similar kinematic constraints on the reconstructed π0 and kaon masses. 2. The NA48/2 experiment The NA48/2 experiment at the CERN SPS was speciﬁcally designed for charge asymmetry measurements in the K± →3π decay modes [6] and collected large samples of charged kaon decays during the 2003–2004 data taking period. The experiment beam line had been designed to deliver simultaneous narrow momentum band K+ and K−beams originating from primary 400 GeV/c protons extracted from the CERN SPS and impinging on a beryllium target. Secondary unseparated beams with central momenta of 60 GeV/c and a momentum band of ±3.8% (rms) were selected and brought to a common beam axis. A fraction of the beam kaons decayed in a ﬁducial decay volume contained in a 114 m long cylindrical evacuated tank. The momenta of charged decay products were measured in a magnetic spectrometer, housed in a tank ﬁlled with helium and placed downstream of the decay volume. The spectrometer was composed of four drift chambers (DCH) and a dipole magnet providing in the horizontal plane a momentum kick ∆p = 120 MeV/c to charged particles. The momentum resolution achieved was σp/p = (1.02 ⊕0.044 · p)% (p in GeV/c). A hodoscope (HOD) consisting of two planes of plastic scintillators, each segmented into 64 strip-shaped counters, followed the spectrometer and provided time measurements for charged particles with a resolution of 150 ps. The HOD surface was logically subdivided into 16 exclusive regions producing fast signals used to trigger the detector readout on charged track topologies. Further downstream was a liquid krypton electromagnetic calorimeter (LKr), an almost homogeneous ionization chamber with an active volume of 7 m3 of liquid krypton, 27X0 deep, segmented transversally into 13248 projective ∼2×2 cm2 cells and with no longitudinal segmentation. The energies of photons were measured with a resolution σE/E = (3.2/ √ E ⊕9.0/E ⊕0.42)% (E in GeV). An iron/scintillator hadronic calorimeter and muon detectors, not used in the present analysis, were located further downstream. A dedicated two-level trigger was used to collect three track decays with a very high eﬃciency [6]. A detailed description of the detector can be found in [7]. 1. Introduction K d h Kaon decays have played a major role in establishing the quark mixing ﬂavour structure of the Standard Model. Radiative kaon decays, dominated by long-distance eﬀects, are particularly suited in testing models describing low-energy quantum chromodynamics (QCD) such as the chiral perturbation theory (ChPT), an eﬀective ﬁeld theory valid below a scale of O(1 GeV). The K± →π±π0e+e−decay, never observed so far, proceeds through virtual photon exchange followed by internal conversion into an electron-positron pair, i.e. K± →π±π0γ∗→π±π0e+e−. The virtual γ∗can be produced by two diﬀerent mechanisms: Inner Bremsstrahlung (IB), where the γ∗is emitted by one of the charged mesons in the initial or ﬁnal state and Direct Emission (DE) where the γ∗is radiated oﬀat the weak vertex. Consequently, the diﬀerential International Conference on Kaon Physics 2019 Journal of Physics: Conference Series International Conference on Kaon Physics 2019 Journal of Physics: Conference Series 1526 (2020) 012004 IOP Publishing doi:10.1088/1742-6596/1526/1/012004 1526 (2020) 012004 IOP Publishing doi:10.1088/1742-6596/1526/1/012004 1526 (2020) 012004 g doi:10.1088/1742-6596/1526/1/012004 1526 (2020) 012004 doi:10.1088/1742-6596/1526/1/012004 suppress photon conversions to an e+e−pair in the upstream material. The ﬁrst level trigger requires coincidences of hits in the two HOD planes in at least two of the 16 square regions. Events with all three tracks hitting the same HOD quadrant, leading to geometrical ineﬃciency, are rejected to ensure high and similar trigger eﬃciencies for both modes. Isolated energy clusters without associated track in the LKr, in time within 5 ns with the mean of the three track times, are identiﬁed as the photon candidates. The minimum photon energy required is 2 GeV. The photon four-momenta are reconstructed assuming they originate from the 3-track vertex. In the signal selection, the two-photon invariant mass is required to be within ±15 MeV/c2 from the nominal PDG [8] π0 mass. The reconstructed invariant mass of the π±π0e+e−system is required to be within ±45 MeV/c2 from the nominal PDG K± mass. In the normalization selection, the same cuts applies to the invariant mass of the γe+e−system (π0 D) and to the π±π0 D system, respectively. The strong kinematic correlation between the reconstructed π0 and kaon masses is exploited, selecting events in a band deﬁned as \|mπ0 −0.42 × mK + 73.2\| < 6 MeV/c2 (masses in MeV/c2), allowing particle identiﬁcation without using the LKr information for electron-pion separation. More than 99% of the normalization simulated events and 96.5% of the signal simulated events satisfy the requirement. The total reconstructed momentum is required to be in the range (54–66) GeV/c, compatible with the beam momentum. In the transverse plane, the reconstructed 3-track vertex position and kaon position at the LKr front face (deﬁned as the energy weighted sum of the non-deviated tracks and photon(s) positions) are required to be consistent with the corresponding beam positions as monitored by K3π events. Two main sources of background are contributing to the signal ﬁnal state: K± →π±π0π0 D (K3πD) when one of the photons is lost, and K± →π±π0 D(γ) (K2πD), when an extra photon combines with the Dalitz photon to mimic a π0 →γγ decay. An additional suppression of the K3πD background events is obtained by requiring the squared invariant mass of the π+π0 system to be greater than 0.12 GeV2/c4, exploiting the larger phase space available in the signal mode. International Conference on Kaon Physics 2019 Journal of Physics: Conference Series International Conference on Kaon Physics 2019 Journal of Physics: Conference Series 1526 (2020) 012004 IOP Publishing doi:10.1088/1742-6596/1526/1/012004 1526 (2020) 012004 doi:10.1088/1742-6596/1526/1/012004 1526 (2020) 012004 doi:10.1088/1742-6596/1526/1/012004 To reject further K2πD background contamination, each of the two possible invariant masses Meeγ are required to be more than 7 MeV/c2 away from the nominal mass of the neutral pion. The background processes contributing to the normalization mode are Kµ3D(K± →µ±νπ0 D) and Ke3D (K± →e±νπ0 D) where the π0 D is correctly reconstructed but the muon (electron) is given a pion mass and therefore can sometimes satisfy all other selection criteria. 6 Samples of 16.3 × 106K2πD and 4919 signal candidates have been selected from a subset of a 1.7×1011 kaon decay exposure in 2003–2004. The background contributions are estimated from simulation. They amount to (10437 ± 119) Kµ3D events and (6851 ± 106) Ke3D events in the normalization mode, corresponding to a total background contamination of (0.106 ± 0.001)%. In the signal mode, they amount to (132 ± 8) events from K3πD, (102 ± 19) events from K2πD and (7 ± 3) events from Ke3D, adding up to a relative background contribution of (4.9 ± 0.4)%. Reconstructed mass distributions are displayed in Figure 1 (Figure 2) for the selected normalization (signal) candidates. Expected background and normalization (signal) simulations, scaled to the number of observed candidates, show a good agreement with the data distributions. 3.1. Candidate selections and background estimates 3.1. Candidate selections and background estimates Both signal and normalization candidates are reconstructed from three charged tracks (two same-sign tracks and one opposite-charge track) forming a common vertex in the ﬁducial decay volume. The tracks are required to be in time within 5 ns of each other using the HOD time of the hit associated to the track and their impact point should lie within the geometrical acceptance of the drift chambers (radial distance to the beam line axis, monitored by fully reconstructed K± →π±π+π−(K3π) events, larger than 12 cm). The track momenta are required to be in the range (2–60) GeV/c and any track-to-track distance at DCH1 should be larger than 2 cm to 2 3.2. Branching ratio measurement ± g Branching Ratio of the K± →π±π0e+e−decay mode is obtained using the expression: The Branching Ratio of the K± →π± The Branching Ratio of the K± →π±π0e+e−decay mode i BR(K± →π±π0e+e−)/BR(K± →π±π0) = (Ns −Nbs) (Nn −Nbn) · An × εn As × εs · Γ(π0 D) Γ(π0γγ) (1) (1) where Ns,bs,n,bn are the number of signal, background to signal, normalization and corresponding background events. As,n and εs,n are the acceptances and trigger eﬃciencies of the signal and 3 International Conference on Kaon Physics 2019 1526 (2020) 012004 O g doi:10.1088/1742-6596/1526/1/012004 Journal of Physics: Conference Series 0 500 1000 1500 2000 2500 3000 3500 4000 x 10 3 120 125 130 135 140 145 150 Data K2πD Ke3D x 50 Kµ3D x 50 mγe+e−[GeV/c2] 0 500 1000 1500 2000 2500 3000 3500 x 10 3 450 460 470 480 490 500 510 520 530 540 Data K2πD Ke3D x 50 Kµ3D x 50 mπ±π0 D [GeV/c2] 1 10 10 2 10 3 10 4 10 5 10 6 0 20 40 60 80 100 120 140 Data K2πD Ke3D Kµ3D me+e−[GeV/c2] Figure 1. Normalization candidates. Left: reconstructed γe+e−mass. Middle: reconstructed π±π0 D mass. Right: reconstructed e+e−mass. The selection requires me+e−> 10 MeV/c2. Dots correspond to data candidates, stacked histograms are, from bottom to top, the expected Kµ3D (green) and Ke3D (blue) backgrounds multiplied by a factor of 50 to be visible. The normalization simulation (red) includes radiative eﬀects. 0 500 1000 1500 2000 2500 3000 3500 4000 x 10 3 120 125 130 135 140 145 150 Data K2πD Ke3D x 50 Kµ3D x 50 mγe+e−[GeV/c2] 0 500 1000 1500 2000 2500 3000 3500 x 10 3 450 460 470 480 490 500 510 520 530 540 Data K2πD Ke3D x 50 Kµ3D x 50 mπ±π0 D [GeV/c2] 1 10 10 2 10 3 10 4 10 5 10 6 0 20 40 60 80 100 120 140 Data K2πD Ke3D Kµ3D me+e−[GeV/c2] 100 120 14 [GeV/c2] 60 80 00 0 0 me+e−[GeV/c2] Figure 1. Normalization candidates. Left: reconstructed γe+e−mass. Middle: reconstructed π±π0 D mass. Right: reconstructed e+e−mass. The selection requires me+e−> 10 MeV/c2. Dots correspond to data candidates, stacked histograms are, from bottom to top, the expected Kµ3D (green) and Ke3D (blue) backgrounds multiplied by a factor of 50 to be visible. The normalization simulation (red) includes radiative eﬀects. 1526 (2020) 012004 doi:10.1088/1742-6596/1526/1/012004 Within the current statistics, the data sample analyzed has no sensitivity to the DE and INT contributions that would be dominant in the upper part of the mee spectrum (Figure 2). However a global analysis of the 3d-space (q2, T ∗ π, E∗ γ) where q2, T ∗ π, E∗ γ are the e+e−mass squared, the charged pion kinetic energy and the virtual photon energy in the kaon rest frame, respectively, allows to measure the fractions M/IB = (1.14 ± 0.43stat)%, to be compared to the prediction (1.41 ± 0.14ext)% and, with a limited accuracy, (IB-E)/IB = (−0.14±0.36stat)%, to be compared to the prediction (−0.39±0.28ext)%. The external errors stem from the uncertainties of the measurements used as input in the predictions. Asymmetries between K+ and K−partial rates give access to the weak (or beyond Standard Model) phases that change sign under charge conjugation, unlike the strong phase that governs the ﬁnal state interaction of the pion system. The simplest CP-violating asymmetry is the charge asymmetry of the integrated partial rates, ACP , measured from the statistically independent measurements of K+ and K−branching ratios, as ACP = (−2.84 ± 1.55)%, translated to a single-sided upper limit of \|ACP \| < 4.82% at 90% CL. Similarly, other asymmetries can be built from partially integrated K+ and K−partial rates over the angular variable φ, the angle between the dipion and dilepton planes in the kaon rest frame. Two CP-violating asymmetries and one long-distance P-violating asymmetry [5] have been evaluated, all consistent with zero and resulting in single-sided upper limits \|Aφ CP \| < 1.9% at 90% CL and \|A(L) P \| < 1.8% at 90% CL . 1526 (2020) 012004 doi:10.1088/1742-6596/1526/1/012004 1526 (2020) 012004 doi:10.1088/1742-6596/1526/1/012004 implementation of radiative eﬀects. The agreement between data and simulation can be seen from the me+e−distribution of Figure 1. The MC simulations for the diﬀerent π±π0e+e− contributions IB, DE, and the electric interference IB-E, have been generated separately according to the theoretical description given in [2][3] neglecting the magnetic interference. The As acceptance of 0.662(1)% has been obtained from a weighted average of the single component acceptances, using as weights the relative contributions with respect to IB computed in [2][3]. As radiative corrections to the π±π0e+e−mode are not considered in [2][3], the signal MC simulation included the real photon(s) emission as implemented in the PHOTOS package. The total branching ratio is obtained as : (2) BR(K± →π±π0e+e−) = (4.237 ± 0.063stat ± 0.033syst ± 0.126ext) × 10−6 (2) where systematic errors include uncertainties on acceptance, trigger eﬃciencies and radiative corrections. The external error originating from the π0 D branching ratio uncertainty is the dominant error in the present measurement obtained with an overall precision of 2.971%. 3.3. Kinematic space study and asymmetries 3.3. Kinematic space study and asymmetries The obtained BR measurement is in very good agreement with the theoretical predictions [2][3] of 4.18 × 10−6 for IB only and 4.23 × 10−6 including all structure dependent contributions but is not precise enough to distinguish between them. Within the current statistics, the data sample analyzed has no sensitivity to the DE and INT contributions that would be dominant in the upper part of the mee spectrum (Figure 2). However a global analysis of the 3d-space (q2, T ∗ π, E∗ γ) where q2, T ∗ π, E∗ γ are the e+e−mass squared, the charged pion kinetic energy and the virtual photon energy in the kaon rest frame, respectively, allows to measure the fractions M/IB = (1.14 ± 0.43stat)%, to be compared to the prediction (1.41 ± 0.14ext)% and, with a limited accuracy, (IB-E)/IB = (−0.14±0.36stat)%, to be compared to the prediction (−0.39±0.28ext)%. The external errors stem from the uncertainties of the measurements used as input in the predictions. p y y The obtained BR measurement is in very good agreement with the theoretical predictions [2][3] of 4.18 × 10−6 for IB only and 4.23 × 10−6 including all structure dependent contributions but is not precise enough to distinguish between them. 3.2. Branching ratio measurement ± 0 100 200 300 400 500 600 700 120 125 130 135 140 145 150 Data Signal IB Ke3D K2πDγ K3πD mγγ [GeV/c2] 0 100 200 300 400 500 600 450 460 470 480 490 500 510 520 530 540 Data Signal IB Ke3D K2πDγ K3πD mπ±π0e+e−[GeV/c2] 1 10 10 2 10 3 0 20 40 60 80 100 120 140 Data Signal IB Ke3D K2πDγ K3πD me+e−[GeV/c2] Figure 2. Signal candidates. Left: reconstructed γγ mass. Middle: reconstructed π±π0e+e− mass. Right: reconstructed e+e−mass. Full dots correspond to data candidates, stacked histograms are, from bottom to top, the expected K3πD (green), K2πD (light blue) and Ke3D (dark blue) backgrounds and signal IB (red) estimated from simulation. mπ±π0e+e−[Ge mπ±π0e+e− Figure 2. Signal candidates. Left: reconstructed γγ mass. Middle: reconstructed π±π0e+e− mass. Right: reconstructed e+e−mass. Full dots correspond to data candidates, stacked histograms are, from bottom to top, the expected K3πD (green), K2πD (light blue) and Ke3D (dark blue) backgrounds and signal IB (red) estimated from simulation. normalization modes. The trigger eﬃciency (εn), is measured using control data samples. It is also evaluated on simulated samples and found to be well reproduced (εn = 98.27(1)%). Because of the very limited data statistics, simulated signal samples are therefore used to evaluate the signal trigger eﬃciency (εs = 98.34(2)%) . The normalization mode branching ratio BR(K± →π±π0) = (20.67 ± 0.08) × 10−2 is obtained from the PDG [8] world average. The acceptances of the signal, the normalization and the background channels are computed using a GEANT3-based [9] Monte Carlo (MC) simulation which includes full detector geometry and material description, stray magnetic ﬁelds, accurate beam line geometry, local detector imperfections and time variations of the above throughout the data taking period. The An acceptance of 3.981(2)% is computed using the implementation of K± →π±π0 according to [10] followed by π0 D according to the most recent calculations [11], including the best current 4 International Conference on Kaon Physics 2019 Journal of Physics: Conference Series 1526 (2020) 012004 IOP Publishing doi:10.1088/1742-6596/1526/1/012004 1526 (2020) 012004 g doi:10.1088/1742-6596/1526/1/012004 4. Summary h ﬁ b The ﬁrst observation of the K± →π±π0e+e−decay has been reported by the NA48/2 collaboration [12] with a branching ratio BR(K± →π±π0e+e−) = (4.24 ± 0.14) × 10−6 in agreement with the theoretical predictions [2][3]. The relative contribution M/IB has been evaluated from a 3d-analysis of the kinematic space as (1.14 ± 0.43stat)%. Several P- and CP- violating asymmetries have been set as single-sided upper limits of ∼2% at 90% CL. Further studies of this rich mode could bring more information on the internal structure of the decay process, in a later data taking period of NA62 including a dedicated trigger stream. 5 1526 (2020) 012004 IOP Publishing doi:10.1088/1742-6596/1526/1/012004 International Conference on Kaon Physics 2019 International Conference on Kaon Physics 2019 Journal of Physics: Conference Series Acknowledgments g The author is most grateful to the local organisers of the KAON 2019 conference for making this contribution possible and for their support and hospitality. References [1] Pichl H 2001 Eur. Phys. J. C 20 371 [1] Pichl H 2001 Eur. Phys. J. C 20 371 [3] Cappiello L, Cat`a O and D’Ambrosio G 2018 Eur. Phys. J. C 78 265 [4] Batley J R et al. [NA48/2 Collaboration] 2010 Eur. Phys. J. C 68 75 [5] Cat`a O, 2019 these proceedings [6] Batley J R et al. [NA48/2 Collaboration] 2007 Eur. Phys. J. C [6] Batley J R et al. [NA48/2 Collaboration] 2007 Eur. Phys. J. C 52 875 2007 Nucl. Instrum. Methods A 574 443 nti V et al. 2007 Nucl. Instrum. Methods A 574 443 [ ] [8] Tanabashi M et al. [Particle Data Group] 2018 Phys. Rev. D 98 030001 [9] GEANT3 Detector Description and Simulation Tool 1994 CERN Program Library W5013 [ ] y [11] Husek T, Kampf K and Novotny J 2015 Phys. Rev. D 92 054027 [11] Husek T, Kampf K and Novotny J 2015 Phys. Rev. D 92 054027 11] Husek T, Kampf K and Novotny J 2015 Phys. Re [12] Batley J R et al. [NA48/2 Collaboration] 2019 Phys. Lett. B 788 552 6
https://openalex.org/W4392843410	https://mentalhealth.bmj.com/content/ebmental/27/1/e300844.full.pdf	English	null	Waiting-list interventions for children and young people using child and adolescent mental health services: a systematic review	BMJ mental health	2,024	cc-by	7,877	WHAT THIS STUDY ADDS ⇒Waiting list interventions have the potential to support children, young people and their families while waiting for mental health assessment and/or treatment. ⇒Psychoeducation lends itself to being offered as a waiting list intervention. 3Institute of Mental Health, School of Medicine, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, UK 4NIHR Nottingham Biomedical Research Centre, Institute of Mental Health, University of Nottingham, Nottingham, UK, University of Nottingham, Nottingham, UK copyright. on October 23 http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from y or children and young escent mental health w Kapil Sayal ‍ ‍ ,3 Charlotte L Hall ‍ ‍ 4 in 2020, although rates have since remained more stable.2 This is reflected in referrals to CAMHS, where a 109% increase in referrals has been seen between April 2019 (31 720 referrals) and April 2022 (66 389 referrals).3 Waiting list times may influence engagement with services, deterring fami- lies from seeking help4 and negatively impacting engagement with therapy.5–7 An exacerbation of mental and physical health issues may also be expe- rienced while on waiting lists.7 The James Lind Alliance (JLA) identified which interventions are effective in supporting children and young people on waiting lists as a top-10 priority (https://www. jla.nihr.ac.uk/priority-setting-partnerships/Mental- health-in-children-and-young-people/top-10-prior- ities.htm). Over the past 20 years, there has been an increasing body of research on waiting list strat- egies within mental health services. It has been recommended that interim interventions (such as bibliotherapy and relaxation training) should be WHAT IS ALREADY KNOWN ON THIS TOPIC ⇒Waiting lists for child and adolescent mental health services are growing worldwide, therefore there is a need to understand how referred patients on waiting lists can be better supported. WHAT THIS STUDY ADDS ⇒Waiting list interventions have the potential to support children, young people and their families while waiting for mental health assessment and/or treatment. ⇒Psychoeducation lends itself to being offered as a waiting list intervention. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ⇒Waiting list interventions show promise in supporting children and young people accessing child and adolescent mental health services; further research is warranted to further understand the benefits to the service, the patient and how best to develop and implement these in practice. copyright. y g p j p j y Althea Z Valentine ‍ ‍ ,1 Sophie S Hall ‍ ‍ ,2 Kapil Sayal ‍ ‍ ,3 Charlotte L Hall ‍ ‍ 4 Althea Z Valentine ‍ ‍ ,1 Sophie S Hall ‍ ‍ ,2 Kapil Sayal ‍ ‍ ,3 Charlotte L Hall ‍ ‍ 4 copyright. on October 23, 2024 by guest. Protected by http://mentalhealth.bmj.com/ ublished as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from ABSTRACT Question Children and young people experience delays in assessment and/or treatment within mental health services. The objective of this systematic review, funded by the Emerging Minds Network, was to explore the current evidence base for mental health waiting list interventions to support children and young people. Study selection and analysis A literature search was conducted in MEDLINE, PsycINFO, Web of Science and the Cochrane databases from 2000 to 2023 (last searched October 2023). Included studies described interventions to support children and young people and/or their family while on a waiting list for child and adolescent mental health services. Titles and abstracts were screened independently by two reviewers, data were extracted by one reviewer, confirmed by a second and a narrative synthesis was provided. ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi. org/10.1136/bmjment-2023- 300844). WHAT IS ALREADY KNOWN ON THIS TOPIC ⇒Waiting lists for child and adolescent mental health services are growing worldwide, therefore there is a need to understand how referred patients on waiting lists can be better supported. ⇒Waiting lists for child and adolescent mental health services are growing worldwide, therefore there is a need to understand how referred patients on waiting lists can be better supported. 1NIHR MindTech MedTech Co-operative, Institute of Mental Health, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, UK 2University of Nottingham, Nottingham Clinical Trials Unit, Nottingham, UK 3Institute of Mental Health, School of Medicine, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, UK 4NIHR Nottingham Biomedical Research Centre, Institute of Mental Health, University of Nottingham, Nottingham, UK, University of Nottingham, Nottingham, UK 1NIHR MindTech MedTech Co-operative, Institute of Mental Health, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, UK Study selection and analysis A literature search was conducted in MEDLINE, PsycINFO, Web of Science and the Cochrane databases from 2000 to 2023 (last searched October 2023). Included studies described interventions to support children and young people and/or their family while on a waiting list for child and adolescent mental health services. Titles and abstracts were screened independently by two reviewers, data were extracted by one reviewer, confirmed by a second and a narrative synthesis was provided. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY Findings Eighteen studies including 1253 children and young people were identified. Studies described waiting list interventions for autism spectrum disorders, eating disorders, generic conditions, transgender health, anxiety/ depression, self-harm and suicide and behavioural issues. Many interventions were multicomponent; 94% involved psychoeducation, other components included parental support, bibliotherapy and coaching. Duration of the interventions ranged from a single session to over a year; 66% involved face-to-face contact. All studies demonstrated benefits in terms of improved clinical outcomes and/or feasibility/acceptability. Evidence for service outcomes/efficiency was largely unexplored. Limitations of the underpinning research, such as sample size and low-quality papers, limit the findings. ⇒Waiting list interventions show promise in supporting children and young people accessing child and adolescent mental health services; further research is warranted to further understand the benefits to the service, the patient and how best to develop and implement these in practice. Correspondence to Dr Althea Z Valentine, NIHR MindTech MedTech Co- operative, Institute of Mental Health, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, NG7 2RD, UK; althea.valentine@nottingham. ac.uk in 2020, although rates have since remained more stable.2 This is reflected in referrals to CAMHS, where a 109% increase in referrals has been seen between April 2019 (31 720 referrals) and April 2022 (66 389 referrals).3 Waiting list times may influence engagement with services, deterring fami- lies from seeking help4 and negatively impacting engagement with therapy.5–7 An exacerbation of mental and physical health issues may also be expe- rienced while on waiting lists.7 The James Lind Alliance (JLA) identified which interventions are effective in supporting children and young people on waiting lists as a top-10 priority (https://www. jla.nihr.ac.uk/priority-setting-partnerships/Mental- health-in-children-and-young-people/top-10-prior- ities.htm). Limitations of the underpinning research, such as sample size and low-quality papers, limit the findings. Received 24 July 2023 Accepted 3 December 2023 Received 24 July 2023 Accepted 3 December 2023 Received 24 July 2023 Accepted 3 December 2023 Conclusions There is limited research exploring waiting list interventions, however, the findings from small-scale studies are promising. Further research using robust study designs and real-world implementation studies are warranted. October 23, 2024 by guest. Protected by ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi. org/10.1136/bmjment-2023- 300844). Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 copyright. on October 23, 2024 by guest. Protected http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from Open access Systematic review BACKGROUND h ld The following were excluded: waiting list management from a service perspective, models for healthcare delivery, appointment scheduling or improving access, opinions on general service satisfaction or clinical changes not linked to waiting list interventions, interventions that could be a waiting list intervention but are not tested as such, preventative trials not at the point of referral. Comparators The intervention must have been used to support participants while on a waiting list for assessment or treatment in a mental health service; any comparators were considered. No exclusion criteria. Outcomes Outcomes included clinical outcomes, service efficiencies and user impact, acceptability and reported barriers and facilitators to engagement. No exclusion criteria. Types of sources of evidence Evidence sources included qualitative and quantitative research studies and conference abstracts that reported clinical, cost-effectiveness and/or perceptions (feasibility/ usability). There were no restrictions on the study design. Non-peer-review articles and study protocols were excluded. BMJ Ment Health: first published as 10.1136/bmjment- copyright. http://menta BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from copyright. on October 23, 2024 by gue http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from copyright. on October 23, 2024 by guest. Protected by http://mentalhealth.bmj.com/ th: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from The following were excluded: waiting list management from a service perspective, models for healthcare delivery, appointment scheduling or improving access, opinions on general service satisfaction or clinical changes not linked to waiting list interventions, interventions that could be a waiting list intervention but are not tested as such, preventative trials not at the point of referral. No exclusion criteria. Types of sources of evidence Evidence sources included qualitative and quantitative research studies and conference abstracts that reported clinical, cost-effectiveness and/or perceptions (feasibility/ usability). There were no restrictions on the study design. Non-peer-review articles and study protocols were excluded. CAMHS, Child, “mental health” or depress). Subject/Medical Subject Headings related to mental health (eg, Mental Health/ Services/ Disorders, Pediatrics, Child and Adolescent Psychi- atry/Psychology/Psychotherapy/Psychopathology). Following data extraction, backward and forward citation chasing was conducted using a citationchaser11 (https://estech.shinyapps.io/ citationchaser/) looking for all records citing (forward citation chasing) or referenced (backward citation chasing) in one or more of the included articles, this search was updated October 2023. lists rather than interim patient-facing interventions. BACKGROUND h ld Children, young people and their families report long waiting lists for assessment, diagnosis and treat- ment once referred to child and adolescent mental health services (CAMHS) in the UK. A survey of almost 14 000 young people found that 44% waited more than a month for mental health support, 26% of which have attempted to take their own life while waiting for support.1 Probable mental health disor- ders in those aged 17–19 years increased from 1 in 10 (10.1%) in 2017 to 1 in 4 (25.7%) in 2022. In those aged 7–16 years, there was an increase from one in nine (12.1%) in 2017 to one in six (16.7%) Over the past 20 years, there has been an increasing body of research on waiting list strat- egies within mental health services. It has been recommended that interim interventions (such as bibliotherapy and relaxation training) should be considered for patients facing long waiting times, as well as improving the administrative manage- ment of waiting lists, for example, using clear eligi- bility criteria, screening and opting in.8 Research has focused mainly on service initiatives to manage To cite: Valentine AZ, Hall SS, Sayal K, et al. BMJ Ment Health 2024;27:1–6. © Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ. To cite: Valentine AZ, Hall SS, Sayal K, et al. BMJ Ment Health 2024;27:1–6. Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 1 Open access Open access Table 1 Inclusion and exclusion criteria PICO criteria Inclusion criteria Exclusion criteria Population Children and young people (up to 18 years) referred to a specialist mental health service for assessment or treatment of any mental health disorder. Participants may also be the families/carers or healthcare providers of these participants. No exclusion criteria. Intervention We defined ‘waiting list interventions’, as an intervention used to support participants and/or their family while on a waiting list for mental health assessment, diagnosis and/ or treatment. There were no restrictions on the frequency, timing, geographical location or healthcare setting, or those administering the intervention, but these details were noted in the data extraction as important features. BACKGROUND h ld A recent systematic review identified 20 articles, including studies of mental health services for children and young people (n=10), family or adult (n=4), primary care (n=3) and university coun- selling (n=3). The paper detailed strategies services have used to manage waiting list demands including demand management approaches such as walk-in models, triage, multidisciplinary care, patient-led approaches and service delivery changes.9 To improve service delivery and better support children and young people on waiting lists, there is a need to understand what waiting list interventions are currently offered and the evidence base to support their use; we conducted a systematic review to explore this. The retrieval method was the same across all search methods. All identified citations were collated and uploaded into EndNote V.x9.3.3 (Clarivate Analytics, Pennsylvania, USA) and 1804 duplicates removed using Endnote automated tools. Following pilot testing the study selection, all titles and abstracts were screened independently against the inclusion criteria in Endnote (AZV) and following data importation to Excel (CLH/SSH). Two researchers independently reviewed the full texts of selected arti- cles and the reason for exclusion was noted. Any disagreements that arose were resolved through discussion. The results of the search are presented in a Preferred Reporting Items for System- atic Reviews and Meta-Analyses (PRISMA) flow diagram12 (see figure 1). OBJECTIVE The objectives of the review were to: (a) explore, summarise and assess the quality of the available peer-reviewed evidence- base for interventions for children and young people on waiting lists for mental health services; (b) provide an overview of the characteristics, nature and diversity of the interventions and (c) explore the evidence in terms of outcomes of interest including clinical outcomes, service efficiencies and user impact, accept- ability and reported barriers and facilitators to engagement. In doing so, we identify gaps, deficiencies and trends in the current evidence to help underpin and inform future research. Data were extracted using a standardised form by one researcher (AZV) and verified for accuracy by one of two reviewers (CLH and SSH), discrepancies were resolved via discussion. The first three papers were used to pilot the data extraction tool, minor modifications were made to ensure that relevant data were collected (eg, including the percentage of eligible participants recruited). Extracted data included study and population characteristics, descriptions of study aims and intervention and barriers and facilitators. Outcomes of interest were clinical (any measure and timepoint), service efficiency (eg, reduction in waiting times, intervention costs, etc) and user impact (feasibility and acceptability). Missing data were recorded and authors were not contacted for additional infor- mation. Because of the heterogeneity of the included studies, a narrative synthesis was used. Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 Quality Th The quality appraisal revealed most studies were of low quality. Although there were four randomised controlled trials (RCTs), only one met the prespecified power sample size and was given a OCEBM level 2 quality rating.22 However, the sample size was small (n=60) and there was insufficient information on how the power calculation was performed.22 This study along with another small study including 51 parents/caregivers and 36 young people24 were designed to assess effectiveness. The remaining RCTs were designed to assess feasibility/acceptability, and thus were not fully powered14 21; these were downgraded from possible 2 to 3 ratings. We noted the importance of these studies to inform the future development of efficacy RCTs. From the remaining papers, all studies were classified as OCEBM level 3–5, most described an evaluation of implementation within a single service, comparing scores at baseline and postinterven- tion. Typically, the studies did not have a comparator group. ober 23, 2024 by guest. Protected by Study selection and analysis Protected by http://mentalhealth.bmj.com/ MJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from Figure 1 Flow diagram showing the selection of reports included in the review. WLI, waiting list intervention. Figure 1 Flow diagram showing the selection of reports included in the review. WLI, waiting list intervention. the appraisal, which was verified by a second reviewer (SSH), any disagreements were resolved via discussion. Population All participants were on waiting lists for mental health services delivered in healthcare settings relevant to the country of the study. Five were conducted in the USA, four in Australia, four in the UK, three in Canada and two in Ireland. The reasons for being on a waiting list included: autism spectrum disorders (ASD; n=5), eating disorders (n=4), generic CAMHS referrals (n=3), transgender health (n=2), anxiety/depression (n=2), self-harm/suicide (n=1) and behavioural issues (n=1). All ages of children were included in the review from under 5 to 18 years. Those under 5 were all referred to CAMHS for ASD or generic reasons. Where reported, all studies included a mix of genders, with ASD and behavioural issues involving predominantly male participants and eating disorders, anxiety and depression, predominantly female participants. Most studies were small with 7 studies involving fewer than 20 participants and 7 studies between 21 and 100 participants. The larger studies included one for 125 families of adolescents with self-harm or suicidality behaviours, one for 268 families on eating disorders waiting lists and the two transgender health studies including 142 and 194 participants, respectively. Recruitment was an issue across multiple studies and small sample size was frequently mentioned as a limitation of the research. FINDINGS A total of 5078 studies were identified, imported into EXCEL and full texts read for relevance (n=93), if not relevant the reason for exclusion was noted (see figure 1), 18 papers were identified as meeting the eligibility criteria.13–30 The character- istics of the 18 included studies are presented in online supple- mental table 2 and online supplemental table A1. on October 23, 2024 by guest. Protected by lth.bmj.com/ Study selection and analysis The objectives, inclusion criteria and methods for this review were specified in advance (table 1), following appropriate guide- lines10 (see online supplemental file 1 for the protocol). Rele- vant terms were noted following a limited database search, from which a comprehensive list was created by clinical and academic experts. The main search was run in three databases MEDLINE (Ovid), PsycINFO (Ovid) and Web of Science, restricted by date from 2000 to 2023 to ensure interventions were relevant, and by those published in English. The search was initially under- taken on 10 September 2021 and updated October 2023. A search was also run in Cochrane Trials in September 2021 only. Free-text search terms related to waiting (eg, “wait”, wait adj5 initiative, wait* intervention, wait* adj time, Wait* adj5 length, Wait* adj5 duration, Access adj5 delay, Wait* adj5 access) and children and young people mental health (eg, psych, behav, ctober 23, 2024 by guest. Protected by A rapid appraisal of the level of evidence was assessed using the Oxford Centre for Evidence-Based Medicine (OCEBM; https://www.cebm.ox.ac.uk/resources/levels-of-evidence/ocebm- levels-of-evidence). The levels of evidence ranged from 1 to 5, where 1 was the highest quality. One reviewer (CLH) conducted 2 Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 copyright. on October 23, 2024 by guest. Protected b http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from Open access Figure 1 Flow diagram showing the selection of reports included in the review. WLI, waiting list intervention. copyright. BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downlo copyright. http:// BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from 3 Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 the appraisal, which was verified by a second reviewer (SSH), any disagreements were resolved via discussion. FINDINGS A total of 5078 studies were identified, imported into EXCEL and full texts read for relevance (n=93), if not relevant the reason for exclusion was noted (see figure 1), 18 papers were identified as meeting the eligibility criteria.13–30 The character- istics of the 18 included studies are presented in online supple- mental table 2 and online supplemental table A1. Quality The quality appraisal revealed most studies were of low quality. Study selection and analysis Although there were four randomised controlled trials (RCTs), only one met the prespecified power sample size and was given a OCEBM level 2 quality rating.22 However, the sample size was small (n=60) and there was insufficient information on how the power calculation was performed.22 This study along with another small study including 51 parents/caregivers and 36 young people24 were designed to assess effectiveness. The remaining RCTs were designed to assess feasibility/acceptability, and thus were not fully powered14 21; these were downgraded from possible 2 to 3 ratings. We noted the importance of these studies to inform the future development of efficacy RCTs. From the remaining papers, all studies were classified as OCEBM level 3–5, most described an evaluation of implementation within a single service, comparing scores at baseline and postinterven- tion. Typically, the studies did not have a comparator group. Population All participants were on waiting lists for mental health services delivered in healthcare settings relevant to the country of the study. Five were conducted in the USA, four in Australia, four in the UK, three in Canada and two in Ireland. The reasons for being on a waiting list included: autism spectrum disorders (ASD; n=5), eating disorders (n=4), generic CAMHS referrals (n=3), transgender health (n=2), anxiety/depression (n=2), self-harm/suicide (n=1) and behavioural issues (n=1). All ages of children were included in the review from under 5 to 18 years. Those under 5 were all referred to CAMHS for ASD or generic reasons. Where reported, all studies included a mix of genders, with ASD and behavioural issues involving predominantly male participants and eating disorders, anxiety and depression, predominantly female participants. Most studies were small with 7 studies involving fewer than 20 participants and 7 studies between 21 and 100 participants. The larger studies included one for 125 families of adolescents with self-harm or suicidality behaviours, one for 268 families on eating disorders waiting lists and the two transgender health studies including 142 and 194 participants, respectively. Recruitment was an issue across multiple studies and small sample size was frequently mentioned as a limitation of the research. Intervention type and duration The interventions varied considerably and most of the interven- tions were multicomponent. All but one14 of the interventions involved at least some psychoeducation aspects, many involved Figure 1 Flow diagram showing the selection of reports included in the review. WLI, waiting list intervention. copyright. on October 23, 2024 by guest. Methodology adopted The research design varied considerably with two RCTs and two randomised controlled feasibility studies, a further five feasibility studies were conducted which were generally quasi-experimental or mixed methods, other research designs included service evalu- ations, action research and qualitative studies. Parents reported increased understanding and/or greater self- efficacy16 23 as well as feeling empowered.24 28 In a pilot study using a brief solution-focused approach, clinicians reported that they were highly satisfied with the waiting list intervention and enthusiastic about the effectiveness of the pilot.25 copyright. on October 23, 2024 by guest. Protec http://mentalhealth.bmj.com/ 2024. Downloaded from Limitations of interventions were also noted in terms of enablers and barriers, with some families feeling they would like more frequent and intensive intervention,23 an advice line for support, a parental support group and a shorter waiting time.13 Within the psychoeducation elements, there was mixed feed- back with some families feeling that the information was too generic, and some families did not find the content as helpful as expected.26 27 Some children and young people reported difficul- ties in attending the intervention because it was held during the school day.21 Low recruitment rate was noted as a barrier in some studies,17 21 with authors postulating that this may be because of parental disempowerment and fear of losing their place on the waiting list or that support may be delayed because of their participation.26 However, once participants were recruited, waiting list interventions generally had low drop-out rates.13 21 22 Parent-related outcomes There were contradictory findings on whether the waiting list interventions improved parental stress14 23 and parental depres- sion and anxiety.15 18 21 27 29 Improvements were noted in quality 17 DISCUSSION This review considered the published literature for waiting list interventions for children and young people and families waiting for mental health assessment and/or treatment. It aimed to (a) explore, summarise and assess the quality of the available peer- reviewed evidence base for interventions for children and young people on waiting lists for mental health services; (b) provide an overview of the characteristics, nature and diversity of the interventions and (c) explore the evidence in terms of outcomes of interest including clinical outcomes, service efficiencies and user impact, acceptability and reported barriers and facilitators to engagement. Behavioural changes were assessed in three of the four eating disorders studies in terms of child weight and eating disorder symptoms. A positive impact was noted in two studies,17 26 with significant weight gains reported in both studies; one study26 additionally noted a decrease in eating disorder behaviours. No impact on eating disorder symptoms was found in another.24 ober 23, 2024 by guest. Protected by User impact In terms of user impact, there were high ratings in terms of parental acceptability, satisfaction and/or feasibility.13–15 20–22 27 28 30 Parents reported increased understanding and/or greater self- efficacy16 23 as well as feeling empowered.24 28 In a pilot study using a brief solution-focused approach, clinicians reported that they were highly satisfied with the waiting list intervention and enthusiastic about the effectiveness of the pilot.25 In terms of user impact, there were high ratings in terms of parental acceptability, satisfaction and/or feasibility.13–15 20–22 27 28 30 Service efficiencies Service efficiencies providing parental support (n=11) and/or provided a self-help manual, video or other reading material for parents/young people (n=10). Coaching (n=3) and referrals or direction to additional support (n=2) were also used. Most interventions (n=12) involved some face-to-face contact and were aimed at parents alone (n=11) or parents alongside children and young people (n=5). Only one study15 gave children and young people the option to complete the intervention with or without their parent/carer (depending on their preference). The duration varied from a single session to >12 sessions. Several studies were a single-session intervention with follow-up support provided where required. Three service efficiencies were reported. McGarry et al found that when a brief consultation and advice appointment was provided as a waiting list intervention, fewer families dropped out compared with those who received treatment as usual.22 In a service report of three different methods of family-based treat- ment for eating disorders, the authors suggested that there was a reduced burden on staff and increased efficiency of appoint- ments, potentially reducing waiting lists, however, no data were given to support this.28 The service evaluation of the First Assess- ment Single-Session Triage intervention for transgender children and young people found a reduction in wait time from 14 to 4 months19 after implementing the intervention. Costs were mentioned in four papers, but these were not related to the economic assessment of the intervention. Two studies reported that the intervention was cost-neutral to participants but did not discuss how they determined this,14 23 one study reported that it was a low-cost intervention26 and another that they provided play materials20 but none provided details on costs or informa- tion about cost savings that would be useful in terms of moni- toring service efficiencies or intervention costs. copyright. on October 23, 2024 by guest. Protected by http://mentalhealth.bmj.com/ th: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from Child-related outcomes The Strengths and Difficulties Questionnaire (SDQ) was used in two studies.13 22 One study found significant reductions on the conduct, emotional and hyperactivity subscales which demon- strated a positive effect of the intervention.13 The other study showed a significant improvement on the mean total SDQ score between baseline and 6-month follow-up.24 Most studies used parent-reported measures for outcomes relating to the child, only four studies used child or young person self-report measures, or included both parental and child self-report measures.15 18 21 24 on October 23, 2024 by guest. Protected by lth.bmj.com/ Aims of reviewed studies All 18 papers aimed to evaluate the waiting list intervention. In seven, this was generally in terms of effectiveness and parental satisfaction. Five papers aimed to assess feasibility and accept- ability of the interventions. The remaining papers were to explore the impact on mental health, to document the creation and implementation of a waiting list intervention, to improve parental knowledge and self-efficacy, to support families and to reduce the waiting list. Intervention type and duration The interventions varied considerably and most of the interven- tions were multicomponent. All but one14 of the interventions involved at least some psychoeducation aspects, many involved Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 3 Open access copyright. on October 23, 2024 by gue http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from Key findings (evidence established) Clinical outcomes The clinical outcomes varied considerably across studies, the main clinical outcomes were changes in child symptoms or behaviours and parental anxiety, depression and stress. Further details about the outcomes in individual studies are presented in the online supplemental file 1. Overall, clinical outcomes were limited in terms of the small sample sizes. Outcomes of interest f db k User feedback in general suggests high levels of satisfaction. Parents appreciated receiving support while on waiting lists, with some evidence of lower attrition to the targeted interven- tion. Given the exploratory nature of many of the studies, few studies reported on clinical outcomes, and these presented mixed results. Nevertheless, several interventions had some supporting evidence for their clinical effectiveness.13 15 17 18 21 22 24 26 27 29 Additionally, the limitations of any review are relevant in that sources of information may be omitted, this is particularly so because of only using three databases, it is possible that searching additional nursing databases would yield further relevant studies. It was somewhat surprising that a limited number of conditions were identified and perhaps not those which were expected, for example, conditions such as ADHD frequently have a long wait to diagnosis but were not identified in this study. Further research using search terms for conditions such as ADHD may be bene- ficial. Although we included a variety of search terms and used a comprehensive search strategy to identify the most relevant terms, the search was hindered by the vast number of studies including waitlist control groups; a future systematic review should consider revision of search terms to increase sensitivity. Most studies included psychoeducation as a waiting list inter- vention. This is not surprising as psychoeducation is often a key element of many therapies and can be delivered with minimal therapeutic input. A few studies suggested psychoeducation involved minimal costs, but no studies provided detailed cost- analysis. For some families, the psychoeducation provided was sufficient support so that they did not require further input, thus potentially reducing waiting lists for targeted services. However, feedback on psychoeducation varied with some fami- lies feeling that the generic information provided in videos or books was not relevant to their specific family circumstances and therefore deemed less useful than more tailored information provided by healthcare practitioners. It is not possible from the few studies evaluated to assess this in detail, but future research could consider the impact of information dissemination via healthcare practitioners and other sources. Most interventions involved some element of face-to-face contact. Outcomes of interest f db k This may change following the surge in digital technology in response to the global pandemic and changes in healthcare; the very limited evidence in this review suggests that this may be useful for families on waiting lists and help with service availability across geographic locations20 as may the telephone.23 Outside of waiting list inter- vention, research has demonstrated that remotely delivered interventions may be both clinically and cost-effective, reducing the need for input from the small pool of highly trained specialist therapists.31 However, adverse events can occur, underlining the need for face-to-face support during the waitlist period or adequate remote monitoring of potential adverse events. Limitations h l The limitations of the underpinning research limit our ability to draw definitive conclusions through this review, particularly as most of the primary studies are small in nature, and feasibility rather than definitive trials. Several studies reported difficulties in terms of recruitment, this could have led to selection bias in that parents/children and young people who participated were more likely to believe that the intervention would work and consequently more likely to benefit.13 17 Some studies reported that recruitment was limited by the exploratory nature of the study.14 It may be possible that recruitment was difficult because families were concerned that they might restrict their chances of future intervention or lose their place on the waiting list, this issue needs to be explored further and be clear for future research and clinical practice.26 If waiting list interventions are to be imple- mented within services, there will need to be clear communica- tion around the role and purpose of the waiting list interventions to remove this barrier. We recommend that this communication is co-produced with patient and public involvement. Characteristics, nature and diversity of the interventions Characteristics, nature and diversity of the interventions Existing waiting list interventions largely fall within recom- mendations made in previous research, such as bibliotherapy and relaxation training,8 alongside coaching interventions for families living with ASD. The majority of papers in the review used psychoeducation approaches. Most waiting list interven- tions were brief (five or fewer sessions), indicating single-session interventions as a possible avenue for further exploration. Future research may consider whether a stepped-care approach, whereby psychoeducation is offered to all children and young people and/or families on waiting lists, reduces waiting times and subsequent length of treatment. Most waiting list interven- tions were aimed at the parent, future child-led research may be beneficial, particularly for conditions such as eating disorders and transgender health support which tended to include older adolescents. copyright. on October 23, 2024 by guest. Protected by http://mentalhealth.bmj.com/ irst published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from copyright. on October 23, 2024 by guest. Protected by http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from copyright. on October 23, 2024 by gue http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from Sample size was a common issue and recruitment problems were frequently reported as barriers to engagement. Nevertheless, these papers indicate the potential to use the waiting list period to offer an active intervention. Sample size was a common issue and recruitment problems were frequently reported as barriers to engagement. Nevertheless, these papers indicate the potential to use the waiting list period to offer an active intervention. to be a growing research focus in this area. For example, the Online Parent Training for the Initial Management of ADHD referral trial is still ongoing, however, the findings will be critical to inform on the clinical and cost-effectiveness, as well as accept- ability and feasibility, of using an online approach as a waiting list initiative.32 Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 Evidence base This review found limited evidence on waiting list interventions; only 18 papers were identified, and the quality of study design was often weak/moderate and often exploratory in nature. of life and parent self-efficacy,17 as well as a reduction in the proportion of families with unhealthy family functioning.18 Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 4 Open access copyright. on October 23 http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from 7 Punton G, Dodd AL, McNeill A. ’You’re on the waiting list’: an interpretive phenomenological analysis of young adults’ experiences of waiting lists within mental health services in the UK. PLoS One 2022;17:e0265542. Charlotte Sanderson, Florence Day, Nikki Chapman, Jen Martin and Bethan Davies in conducting this research. 7 Punton G, Dodd AL, McNeill A. ’You’re on the waiting list’: an interpretive phenomenological analysis of young adults’ experiences of waiting lists within mental health services in the UK. PLoS One 2022;17:e0265542. Contributors CLH and AZV designed the study and protocol on which KS provided feedback. AZV coordinated the day-to-day project management and wrote the manuscript draft under the supervision of CLH. SSH completed the second reviewing of data. CLH is the guarantor, accepts full responsibility for the work and conduct of the study, had full access to the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors contributed to and have approved the final manuscript. 8 Ní Shiothcháin A, Michael M. Waiting list management and initiatives. Irish Psychologist 2009;35. 8 Ní Shiothcháin A, Michael M. Waiting list management and initiatives. Irish Psychologist 2009;35. y g 9 Thomas KA, Schroder AM, Rickwood DJ. A systematic review of current approaches to managing demand and waitlists for mental health services. MHRJ 2021;26:1–17. 10 Centre for Reviews and Dissemination. Systematic reviews: CRD’s guidance for undertaking reviews in health care; 2009. Available: www.york.ac.uk/media/crd/ Systematic_Reviews.pdf Funding This research was funded by the Emerging Minds Network. Emerging Minds is a UK Research & Innovation-funded research network which aims to facilitate research which has the potential to reduce the prevalence of mental health problems experienced by children and young people. 11 Haddaway N, Grainger MJ, Gray CT. Citationchaser: an R package and shiny App for forward and backward citations chasing in academic searching. 2021. 12 Page MJ, McKenzie JE, Bossuyt PM, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ 2021;372:71. 13 Johnston T, Titman P. A health visitor-led service for children with behavioural problems. Community Pract 2004;77:90–4. Disclaimer The funders had no role in the study design, collection, analysis or interpretation of the data, writing the manuscript or the decision to submit the paper for publication. 14 Bernie C, Williams K, Graham F, et al. copyright. on October 23 http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from Coaching while waiting for autism spectrum disorder assessment: a pilot feasibility study for a randomized controlled trial on occupational performance coaching and service navigation. J Autism Dev Disord 2023;53:2905–14. Competing interests CLH acknowledges the support of the NIHR Nottingham Biomedical Research Centre and NIHR MindTech MedTech Co-operative and the support of the UK Research and Innovation (UKRI) Digital Youth Programme award (Medical Research Council project reference MR/W002450/1), which is part of the AHRC/ESRC/MRC Adolescence, Mental Health and the Developing Mind programme. 15 Ching BC, Bennett SD, Morant N, et al. Growth mindset in young people awaiting treatment in a paediatric mental health service: a mixed methods pilot of a digital single-session intervention. Clin Child Psychol Psychiatry 2023;28:637–53. Patient consent for publication Not applicable. Patient consent for publication Not applicable. 16 Connolly M, Gersch I. A support group for parents of children on a waiting list for an assessment for autism spectrum disorder. Educational Psychology in Practice 2013;29:293–308. Ethics approval Not applicable. Ethics approval Not applicable. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. 17 Couturier J, Sami S, Nicula M, et al. Examining the feasibility of a parental self-help intervention for families awaiting pediatric eating disorder services. Int J Eat Disord 2023;56:276–81. Data availability statement Data created during this research are openly available from the University of Nottingham data repository at https://doi.org/10. 17639/nott.7375. Data availability statement Data created during this research are openly available from the University of Nottingham data repository at https://doi.org/10. 17639/nott.7375. 18 Dahlgren Allen S, Tollit MA, McDougall R, et al. A waitlist intervention for transgender young people and psychosocial outcomes. Pediatrics 2021;148. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. 19 Eade DM, Telfer MM, Tollit MA. Implementing a single-session nurse-led assessment clinic into a gender service. CONCLUSIONS AND CLINICAL IMPLICATIONS This review explored and summarised the available peer- reviewed evidence base for interventions for children and young people on waiting lists for mental health services. It was found that waiting list interventions are being used in some mental health services internationally to help support young people. The limited available evidence indicates some promise of clinical effectiveness and positive user impact for waiting list interven- tions, there was limited evidence of service efficiencies. In the present review, we have found limited published evidence about user impact (acceptability and feasibility), which has largely focused on the views of parents and clinicians. There needs to be more pragmatic trials and/or service evaluations to evaluate the real-world user impact particularly taking into consideration the views of children and young people. Future research should evaluate waiting list interventions using RCTs and/or gather real- world data on benefits to patients and service efficiencies. on October 23, 2024 by guest. Protected by alth.bmj.com/ ober 23, 2024 by guest. Protected by Twitter Sophie S Hall @Hall_S_Sophie The purpose of this paper was to look at the existing evidence for waiting list interventions in CAMHS. As a result, we did not include study protocols. However, given the increasing pressure on CAMHS resources and the need to support families while they are waiting for assessment and treatment, there is likely Acknowledgements We would like to thank the children, young people and their families who provided study feedback through Young Minds and the NHS staff who supported the research. We would like to acknowledge the support of Beverley Brown, Roanna Coles, Caitlin McKenzie, Boliang Guo, Chris Hollis, Valentine AZ, et al. BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844 5 copyright. on October 23, 2024 by guest. Protected http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from ORCID iDs Althea Z Valentine http://orcid.org/0000-0003-2129-9278 Sophie S Hall http://orcid.org/0000-0002-9847-8269 Kapil Sayal http://orcid.org/0000-0002-2050-4316 Charlotte L Hall http://orcid.org/0000-0002-5412-6165 Althea Z Valentine http://orcid.org/0000-0003-2129-9278 Sophie S Hall http://orcid.org/0000-0002-9847-8269 Kapil Sayal http://orcid.org/0000-0002-2050-4316 Charlotte L Hall http://orcid.org/0000-0002-5412-6165 Althea Z Valentine http://orcid.org/0000-0003-2129-9278 Sophie S Hall http://orcid.org/0000-0002-9847-8269 25 Terry J. Brief intervention: a pilot initiative in a child and adolescent mental health service. Mental Health Practice 2003;6:18–9. p p g Kapil Sayal http://orcid.org/0000-0002-2050-4316 26 Wade T, Byrne S, Fursland A, et al. Is guided self-help family-based treatment for parents of adolescents with anorexia nervosa on treatment Waitlists feasible? A pilot trial. Int J Eat Disord 2022;55:832–7. y g Charlotte L Hall http://orcid.org/0000-0002-5412-6165 27 Beard C, Beckham E, Solomon A, et al. A pilot feasibility open trial of an interpretation bias intervention for parents of anxious children. Cogn Behav Pract 2022;29:860–73. copyright. on October 23 http://mentalhealth.bmj.com/ BMJ Ment Health: first published as 10.1136/bmjment-2023-300844 on 1 February 2024. Downloaded from Transgend Health 2018;3:43–6. 20 Kunze MG, Machalicek W, Wei Q, et al. Coaching via telehealth: caregiver-mediated interventions for young children on the waitlist for an autism diagnosis using single- case design. J Clin Med 2021;10:1654. g 21 Loucas CE, Sclare I, Stahl D, et al. Feasibility randomized controlled trial of a one-day CBT workshop (’DISCOVER’) for 15- to 18-year-olds with anxiety and/or depression in clinic settings. Behav Cogn Psychother 2020;48:142–59. g g y 22 McGarry J, McNicholas F, Buckley H, et al. The clinical effectiveness of a brief consultation and advisory approach compared to treatment as usual in child and adolescent mental health services. Clin Child Psychol Psychiatry 2008;13:365–76. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. y y y 23 Rivard M, Morin M, Mercier C, et al. Social validity of a training and coaching program for parents of children with autism spectrum disorder on a waiting list for early behavioral intervention. J Child Fam Stud 2017;26:877–87. 24 Spettigue W, Maras D, Obeid N, et al. 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BMJ Ment Health 2024;27:1–6. doi:10.1136/bmjment-2023-300844
https://openalex.org/W4205218821	https://jcope.uobaghdad.edu.iq/index.php/jcope/article/download/1218/1050	Arabic	null	The Effect of Specific Regions Exercises on Performance Endurance Development of Youth Soccer Players	Al-tarbiyaẗ al-riyādiyyaẗ/Al-tarbiyaẗ al-riyādiyyaẗ	2,021	cc-by	4,009	( 1 ) طالب دراسات عليا (الماجستير ،) .جامعة بغداد، كلية التربية البدنية وعلوم الرياضة( karraribrahim95@gmail.com ) Karrar Ibrahim Bader, Post Graduate Student (Master), University of Baghdad, College of Physical Education and Sport Sciences, (karraribrahim95@gmail.com) (+9647712395261). ( 2 ) أستاذ مساعد ، دكتوراه تربية رياضية، جامعة بغداد، كلية التربية البدنية وعلوم الرياضة( mohammedthrowing1@gmail.com ). Muhammad Jasim Othman, Assist Prof (PH.D), University of Baghdad, College of Physical Education and Sport Sciences, (mohammedthrowing1@gmail.com) (+9647709665063). ال مستخلص ال مستخلص تعد كرة القدم واحدة من االلعاب التي تتطلب التحمل والسرعة في االداء وقد هدفت الدراسة الى أعداد تمرينات المناطق المحددة لتطوير تحمل االداء بكرة القدم لالعبين الشباب ، اذ تم تطبيق الدراسة على عينه ( يمثلون شباب نادي القوة الجوية الرياضي في بغداد للموسم2020 / 2021 () وبأعمار17 - 19 ) سنة وعددهم ( 20 ( ) العبا قسموا الى مجموعتين تجريبية وضابطة من خالل اعداد تمرينات المناطق المحددة لمدة8 ) اسابيع يحتوي على تمارين تطوير قدرة تحمل االداء لالعبين الشباب بكرة القدم ، بعد ذلك استخدم الوسائل االحصائية التي تناسب البحث ثم عرض وم ناقشة النتائج وقد اعطى الباحث اهم االستنتاجات التي تناسب هذا الدراسة ومن خالل استنتاجات البحث تبين ان التمرينات التي وضعها الباحث قد أثرت بشكل ايجابي على نتائج االختبارات . البعدية ولصالح المجموعة التجريبية اا :الكلمات المفتاحية االجهزة واالدوات المساعد ة، التحمل الخاص، فئة الشباب، كرة القدم، تدريب المناطق .المحددة مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . ان أسللللللوب اللعلللللب الحلللللديث فلللللي لعبلللللة كلللللرة القلللللدم لللللله مميلللللزات عديلللللدة منهلللللا السلللللرعة فلللللي األداء ونقلللللل الكلللللرات وقلللللد جلللللاء ذللللللك نتيجلللللة التركيلللللز عللللللى الجوانلللللب البدنيلللللة والمهاريلللللة التلللللي ملللللن شلللللأنها تطلللللوير مسلللللتو االداء واصلللللبح أداء المهلللللا رات يتميلللللز بالتحملللللل والقلللللوة والسلللللرع ة ، اذ يعلللللد التحملللللل الخلللللاص والمتمثلللللب بتحملللللل االداء ملللللن القلللللدرات المهملللللة فلللللي لعبللللة كلللللرة القلللللدم اذ تأخلللللذ حيلللللزا كبيلللللرا فلللللي الوحلللللدات التدريبيللللة وخاصلللللة فلللللي فتلللللرة االعلللللداد الخلللللاص وتتمثلللللل أهميلللللة هلللللذ ه القللللدرات مللللن خللللالل الحركللللة المسللللتمرة فللللي المبللللاراة و مواجهللللة متغيللللرات اللعللللب بك للللاءة عاليللللة دون الهبللللوط فللللي المسللللتو واداء ا لعديللللد مللللن الواجبللللات داخللللل ا لمبللللاراة . اذ ان اسللللتخدام تمرينللللات المنللللاطق المحللللددة فهللللي تعللللد واحللللدة مللللن األسللللاليب التدريبيلللللة الحديثلللللة القليللللللة االسلللللتخدام فلللللي ال ئلللللات العمريلللللة فلللللي العلللللراق لملللللا لهلللللا تلللللاثير أيجلللللابي فلللللي اكتسلللللاب المهلللللارات األساسللللية واتقانهللللا وتطللللوير القللللدرات البدنيللللة وال مهاريللللة لالعبللللين والتلللللي تقللللوم علللللى أسللللا تمرينللللات مشللللابهة لمللللا يحلللللدث فللللللي المباريللللللات فضللللللال عللللللن ان هللللللذه التمرينللللللات تعللللللود الالعبللللللين فللللللي اكتسللللللاب العللللللادات الجيللللللدة وتزيللللللد التواصللللللل بللللللين الالعبين واتخاذ القرارات السريعة في الوقت المناسب وتحسين نوعية المناولة في المساحات الصغيرة. ير ي و ين و ي ب و ي و ير رر ابين و مللللللن خللللللالل اطللللللالل الباحللللللث ميللللللدانيا علللللللى تمرينللللللات و مباريللللللات فللللللرق ال ئللللللات العمريللللللة فللللللي بغللللللداد وااللتقللللللاء بلللللبعض الملللللدربين واإلطلللللالل عللللللى الدراسلللللات التلللللي تخلللللص كلللللرة القلللللدم فقلللللد وجلللللد هنلللللاك ضلللللعل فلللللي التحملللللل الخلللللاص والتللللي للللله أهميللللة كبيللللرة فللللي لعبللللة كللللرة القللللدم وكللللذلك الللللبط ء فللللي أداء المهللللارات والتللللي تعللللد مللللن اساسلللليات كللللرة القللللدم ، اذ يعلللللزو الباحلللللث سللللللبب ذلللللل ك ان اغلللللللب األنديلللللة تسللللللتخدم تمرينلللللات المنللللللاطق المحلللللددة باألسلللللللوب التقليلللللدي المعللللللرو ل واللعللللب فريللللق ضللللد فريللللق علللللى الملعللللب بالكامللللل فللللي نهايللللة الوحللللدة التدريبيللللة او تسللللتخدم لتكملللللة االحمللللاء والتسللللتخدمها لتطلللللوير النلللللواحي البدنيلللللة كأسللللللوب أساسلللللي لتطلللللوير مسلللللتويات الالعبلللللين ،للللللذا ارتلللللأ الباحلللللث أ سلللللتخدام أسللللللوب جديلللللد في تدريب المناطق المحددة بأضافة أجهزة وأدوات تدريبية في التمرينات .لتطوير تحمل األداء أ ،ومللللللن الدراسلللللللات التلللللللي تناوللللللللت موضللللللول الدراسلللللللة،دراسلللللللة اثيلللللللر عصللللللام عبلللللللد اللللللللرزاق (اثيلللللللر2019 ) تلللللللاثير التمرينلللللات التنافسلللللية المصلللللغرة فلللللي تطلللللوير بعلللللض أنلللللوال السلللللرل لالعبلللللي انديلللللة ا لدرجلللللة الممتلللللازة بكلللللرة القلللللدم ، اسلللللتخدم الباحلللللث الملللللنهج التجريبلللللي ب( تصلللللميم المجملللللوعتين المتكلللللافئتين وهملللللا الضلللللابطة والتجريبيلللللة عللللللى عينللللله مكونللللله ملللللن25 ) العبلللللا وهلللللم نلللللادي بغلللللداد بكلللللرة القلللللدم، اهلللللم النتلللللائج التلللللي وصلللللل اليهلللللا هلللللي وجلللللود فلللللروق معنويلللللة ذات داللللللله حصلللللائية لصللللالح االختبلللللارات البعديلللل ة فلللللي (السلللللرعة الحركية،والسللللرعة االنتقاليلللللة) للمجموعللللة التجريبيلللللة . ABSTRACT The Effect of Specific Regions Exercises on Performance Endurance Development of Youth Soccer Players Soccer is considered on the sports that required endurance, speed of performance. The study aimed at designing exercises for developing performance endurance in youth soccer players. The researcher was applied on (20) youth soccer players rom Al Jawia sport club (2020 – 2021) aged (17 – 19) years old divided into two equal groups. The program was applied for (8) weeks that consisted of exercises for developing performance endurance in youth soccer players. The data was collected and treated using proper statistical operations to conclude that these exercises positively affect posttests. p Keywords: aiding tools and apparatuses, special endurance, youth, soccer, training. 116 مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . املللللا دراسللللة خللللللل جلللللالل ، بللللراهيم الشللللمري(خلل2017 ) تللللاثير تمرينللللات حيللللازة الكللللرة عللللللى وفللللق المنللللاطق المحللللددة فللللي بعللللض القللللدرات الحركيلللللة والمهلللللللارات األساسلللللللية بكلللللللرة القلللللللدم للناشلللللللئين ، اسلللللللتخدم الباحلللللللث الملللللللنهج التجريبلللللللي ذا تصلللللللميم المجموعلللللللة التجريبيلللللللة ( الواحللللدة ، واختللللار الباحللللث عينللللة البحللللث بالطريقللللة العمديلللللة مللللن الالعبللللين الناشللللئين لنللللادي ديللللالى والبللللال علللللددهم16 ) ( العبللللللاص مللللللن اصللللللل28 ، ) العبللللللاص اهللللللم النتللللللائج التللللللي وصللللللل هللللللي وجللللللود فللللللروق معنويللللللة بللللللين نتللللللائج االختبللللللار القبللللللل ي والبعلللللدي ولصلللللالح البعلللللدي لعينلللللة البحلللللث فلللللي كافلللللة اختبلللللارات المهلللللارات األساسلللللية التلللللي تناولهلللللا البحلللللث. دراسلللللة محملللللد ،جاسلللللم محملللللد (محملللللد2017 ) تلللللاثير تمرينلللللات بدنيةلللللللللل مهاريلللللة خاصلللللة داخلللللل مسلللللاحات محلللللددة لتطلللللوير تحملللللل السلللللرعة ، لالعبلللللين الشلللللباب بكلللللرة القلللللدم اسلللللتخدم الباحلللللث الملللللنهج التجريبلللللي ب تصلللللميم المجملللللوعتين المتكلللللافئتين وهملللللا الضلللللابطة ( والتجريبيلللللة عللللللى عينللللله مكونللللله ملللللن25 ( ) العبلللللا وهلللللم شلللللباب نلللللادي الكهربلللللاء بكلللللرة القلللللدم للموسلللللم الرياضلللللي2016 - 2017 )، اهلللللم النتلللللائج التلللللي وصلللللل اليهلللللا هلللللي تلللللاثير معنلللللوي فلللللي متغيلللللر تحملللللل السلللللرعة بلللللين المجملللللوعتين الضلللللابطة والتجريبية ولصالح المجموع ة التجريبية في االختبارات البعدية. ومللللللن هنللللللا تكمللللللن أهميللللللة البحللللللث العلميللللللة مللللللن خللللللالل اسللللللتخدام تمرينللللللات جديللللللدة، ومنهللللللا تمرينللللللات المنللللللاطق ومللللللن هنللللللا تكمللللللن أهميللللللة البحللللللث العلميللللللة مللللللن خللللللالل اسللللللتخدام تمرينللللللات جديللللللدة، ومنهللللللا تمرينللللللات المنللللللاطق المحلللللللددة باسلللللللتخدام أجهلللللللزة وأدوات تدريبيلللللللة فلللللللي التحملللللللل الخلللللللاص وسلللللللرعة أداء بعلللللللض المهلللللللارات األساسلللللللية لالعبلللللللين الشلللللباب بكلللللرة القللللللدم ملللللن خلللللالل التللللللدرب عللللللى م واقلللللل مشللللللابهة لمواقلللللل اللعلللللب واالسللللللت ادة م نهلللللا فلللللي المباريللللللات . و ان 117 مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . الغلللللرض ملللللن هلللللذه البحلللللث هلللللو اعلللللداد تمرينلللللات المنلللللاطق المحلللللددة لالعبلللللين الشلللللباب بكلللللرة القلللللدم فضلللللال علللللن التعلللللرل تاثير. تمرينات المناطق المحددة في تطوير متغير تحمل األداء الطريقة واألدوات: (ميلللثم ، 2018،ص53 )  الهدل من االختبار: قيا قدرة تحمل االداء.لد العبي كرة القدم الشباب  :توصللللليل االختبلللللار يبلللللدأ االختبلللللار ملللللن المربلللللع رقلللللم( 1 ) عنلللللد سلللللمال الصلللللافرة يقلللللوم الالعلللللب بتنطللللليط الكلللللرة( 35 ) مللللرة وبعللللد اكمللللال ذلللللك يقللللوم بالدحرجللللة خللللار المربللللع لمسللللافة( 10 ) تللللر م ليلعللللب الكللللرة مناولللللة ارضللللية الللللى الزميللللل فلللللي المربلللللع المقابلللللل لللللله ليقلللللوم بعلللللدها بلللللالركض جانبلللللا لمسلللللافة ( 10 ) تلللللر م لينتقلللللل بعلللللدها للقيلللللام بلللللالركض بلللللين ( 4 ) شلللللواخص المسللللللافة بلللللين قمللللللع واخلللللر ( 3 ) متللللللر وحلللللين االنتهللللللاء مللللللن ذللللللك يعللللللود ليتسللللللم الكللللللرة ملللللن زميللللللله ويقللللللوم بالللللدوران ولعللللب الكللللرة وتغييللللر اتجللللاه اللعللللب بمناولللللة عاليللللة طويلللللة فللللي الجانللللب االخللللر ليللللركض بعللللدها ليقللللوم بللللالق ز بكلتلللللللا القلللللللدمين ملللللللن فلللللللوق ( 5 ) شلللللللواخص المسلللللللافة بلللللللين شلللللللاخص واخلللللللر( 1 )تلللللللر م ويلللللللركض لمهاجملللللللة الكلللللللرات المرفوعللللة مللللن كللللل جانللللب بللللالرأ مللللن خللللالل اقمللللال موجللللودة مقابللللل كللللل عمللللود للمرمللللى وبمسللللافة مشللللابهة لحللللدود ،منطقلللللة الجلللللزاء وع نلللللدما يكملللللل ذللللللك يعلللللود مسلللللرعا ليقلللللوم بدحرجلللللة الكلللللرة بلللللين( 5 ) شلللللواخص المسلللللافة بلللللين كلللللل شللللللاخص( 1.5 ) تللللللر م فيلعللللللب مناولللللللة جداريللللللة للمللللللدرب ليعللللللود للعللللللب الكللللللرة فللللللي ن لللللل اتجللللللاه اللعللللللب الللللللى الزميللللللل الموجللللود فللللي المربللللع المواجلللله للللله ليتحللللول بعللللدها الللللى تسللللديد الكللللرات الخملللل الموجللللودة علللللى حافللللة منطقللللة الجللللزا ء :االختباااار المساااتخدم فاااي البحاااث اختباااار مياااثم داعاااي الحاااق لتحمااال االداء لااادى العباااي كااارة القااادم الشاااباب . الطريقة واألدوات: اسللللللللتخدم الباحثلللللللل ان المللللللللنهج التجريبللللللللي لمالئمتلللللللله طبيعللللللللة المشللللللللكلة المللللللللراد حلهللللللللا لتحقيللللللللق اهللللللللدال البحللللللللث وفروضلللله، و تلللللم تصلللللميم المجملللللوعتين المتكلللللافئتين وهمللللل ا الضلللللابطة والتجريبيللللة ، تكلللللون مجتملللللع البحلللللث ملللللن العبلللللي انديلللللة ( بغللللللداد المشللللللاركين فللللللي دوري الشللللللباب بكللللللرة القللللللدم بأعمللللللار17 - 19) سللللللنة وال مسللللللجل ين رسللللللميا فللللللي سللللللجالت ا التحللللللاد ( العراقللللللي المركللللللزي لكللللللرة القللللللدم للموسللللللم2020 - 2021 ) ( والبللللللال عللللللددهم10 ( ) انديللللللة مجمللللللول العبيهللللللا250 ) العبللللللاص ، وتللللم اختيللللار عينللللة البحللللث عشللللوائياص باسللللتخدام طريقللللة القرعللللة لتحد يللللد النللللادي الللللذي يمثللللل عينللللة البحللللث ، و اسلللل رت نتللللائج القرعللللة علللللن تمثيلللللل شلللللباب نلللللادي القللللوة الجويلللللة كعينلللللة للبحلللللث اذ ( بلللللل عللللدد عينلللللة البحلللللث26 ( ) العبلللللاص تلللللم اسلللللتبعاد6 ) العبللللين ملللللنهم( ، 4) العبلللللين للتجربلللللة االسلللللتطالعية و( 2 ) العبلللللين حللللرا المرملللللى وبلللللذلك ( بلللللل علللللدد العينلللللة20 ) العبلللللاص ( وتللللللم تقسلللللليمهم بالتسللللللاوي لمجمللللللوعتين تجريبيللللللة وضللللللابطة لكللللللل مجموعللللللة10 ) العبللللللين، اذ تبللللللل نسللللللبة عينللللللة البحللللللث ( 10 )% .من المجتمع الكلي ( الجدول1 ) ( يبين الوسط الحسابي واالنحرال المعياري لالختبارات القبلية وقيمةt ) المحسوبة وداللتها للمجموعتين )الضابطة والتجريبية (التكافؤ االختبار المجموعة التجريبية المجموعة الضابطة T المحسوبة مستو الخطأ الداللة ±ل ±ل اختبار ميثم داعي الحق لتحمل األداء لد العبي كرة القدم الشباب 1.1610 .01663 1.1630 .01889 -.251 .804 غير معنوي معنوي< ( 0.05 ( ) عند درجة حرية18 ( ) وتحت مستوى داللة0.05 ) :االختباااار المساااتخدم فاااي البحاااث اختباااار مياااثم داعاااي الحاااق لتحمااال االداء لااادى العباااي كااارة القااادم الشاااباب . (ميلللثم ، 2018،ص53 ) ااا :االختباااار المساااتخدم فاااي البحاااث اختباااار مياااثم داعاااي الحاااق لتحمااال االداء لااادى العباااي كااارة القااادم الشاااباب . الطريقة واألدوات: (ميلللثم ، 2018،ص53 )  الهدل من االختبار: قيا قدرة تحمل االداء.لد العبي كرة القدم الشباب  :توصللللليل االختبلللللار يبلللللدأ االختبلللللار ملللللن المربلللللع رقلللللم( 1 ) عنلللللد سلللللمال الصلللللافرة يقلللللوم الالعلللللب بتنطللللليط الكلللللرة( 35 ) مللللرة وبعللللد اكمللللال ذلللللك يقللللوم بالدحرجللللة خللللار المربللللع لمسللللافة( 10 ) تللللر م ليلعللللب الكللللرة مناولللللة ارضللللية الللللى الزميللللل فلللللي المربلللللع المقابلللللل لللللله ليقلللللوم بعلللللدها بلللللالركض جانبلللللا لمسلللللافة ( 10 ) تلللللر م لينتقلللللل بعلللللدها للقيلللللام بلللللالركض بلللللين ( 4 ) شلللللواخص المسللللللافة بلللللين قمللللللع واخلللللر ( 3 ) متللللللر وحلللللين االنتهللللللاء مللللللن ذللللللك يعللللللود ليتسللللللم الكللللللرة ملللللن زميللللللله ويقللللللوم بالللللدوران ولعللللب الكللللرة وتغييللللر اتجللللاه اللعللللب بمناولللللة عاليللللة طويلللللة فللللي الجانللللب االخللللر ليللللركض بعللللدها ليقللللوم بللللالق ز بكلتلللللللا القلللللللدمين ملللللللن فلللللللوق ( 5 ) شلللللللواخص المسلللللللافة بلللللللين شلللللللاخص واخلللللللر( 1 )تلللللللر م ويلللللللركض لمهاجملللللللة الكلللللللرات المرفوعللللة مللللن كللللل جانللللب بللللالرأ مللللن خللللالل اقمللللال موجللللودة مقابللللل كللللل عمللللود للمرمللللى وبمسللللافة مشللللابهة لحللللدود ،منطقلللللة الجلللللزاء وع نلللللدما يكملللللل ذللللللك يعلللللود مسلللللرعا ليقلللللوم بدحرجلللللة الكلللللرة بلللللين( 5 ) شلللللواخص المسلللللافة بلللللين كلللللل شللللللاخص( 1.5 ) تللللللر م فيلعللللللب مناولللللللة جداريللللللة للمللللللدرب ليعللللللود للعللللللب الكللللللرة فللللللي ن لللللل اتجللللللاه اللعللللللب الللللللى الزميللللللل الموجللللود فللللي المربللللع المواجلللله للللله ليتحللللول بعللللدها الللللى تسللللديد الكللللرات الخملللل الموجللللودة علللللى حافللللة منطقللللة الجللللزا ء 118 مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . بلللالعودة اللللى الشللللاخص الموجلللود للخلللللل عنلللد تسللللديد كلللل كللللرة وعنلللد اكمللللال عمليلللة التسللللديد يعلللود مسللللرعا اللللى حيللللث .وجود الخط باللون االص ر حيث عندها ينتهي االختبار  :مالحظلللللة:يلللللؤدي الالعلللللب االختبلللللار فلللللي جلللللو لعلللللب كلللللرة قلللللدم مثلللللل علللللادة التنطللللليط للكلللللرة عنلللللد سلللللقوطها للوصلللللول اللللللى علللللدد الملللللرات المطل وبلللللة، و محاوللللللة تسلللللجيل االهلللللدال بلللللالرا والقلللللدم قلللللدر االمكلللللان او عللللللى االقلللللل توجيههلللللا .على المرمى لعب الكرة والمناولة بإتقان، و.يراعي تن يذ االداء بأعلى سرعة ممكنة اا ما .على المرمى لعب الكرة والمناولة بإتقان، و.يراعي تن يذ االداء بأعلى سرعة ممكنة  التسجيل: تعطى محاولة واحدة لكل مختبر ويحسب زمن االداء الكلي بالثواني واجزاؤه ( الشكل1 ) يوضح اختبار قدرة تحمل االداء م اتيح الحركات: حركة الكرة حركة الالعب مع الكرة التهديل الق ز بين الشواخص حركة الالعب بدون كرة ا  التسجيل: تعطى محاولة واحدة لكل مختبر ويحسب زمن االداء الكلي بالثواني واجزاؤه ( الشكل1 ) يوضح اختبار قدرة تحمل االداء ( الشكل1 ) يوضح اختبار قدرة تحمل االداء م اتيح الحركات: حركة الكرة حركة الالعب مع الكرة التهديل الق ز بين الشواخص حركة الالعب بدون كرة 119 119 مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . مجلة التربية الرياضية ( المجلد33 ) ( العدد4 ) سنة1 202 . و بعللللللللد الوقللللللللول علللللللللى جميللللللللع المتغيللللللللرات والظللللللللرول الخاصللللللللة بالبحللللللللث ومللللللللن اجللللللللل تحقيللللللللق البعديللللللللة علللللللللى المجموعلللللة التجريبيلللللة والضلللللابطة والتمرينلللللات المسلللللتخدمة عللللللى المجموعلللللة التجريبيلللللة ، قلللللام الباحلللللث بلللللإجراء االختبلللللارات القبليلللللللللة لعينلللللللللة البحلللللللللث الضلللللللللابطة والتجريبيلللللللللة عللللللللللى ملللللللللدار يلللللللللومي (الثالثلللللللللاء واالربعلللللللللاء ( ) الموافلللللللللق1 - 2 / 12 / 2020 .) السللللللاعة العاشللللللرة صللللللباحاص علللللللى ملعللللللب القللللللوة الجويللللللة الثللللللاني لكللللللرة القللللللدم تبللللللع ذلللللللك جللللللراء التجربللللللة الرئ يسللللللة للبحلللللث ، اذ قلللللام الباحثللللل ان بإ( علللللداد مجموعلللللة ملللللن التمرينللللللات والبلللللال علللللددها16) تملللللرين فلللللي المنلللللاطق المحلللللددة ، و تللللللم ( تطبيللللللق التمرينللللللات بواقللللللع3 ( ) وحللللللدات تدريبيللللللة فللللللي األسللللللبول و لمللللللدة8 ) أسللللللابيع وكللللللان مجمللللللول الوحللللللدات التدريبيللللللة ( 24) وحللللدة تدريبيلللللة و كانللللت أيلللللام الوحللللدات التدريبيلللللة هللللي (ا الحلللللد- الثالثللللاء- االربعلللللاء) فللللي فتلللللرة االعللللداد الخلللللاص و ( يتللللللرواا زملللللللن الوحلللللللدة التدريبيللللللة المخصصلللللللة للتمرينلللللللات35 - 45 ) دقيقللللللة الجلللللللزء اللللللللرئي (للجانللللللب البلللللللدني) لشلللللللباب ( نادي القوة الجوية لل ترة من6 / 12 / 2020 ( ) ولغاية27 / 1 / 2021 . ) و( تراوحلللللللت الشلللللللدة الكليلللللللة للتمرينلللللللات المسلللللللتخدمة ملللللللابين75 للللللللل% 90 ) وذللللللللك بأسلللللللتخدام طريقلللللللة التلللللللدريب% ال تللللللللللري مرت للللللللللع الشللللللللللدة الللللللللللذي يحمللللللللللل صلللللللللل ة مشللللللللللابهة لحالللللللللللة اللعللللللللللب مللللللللللن حيللللللللللث الللللللللللركض والتوقللللللللللل . مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . (احمللللللللللد ،عبدالعزيز2008،ص86 ) و بعلللللللد االنتهلللللللاء ملللللللن تطبيلللللللق م لللللللردات الوحلللللللدات التدريبيلللللللة قلللللللام الباحثللللللل ان بلللللللإجراء االختبلللللللارات البعديلللللللة لعينلللللللة البحلللللللث ( الضلللللللابطة والتجريبيلللللللة فلللللللي يلللللللومي (االحلللللللد واالثنلللللللين) الموافلللللللق31 - 1 / 1 - 2 / 2021 ) السلللللللاعة العاشلللللللرة صلللللللباحاص عللللللللى .ملعب القوة الجوية الثاني لكرة القدم وقد اتبع الباحث شروط وا جراءات االختبارات القبلية ن سها :النتائج ال( جدول2 ) يبين األ وساط الحسابية واالنحرافات المعيارية باالختبارين القبلي والبعدي لمتغير تحمل األداء للمجموعة التجريبية االختبار االختبار القبلي االختبار البعدي ل ل هل قيمةt المحسوبة مستو الخطأ الداللة ±ل ±ل اختبار ميثم داعي الحق لتحمل األداء لدى العبي كرة القدم الشباب 1.1610 .01663 1.1320 .01549 .02900 .00180 16.155 .000 معنوي معنوي< ( 0.05 ( ) عند درجة حرية9 ( ) وتحت مستوى داللة0.05 .) ال( جدول3 ) يبين األوساط الحسابية واالنحرافات المعيارية باالختبارين القبلي والبعدي لمتغير تحمل االداء للمجموعة الضابطة االختبار االختبار القبلي االختبار البعدي ل ل هل قيمةt المحسوبة مستو الخطأ الداللة ±ل ±ل اختبار ميثم داعي الحق لتحمل األداء لدى العبي كرة القدم الشباب 1.1630 .01889 1.1510 .01853 .01200 .00327 3.674 .005 معنوي معنوي< ( 0.05 ( ) عند درجة حرية9 ( ) وتحت مستوى داللة0.05 .) :النتائج ال( جدول2 ) يبين األ وساط الحسابية واالنحرافات المعيارية باالختبارين القبلي والبعدي لمتغير تحمل األداء للمجموعة التجريبية االختبار االختبار القبلي االختبار البعدي ل ل هل قيمةt المحسوبة مستو الخطأ الداللة ±ل ±ل اختبار ميثم داعي الحق لتحمل األداء لدى العبي كرة القدم الشباب 1.1610 .01663 1.1320 .01549 .02900 .00180 16.155 .000 معنوي معنوي< ( 0.05 ( ) عند درجة حرية9 ( ) وتحت مستوى داللة0.05 .) ال( جدول3 ) يبين األوساط الحسابية واالنحرافات المعيارية باالختبارين القبلي والبعدي لمتغير تحمل االداء للمجموعة الضابطة االختبار االختبار القبلي االختبار البعدي ل ل هل قيمةt المحسوبة مستو الخطأ الداللة ±ل ±ل اختبار ميثم داعي الحق لتحمل األداء لدى العبي كرة القدم الشباب 1.1630 .01889 1.1510 .01853 .01200 .00327 3.674 .005 معنوي معنوي< ( 0 05 () عند درجة حرية9 () وتحت مستوى داللة0 05 ) معنوي< ( 0.05 ( ) عند درجة حرية9 ( ) وتحت مستوى داللة0.05 .) 120 مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . ال( جدول4 ) يبين األوساط الحسابية واالنحرافات المعيارية في االختبارات البعدية للمتغير تحمل االداء وللمجموعتين التجريبية والضابطة االختبار المجموعة التجريبية المجموعة الضابطة قيمةt المحسوبة مستو الخطأ الداللة ±ل ±ل اختبار ميثم داعي الحق لتحمل األداء لد العبي كرة القدم الشباب 1.1320 .01549 1.1510 .01853 -2.488 .023 معنوي معنوي< ( 0.05 ( ) عند درجة حرية18 ( ) وتحت مستوى داللة0.05 ) مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . مناقشة النتائج: مناقشة النتائج: ( أظهللللللرت نتللللللائج الجللللللدول2 ) معنويللللللة ال للللللروق بللللللين االختبللللللارين القبلللللللي والبعللللللدي ولصللللللالح االختبللللللار البعللللللدي لمتغيلللللر تحملللللل االداء للمجموعللللللة التجريبيلللللة .ويعللللللزو الباحثللللل ان أسللللللباب ذللللللك الللللللى اسلللللتخدام تمرينللللللات المنلللللاطق المحللللللددة للمجموعللللللة التجريبيللللللة وكانللللللت التمرينللللللات مدروسللللللة وفللللللق أسلللللل علميللللللة وضللللللوابط تدريبيللللللة حديثللللللة مللللللع مراعللللللاة قابليللللللات وامكانيلللللات ال العبلللللين مملللللا أد اللللللى ت اعلللللل الالعبلللللين ملللللع التمرينلللللات والتلللللدريب المسلللللتمر لملللللدة شلللللهرين اذ ملللللن المنطلللللق ان يحصلللللل تطلللللور للمجموعلللللة التجريبيلللللة وهلللللذا ملللللاظهر واضلللللحاص فلللللي متغيلللللر تحملللللل األداء اذ كانلللللت طبيعلللللة التمرينلللللات ذات مواقللللللل ومثيللللللرات مشللللللابهه لمللللللا يحللللللدث فللللللي المباريللللللات وفرضللللللت علللللللى الالعبللللللين اللعللللللب بسللللللرعة دون القصللللللوية ودون تلللللأخير الكلللللرة وحركلللللة مسلللللتمرة فلللللي الملعلللللب مملللللا أد اللللللى احلللللداث تكيلللللل للللللد الالعبلللللين وهلللللذا يت لللللق ملللللع (ياسلللللر ،هاشللللللم2010،ص13 ) " تحمللللللل األداء بصلللللل ة عامللللللة علللللللى انلللللله احللللللد الصلللللل ات البدنيللللللة المركبللللللة تعبللللللر عللللللن مكانيللللللة الالعلللللب عللللللى مقاوملللللة التعلللللب فلللللي اثنلللللاء األداء الحركلللللي او ا لمهلللللاري والتلللللي يعتملللللد عللللللى مسلللللتو شلللللدة العملللللل الملللللراد " تن يذة واالنجاز المطلوب في ان واحد مع ضرورة تن يذ األداء بأقل وقت ممكن و( أظهللللللرت نتللللللائج الجللللللدول3 ) معنويللللللة ال للللللروق بللللللين االختبللللللارين القبلللللللي والبعللللللدي ولصللللللالح االختبللللللار البعللللللدي لمتغيللللللر تحمللللللل االداء للمجموعللللللة ال ضللللللابطة ،ويللللللر البا ان ثلللللل ح ان التطللللللور الحاصللللللل فللللللي متغيللللللرات التحمللللللل الخللللللاص للمجموعللللللة الضلللللابطة يرجلللللع اللللللى الكثيلللللر ملللللن األسلللللباب أهمهلللللا تلللللدريبات ال ريلللللق كانلللللت مسلللللتمرة وللللللم تحلللللدث فتلللللرة انقطلللللال بسلللللبب االسلللللتعداد اللللللى دوري الشلللللباب والسلللللبب الثلللللاني يعلللللود اللللللى أسللللللوب الملللللدرب اللللللذي اعتلللللاد عليلللللة والخضلللللول اللللللى تلللللدريبات مختل للللللة عللللللن تللللللدريبات المجمو عللللللة التجريبيللللللة ، فضللللللال عللللللن تركيللللللز المللللللدرب علللللللى التللللللدريبات البدنيللللللة القويللللللة باسللللللتخدام مسلللللاحات واسلللللعة ملللللن الملعلللللب وهلللللذا ملللللايتالئم ملللللع اسلللللتمرار االداء ،ويرجلللللع السلللللبب ا خلللللر اللللللى االلتلللللزام فلللللي الحضلللللور للوحللللدات التدريبيللللة والمنافسللللة بللللين الالعبللللين للحصللللول علللللى مكلللللان أساسللللي فللللي التشللللكيلة وهللللذه األمللللور تللللرجح بال ائللللل دة لل ريق.ولكنها لم ترتقي للتطور الحاصل في المجموعة التجريبية وهذا ما اثبتته نتائج االختبارات و( ملللللللن خلللللللالل الجلللللللدول4 ) تظهلللللللر النتلللللللائج لالختبلللللللار البعلللللللدي أفضللللللللية ال لللللللروق بلللللللين المجملللللللوعتين التجريبيلللللللة وال ضللللابطة ولصلللللالح المجموعلللللة التجريبيلللللة ، ويعللللزو الباحثللللل ان أسلللللباب تللللللك ال لللللروق فللللي مت غيلللللر تحملللللل االداء اللللللى طبيعلللللة تمرينلللللات المنلللللاطق المحلللللددة بأسلللللتخدام أجهلللللزة وأدوات تدريبيلللللة التلللللي تعلللللد واحلللللدة ملللللن افضلللللل الوسلللللائل التدريبيلللللة الحديثلللللة التللللي سللللاهمت بشللللكل كبيللللر و كللللان لهللللا دور يجللللابي فللللي تطللللوير مسللللتو الالعبللللين مللللن الناحيللللة البدنيللللة والمهاريللللة مللللن خللللللالل المشللللللاركة ال عالللللللة لالعبللللللين اذ يتجلللللله التللللللدريب نحللللللو اللعللللللب الللللللواقعي مللللللن خللللللالل سللللللرعة اتخللللللاذ القللللللرارات وعللللللدد لمسللللات الكللللرة وتسللللمح لهللللم بحللللل المشللللاكل التللللي تللللواجههم فللللي المباريللللات والغللللرض منهللللا هللللو تكللللرار المواقللللل المسللللتخدمة فلللللي المبلللللاراة بلللللأكبر علللللدد ممكلللللن وتحتلللللوي عللللللى التنلللللول واالثلللللارة والتشلللللويق فلللللي تمريناتهلللللا لالبتعلللللاد علللللن ا للللللروتين اليلللللومي ( أظهللللللرت نتللللللائج الجللللللدول2 ) معنويللللللة ال للللللروق بللللللين االختبللللللارين القبلللللللي والبعللللللدي ولصللللللالح االختبللللللار البعللللللدي لمتغيلللللر تحملللللل االداء للمجموعللللللة التجريبيلللللة .ويعللللللزو الباحثللللل ان أسللللللباب ذللللللك الللللللى اسلللللتخدام تمرينللللللات المنلللللاطق المحللللللددة للمجموعللللللة التجريبيللللللة وكانللللللت التمرينللللللات مدروسللللللة وفللللللق أسلللللل علميللللللة وضللللللوابط تدريبيللللللة حديثللللللة مللللللع مراعللللللاة قابليللللللات وامكانيلللللات ال العبلللللين مملللللا أد اللللللى ت اعلللللل الالعبلللللين ملللللع التمرينلللللات والتلللللدريب المسلللللتمر لملللللدة شلللللهرين اذ ملللللن المنطلللللق ان يحصلللللل تطلللللور للمجموعلللللة التجريبيلللللة وهلللللذا ملللللاظهر واضلللللحاص فلللللي متغيلللللر تحملللللل األداء اذ كانلللللت طبيعلللللة التمرينلللللات ذات مواقللللللل ومثيللللللرات مشللللللابهه لمللللللا يحللللللدث فللللللي المباريللللللات وفرضللللللت علللللللى الالعبللللللين اللعللللللب بسللللللرعة دون القصللللللوية ودون تلللللأخير الكلللللرة وحركلللللة مسلللللتمرة فلللللي الملعلللللب مملللللا أد اللللللى احلللللداث تكيلللللل للللللد الالعبلللللين وهلللللذا يت لللللق ملللللع (ياسلللللر ،هاشللللللم2010،ص13 ) " تحمللللللل األداء بصلللللل ة عامللللللة علللللللى انلللللله احللللللد الصلللللل ات البدنيللللللة المركبللللللة تعبللللللر عللللللن مكانيللللللة الالعلللللب عللللللى مقاوملللللة التعلللللب فلللللي اثنلللللاء األداء الحركلللللي او ا لمهلللللاري والتلللللي يعتملللللد عللللللى مسلللللتو شلللللدة العملللللل الملللللراد " تن يذة واالنجاز المطلوب في ان واحد مع ضرورة تن يذ األداء بأقل وقت ممكن و( أظهللللللرت نتللللللائج الجللللللدول3 ) معنويللللللة ال للللللروق بللللللين االختبللللللارين القبلللللللي والبعللللللدي ولصللللللالح االختبللللللار البعللللللدي لمتغيللللللر تحمللللللل االداء للمجموعللللللة ال ضللللللابطة ،ويللللللر البا ان ثلللللل ح ان التطللللللور الحاصللللللل فللللللي متغيللللللرات التحمللللللل الخللللللاص للمجموعللللللة الضلللللابطة يرجلللللع اللللللى الكثيلللللر ملللللن األسلللللباب أهمهلللللا تلللللدريبات ال ريلللللق كانلللللت مسلللللتمرة وللللللم تحلللللدث فتلللللرة انقطلللللال بسلللللبب االسلللللتعداد اللللللى دوري الشلللللباب والسلللللبب الثلللللاني يعلللللود اللللللى أسللللللوب الملللللدرب اللللللذي اعتلللللاد عليلللللة والخضلللللول اللللللى تلللللدريبات مختل للللللة عللللللن تللللللدريبات المجمو عللللللة التجريبيللللللة ، فضللللللال عللللللن تركيللللللز المللللللدرب علللللللى التللللللدريبات البدنيللللللة القويللللللة باسللللللتخدام مسلللللاحات واسلللللعة ملللللن الملعلللللب وهلللللذا ملللللايتالئم ملللللع اسلللللتمرار االداء ،ويرجلللللع السلللللبب ا خلللللر اللللللى االلتلللللزام فلللللي الحضلللللور للوحللللدات التدريبيللللة والمنافسللللة بللللين الالعبللللين للحصللللول علللللى مكلللللان أساسللللي فللللي التشللللكيلة وهللللذه األمللللور تللللرجح بال ائللللل دة لل ريق.ولكنها لم ترتقي للتطور الحاصل في المجموعة التجريبية وهذا ما اثبتته نتائج االختبارات ا و( ملللللللن خلللللللالل الجلللللللدول4 ) تظهلللللللر النتلللللللائج لالختبلللللللار البعلللللللدي أفضللللللللية ال لللللللروق بلللللللين المجملللللللوعتين التجريبيلللللللة وال ضللللابطة ولصلللللالح المجموعلللللة التجريبيلللللة ، ويعللللزو الباحثللللل ان أسلللللباب تللللللك ال لللللروق فللللي مت غيلللللر تحملللللل االداء اللللللى طبيعلللللة تمرينلللللات المنلللللاطق المحلللللددة بأسلللللتخدام أجهلللللزة وأدوات تدريبيلللللة التلللللي تعلللللد واحلللللدة ملللللن افضلللللل الوسلللللائل التدريبيلللللة الحديثلللللة التللللي سللللاهمت بشللللكل كبيللللر و كللللان لهللللا دور يجللللابي فللللي تطللللوير مسللللتو الالعبللللين مللللن الناحيللللة البدنيللللة والمهاريللللة مللللن خللللللالل المشللللللاركة ال عالللللللة لالعبللللللين اذ يتجلللللله التللللللدريب نحللللللو اللعللللللب الللللللواقعي مللللللن خللللللالل سللللللرعة اتخللللللاذ القللللللرارات وعللللللدد لمسللللات الكللللرة وتسللللمح لهللللم بحللللل المشللللاكل التللللي تللللواجههم فللللي المباريللللات والغللللرض منهللللا هللللو تكللللرار المواقللللل المسللللتخدمة فلللللي المبلللللاراة بلللللأكبر علللللدد ممكلللللن وتحتلللللوي عللللللى التنلللللول واالثلللللارة والتشلللللويق فلللللي تمريناتهلللللا لالبتعلللللاد علللللن ا للللللروتين اليلللللومي 121 مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . مناقشة النتائج: فللللللي أداء التمرينللللللات المعتللللللادة وكسللللللر حللللللاجز الملللللللل للمجموعللللللة التجريبيللللللة ، اذ كانللللللت التمرينللللللات تحتللللللوي علللللللى تطللللللوير اكثلللللللر ملللللللن قلللللللدرة بدنيلللللللة واشلللللللتملت التمرينلللللللات عللللللللى لعلللللللب المنلللللللاوالت والدحرجلللللللة السلللللللريعة بلللللللالكرة والتحلللللللرك دون كلللللللرة والضلللللغط السلللللتعادة الكلللللرة وجلللللرت التلللللدريبات فلللللي ظلللللرول مشلللللاب ،هة للمباريلللللات ، وذكلللللر (موفلللللق مجيلللللد الملللللولى2014 ، ص19 ) " ان اللعللللللب فللللللي مسللللللاحات مصللللللغرة تعنللللللي مشللللللاركة اكبللللللر عللللللدد مللللللن الالعبللللللين باللعللللللب وهللللللو الهللللللدل واالهللللللم ويمللللنح الالعبللللين الخبللللرة االحسللللن مللللن خللللالل اللملللل األكثللللر للكللللرة فللللي اللعللللب فللللي لعللللب واقعللللي فيصللللبح الالعللللب شللللامل التكوين ومت هم لدور زمالئه"من الالعبين . االستنتاجات:  تشللللجيع المللللدربين علللللى اسللللتخدام المنللللاطق المحللللددة كأسلللللوب تللللدريبي لللللم تسللللتخدم فللللي ال ئللللات العمريللللة لمللللا لهللللا مللللن دور فعال و خلق بيئة مناسبة لتطوير الالعبين.  تقنين تمرينات المناطق المحددة واستخدامها في تدريب ال ئات العمرية كافة.  ضرورة االعتماد على المدربين األكا ديميين في تدريب ال ئات العمرية.  أجراء دراسات مشابهة بأستخدام المناطق ال محددة لل ئات العمرية والمتقدمين. المصادر اثيلللللللر عصلللللللام عبلللللللد اللللللللرزاق( 2019 .) تلللللللاثير التمرينلللللللات التنافسلللللللية المصلللللللغرة فلللللللي تطلللللللوير بعلللللللض أنلللللللوال السلللللللرل لالعبلللللي انديلللللة الدرجلللللة الممتلللللازة بكلللللرة ال قلللللدم ، بحلللللث منشلللللور فلللللي مجللللللة ،كليلللللة التربيلللللة الب دنيلللللة وعللللللوم الرياضلللللة/جامعة بغداد. احملللللد عبلللللد العزيلللللز فلللللر( 2008 .) اسلللللتخدام طريقلللللة التلللللدريب ال تلللللري مرت لللللع الشلللللدة فلللللي تطلللللوير التحملللللل الخلللللاص وأثرهللللا فللللي دقللللة اداء بعللللض المهللللارات االساسللللية لالعبللللي كلللللرة القللللدم الشللللباب ، رسللللالة ماجسللللتير ، كليللللة التر بيللللة البدنيلللللة وعلوم ال رياضة ، جامعة بغداد. خللللللل جلللللالل بلللللراهيم الشلللللمري ( 2017 .) تلللللاثير تمرينلللللات حيلللللازة الكلللللرة عللللللى وفلللللق المنلللللاطق المحلللللددة فلللللي بعلللللض القللللللللدرات الحركيللللللللة والمهللللللللارات األساسللللللللية بكللللللللرة القللللللللدم للناشللللللللئين ، رسللللللللالة ماجسللللللللتير، كليللللللللة التربيللللللللة البدن يللللللللة وعلللللللللوم الرياضة/جامعة ديالى. 122 مجلة التربية الرياضية– ( المجلد33 ) – ( العدد4 ) – سنة1 202 . 123 التمرينات المستخدمة في البحث ( التمرين رقم1 ) اسم التمرين : تحمل اداء ( شللللرا التمللللرين: يللللتم التمللللرين علللللى شللللكل مربللللع30 x 35 ) م يحللللدد التمللللرين بواسللللطة شللللواخص يبللللدأ التمللللرين بلللللللالوقول علللللللللى شللللللللكل صلللللللل ين خللللللللار المربللللللللع مجموعللللللللة ضللللللللد مجموعللللللللة وسللللللللحب الالعللللللللب األول بواسللللللللطة الحبللللللللال ( المطاطيلللللة ملللللن قبلللللل الزميلللللل ويعملللللل رفلللللع الركبلللللة لملللللدة5 )ثلللللا بعلللللدها يصللللل ر الملللللدرب لينطللللللق بعلللللدها واللللللدخول للمربلللللع واللعلللللب1 ضلللللد1 ويسلللللتمر التملللللرين لحلللللين اكتملللللال العلللللدد ويصلللللبح اللعلللللب5 ضلللللد5 والتاكيلللللد عللللللى التحلللللرك والمنلللللاوالت . بين الالعبين اسم التمرين الشدة زمن االداء الراحة البينية التكرار المجاميع الراحة بين المجاميع الزمن الكلي تحمل االداء 80 % 3 د 1 د 2 2 2 د د16 ( التمرين رقم2 ) اسم التمرين : تحمل اداء ( شللللرا التمللللرين: يللللتم التمللللرين علللللى شللللكل مربللللع25 x 30 ) م يحللللدد التمللللرين بواسللللطة شللللواخص ويوجللللد علللللى جوانللللب المربللللع أدوات تدريبيللللة ،جانللللب موانللللع والجانللللب االخللللر سلللللم أرضللللي ، يبللللدأ التمللللرين بصللللافرة المللللدرب الللللذي يقللللل خللللللار المربللللللع مللللللع الكللللللرات بلعللللللب5 ضللللللد5 داخللللللل المربللللللع لمللللللدة دقيقتللللللين لعللللللب حللللللر بعللللللدها يصلللللل ر المللللللدرب يخللللللر الالعبللللللين مللللللن المربللللللع بللللللالركض مجموعللللللة تق للللللز علللللللى الموانللللللع والمجموعللللللة األخللللللر علللللللى السلللللللم األرضللللللي ومللللللن ثللللللم . الدخول للمربع مرة أخر وتكملة التمرين لمدة دقيقة واللعب بلمستين اسم التمرين الشدة زمن االداء الراحة البينية التكرار المجاميع الراحة بين الم جاميع الزمن الكلي تحمل االداء 85 % 3 د 45 ثا 2 2 2 د 15 د ( التمرين رقم2 ) اسم التمرين : تحمل اداء ( شللللرا التمللللرين: يللللتم التمللللرين علللللى شللللكل مربللللع25 x 30 ) م يحللللدد التمللللرين بواسللللطة شللللواخص ويوجللللد علللللى جوانللللب المربللللع أدوات تدريبيللللة ،جانللللب موانللللع والجانللللب االخللللر سلللللم أرضللللي ، يبللللدأ التمللللرين بصللللافرة المللللدرب الللللذي يقللللل خللللللار المربللللللع مللللللع الكللللللرات بلعللللللب5 ضللللللد5 داخللللللل المربللللللع لمللللللدة دقيقتللللللين لعللللللب حللللللر بعللللللدها يصلللللل ر المللللللدرب يخللللللر الالعبللللللين مللللللن المربللللللع بللللللالركض مجموعللللللة تق للللللز علللللللى الموانللللللع والمجموعللللللة األخللللللر علللللللى السلللللللم األرضللللللي ومللللللن ثللللللم . الدخول للمربع مرة أخر وتكملة التمرين لمدة دقيقة واللعب بلمستين اسم التمرين الشدة زمن االداء الراحة البينية التكرار المجاميع الراحة بين الم جاميع الزمن الكلي تحمل االداء 85 % 3 د 45 ثا 2 2 2 د 15 د 123
https://openalex.org/W3177542322	https://zenodo.org/records/6371775/files/4%20Sinergi%20Bela%20Negara%20dan%20Kemanusiaan%20dalam%20Kegiatan%20Pengabdian%20Kepada%20Masyarakat%20Prodi%20Teknik%20Mesin%20Unhan%20RI%20di%20RSIA%20Melania%20Cibinong.pdf	Indonesian	null	Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong	Komatika	2,021	cc-by-sa	4,713	Abstrak Kewajiban utama masyarakat akademis di lingkungan Program Studi (Prodi) Teknik Mesin Universitas Pertahanan Republik Indonesia (Unhan RI) adalah melaksanakan Tri Dharma Perguruan Tinggi, yang salah satu komponennya adalah Pengabdian Kepada Masyarakat (PKM). Berada di dalam kelembagaan Kementerian Pertahanan, masyarakat akademis di lingkungan Unhan RI ditekankan untuk menjadi cendekiawan Bela Negara. Oleh karena itu, Prodi Teknik Mesin Unhan RI merancang kegiatan PKM yang tidak hanya bertujuan untuk menerapkan keahlian Teknik Mesin bagi masyarakat umum tetapi juga menyebarluaskan semangat Bela Negara. Puncak dari setiap kegiatan PKM tersebut adalah pelaksanaan sosialisasi Bela Negara dalam rangka memberikan kesadaran pelaksanaan Bela Negara melalui profesi masing-masing. Artikel ini membahas terwujudnya sinergi Bela Negara dan Kemanusiaan melalui kegiatan PKM Prodi Teknik Mesin Unhan RI di Rumah Sakit Ibu dan Anak (RSIA) Melania Cibinong. Tahap pertama kegiatan PKM ini adalah penerapan keahlian Teknik Mesin melalui perbaikan berbagai peralatan pendukung operasional rumah sakit seperti komputer, genset dan alat RO (Reverse Osmosis). Selanjutnya, tahap kedua PKM ini adalah kegiatan sosialisasi Bela Negara pada pimpinan dan karyawan rumah sakit. Melalui kegiatan PKM ini, semangat Bela Negara masyarakat akademis di lingkungan Teknik Mesin Unhan RI terwujud melalui penerapan keahliannya dalam mendukung keberlangsungan pelayanan kemanusiaan terhadap masyarakat. Di lain pihak, pimpinan dan karyawan RSIA Melania Cibinong memiliki kesadaran bahwa Bela Negara dapat diwujudkan melalui tanggung jawab profesinya dibidang pelayanan kemanusiaan. Pemilihan institusi kesehatan sebagai tempat dilaksanakannya PKM Prodi Teknik Mesin Unhan RI menjadi contoh sinergi antara semangat Bela Negara dan Kemanusiaan yang mendukung kehidupan berbangsa dan bernegara yang sehat dan humanis. Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong 17 Linus Yoseph Wawan Rukmono1, Arie Sukma Jaya2* Program Studi Teknik Mesin, Universitas Pertahanan, Sentul, Jawa Barat Email: 1linus.rukmono@idu.ac.id, 2arie.jaya@idu.ac.id Program Studi Teknik Mesin, Universitas Pertahanan, Sentul, Jawa Barat Email: 1linus.rukmono@idu.ac.id, 2arie.jaya@idu.ac.id (Naskah masuk: 30 Maret 2021, direvisi: 19 April 2021, diterima: 20 Mei 2021) Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong Linus Yoseph Wawan Rukmono1, Arie Sukma Jaya2* 1. Perbaikan komputer Komputer merupakan salah satu hasil teknologi yang memudahkan aktivitas manusia. Pada zaman ini, pengenalan akan komputer dapat meningkatkan taraf hidup manusia [9, 10]. Pengenalan tersebut dapat dilakukan melalui pelatihan perawatan ataupun perbaikan komputer [11]. Pada operasional rumah sakit, seperti RSIA Melania Cibinong, komputer merupakan salah satu komponen pendukung yang vital. Namun demikian, karena usia pemakaian komputer yang digunakan di RSIA Melania Cibinong sudah cukup lama, sehingga sering mengalami kendala tidak responsif (hang). Oleh karena itu, perlu dilakukan perawatan pada bagian perangkat keras dan install ulang beberapa perangkat lunak untuk mengatasi kendala lambatnya kerja komputer. Selain itu, perlu dilakukan juga penambahan Random Access Memory (RAM) pada beberapa komputer yang digunakan untuk pendataan pasien. Komputer merupakan salah satu hasil teknologi yang memudahkan aktivitas manusia. Pada zaman ini, pengenalan akan komputer dapat meningkatkan taraf hidup manusia [9, 10]. Pengenalan tersebut dapat dilakukan melalui pelatihan perawatan ataupun perbaikan komputer [11]. Pada operasional rumah sakit, seperti RSIA Melania Cibinong, komputer merupakan salah satu komponen pendukung yang vital. Namun demikian, karena usia pemakaian komputer yang digunakan di RSIA Melania Cibinong sudah cukup lama, sehingga sering mengalami kendala tidak responsif (hang). Oleh karena itu, perlu dilakukan perawatan pada bagian perangkat keras dan install ulang beberapa perangkat lunak untuk mengatasi kendala lambatnya kerja komputer. Selain itu, perlu dilakukan juga penambahan Random Access Memory (RAM) pada beberapa komputer yang digunakan untuk pendataan pasien. Institusi Rumah Sakit Ibu dan Anak (RSIA) Melania Cibinong merupakan rumah sakit Kelas C yang berada di Cibinong, Bogor. RSIA Melania Cibinong memiliki beberapa pelayanan diantaranya perawatan umum, UGD, perawatan anak (pediatrik), laboratorium, radiologi (x-ray), serta perawatan kehamilan dan wanita (obstetri dan gynaecologi) [6- 8]. Mengingat lokasinya yang strategis dengan jumlah pelayanan yang cukup tinggi dan pentingnya pelayanan rumah sakit ini bagi kehadiran kehidupan baru yang sehat, maka keberlangsungan operasional rumah sakit ini menjadi penting. Oleh karena itu, program PKM Prodi Teknik Mesin Unhan RI dilakukan pada institusi kesehatan ini. Pemilihan institusi kesehatan sebagai tempat dilaksanakannya PKM bermaksud untuk memperoleh pengalaman sinergi semangat Bela Negara dan Kemanusiaan. Penerapan keahlian Teknik Mesin pada institusi kesehatan diharapkan dapat mendukung operasional institusi tersebut sehingga selalu menghasilkan pelayanan yang prima. Sinergi dalam PKM ini diharapkan dapat menjadi percontohan bagi bidang ilmu yang lain untuk semakin meningkatkan kolaborasi dalam mewujudkan masyarakat Indonesia yang sejahtera. Kata Kunci: teknik mesin, sinergi, bela negara, kesehatan, kemanusiaan. The synergy of Bela Negara and Humanity in Community Service of the Mechanical Engineering Study Program Unhan RI at RSIA Melania Cibinong Kata Kunci: teknik mesin, sinergi, bela negara, kesehatan, kemanusiaan. Kata Kunci: teknik mesin, sinergi, bela negara, kesehatan, kemanusiaan. Abstract The main responsibility of the academic community of the Mechanical Engineering Study Program (Prodi Teknik Mesin) of the Republic of Indonesia Defense University (Unhan RI) is the implementation of the Tri Dharma of Higher Education, one of which is Community Service (PKM). Within the Ministry of Defense institution, the academic community of Unhan RI is always emphasized to become intellectuals of Bela Negara (the spirit of defending the country). Therefore, the Prodi Teknik Mesin Unhan RI designed PKM activities that not only aim to apply Mechanical Engineering expertise to the community but also spread the spirit of Bela Negara. The culmination of each PKM activity was the implementation of Bela Negara socialization to provide awareness of the implementation of Bela Negara through their own professions. This article discusses the realization of the synergy of Bela Negara and Humanity through PKM activities by Prodi Teknik Mesin Unhan RI at Mother and Child Hospital (RSIA) Melania Cibinong. The first phase of this PKM activity was the application of Mechanical Engineering expertise through the reparation of hospital operational support equipments such as computers, generator set, and RO (Reverse Osmosis) devices. DOI: 10.34148/komatika.v1i1.352 DOI: 10.34148/komatika.v1i1.352 KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong 18 Next, the second stage of this PKM was the socialization of Bela Negara to leaders and employees of the hospital. Through this PKM activity, the spirit of Bela Negara of the Prodi Teknik Mesin Unhan RI academic community was actualized through the implementation of their expertise to support the continuity of humanitarian services to the community. On the other side, the leaders and employees of RSIA Melania Cibinong had an awareness that Bela Negara can be applied through the responsibility of their profession in the field of humanitarian services. II. METODE KEGIATAN Universitas Pertahanan Indonesia Republik Indonesia (Unhan RI) merupakan lembaga pendidikan tinggi yang diselenggarakan oleh pemerintah [1]. Unhan RI secara akademik dibina oleh Kementerian Pendidikan dan Kebudayaan dan secara fungsional dibina oleh Kementerian Pertahanan [2]. Unhan RI merupakan kampus yang menekankan pentingnya Bela Negara bagi masyarakat akademis di lingkungannya [3, 4]. Program studi (Prodi) Teknik Mesin Unhan RI merupakan bagian dari Fakultas Teknik Militer Unhan RI [5]. Prodi Teknik Mesin Unhan RI mendukung visi dan misi Unhan RI untuk mencetak lulusan Sarjana Teknik dibidang Teknik Mesin yang memiliki karakteristik bela negara dan memahami ilmu pertahanan. Oleh karena itu, lingkungan pendidikan Teknik Mesin Unhan RI disesuaikan untuk mencetak kader intelektual bela negara yang menguasai desain, produksi, material, konversi energi, serta mekatronika permesinan untuk pertahanan. Sebagai institusi pendidikan, maka Prodi Teknik Mesin Unhan RI perlu melakukan Tri Dharma Perguruan Tinggi yang salah satunya adalah Pengabdian Kepada Masyarakat (PKM). Prodi Teknik Mesin Unhan RI melakukan PKM di RSIA Melania Cibinong dengan dua bagian utama yaitu kegiatan penerapan keahlian Teknik Mesin dalam perbaikan dan perawatan alat-alat mekanis dan kegiatan sosialisasi Bela Negara. Kegiatan ini menjadi pelaksanaan Tri Dharma perguruan tinggi yang dilakukan para dosen dari Prodi Teknik Mesin Unhan RI. Pihak-pihak yang terlibat dalam kegiatan ini antara lain: a. Teknik Mesin Unhan RI: Pimpinan Program Studi, para dosen, laboran dan staf tata usaha prodi. b. RSIA Melania Cibinong: Pengurus yayasan, suster rumah sakit dan staf rumah sakit. Kata Kunci: teknik mesin, sinergi, bela negara, kesehatan, kemanusiaan. The synergy of Bela Negara and Humanity in Community Service of the Mechanical Engineering Study Program Unhan RI at RSIA Melania Cibinong The selection of a health institution as a place for PKM by the Prodi Teknik Mesin Unhan RI was an example of the synergy between the spirit of Bela Negara and Humanity that supports a healthy and humanist life of the nation and state. Keywords: mechanical engineering, synergy, Bela Negara, health, humanity. A. Kegiatan penerapan keahlian Teknik Mesin Penerapan keahlian Teknik Mesin pada kegiatan PKM di RSIA Melania Cibinong dilakukan melalui beberapa aktivitas antara lain: Penerapan keahlian Teknik Mesin pada kegiatan PKM di RSIA Melania Cibinong dilakukan melalui beberapa aktivitas antara lain: 1. Perbaikan komputer KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 1. Perbaikan komputer Selain itu, sinergi ini juga diharapkan dapat menambah luas penerapan keahlian Teknik Mesin, tidak hanya dalam ranah konvensional yang terkait industri dan rekayasa teknik, tetapi juga ranah yang berkaitan dengan kemanusiaan dan kesehatan. Kegiatan perbaikan komputer ini penting untuk mendukung kelancaran kegiatan administrasi dan digitalisasi pendataan/rekam medis pasien. Kegiatan ini dilakukan secara paralel selama dua minggu pada empat komputer untuk tetap menjaga keberlangsungan operasional pendataan pasien. Gambar 1 menunjukkan salah satu bagian perangkat keras dari komputer yang diperbaiki pada kegiatan ini. KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 DOI: 10.34148/komatika.v1i1.352 KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong 19 3. Perbaikan alat pemurni air Reverse Osmosis (RO) Reverse Osmosis (RO) merupakan metode yang diterapkan secara efektif pada alat pemurni air. Untuk kebutuhan sehari-hari, masyarakat umumnya memperoleh air dari beberapa sumber seperti sumur, ledeng, ataupun sumber air alami. Namun demikian, air yang diperoleh secara langsung dari sumber tersebut memiliki kontaminan dalam kadar yang cukup tinggi sehingga belum layak digunakan sebagai air minum. Dengan metode RO pada alat penjernih air, maka kontaminan yang membuat air memiliki bau, warna, atau rasa yang tidak sedap dapat disaring, sehingga air yang keluar dari alat tersebut dapat dikonsumsi sebagai air minum [16, 17]. Gambar 1. Perangkat Keras Komputer yang Diperbaiki Untuk skala industri, metode RO dapat secara efektif menyaring bakteri dan virus dalam air dengan membran yang memiliki ukuran pori mencapai 0,001 mikron [18]. Gambar 1. Perangkat Keras Komputer yang Diperbaiki 2. Perbaikan dan pelatihan perawatan mesin generator set (genset) 2. Perbaikan dan pelatihan perawatan mesin generator set (genset) Gambar 3. Alat RO pemurni air yang tidak berfungsi Genset merupakan salah satu mesin yang dibutuhkan membangkitkan arus listrik pada saat terjadinya pemadaman arus listrik [12, 13]. Genset RSIA Melania Cibinong, seperti pada Gambar 2, menggunakan bahan bakar solar dan listrik yang dihasilkan dapat digunakan untuk beberapa keperluan penting seperti menjaga keberlangsungan instrumen elektronik pada ruang bersalin dan penerangan rumah sakit. Namun demikian, genset ini mengalami kerusakan sejak tahun 2011 karena kurangnya perawatan dan tidak adanya tenaga ahli yang dapat memperbaikinya. Oleh karena pentingnya fungsi mesin ini, maka perbaikan genset dimasukkan dalam program PKM. KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 1. Perbaikan komputer S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong 20 produk. Dengan adanya mesin cuci tangan otomatis di lingkungan RSIA Melania Cibinong, diharapkan meningkatkan kesadaran karyawan dan pengunjung rumah sakit untuk tetap menjaga kebersihan dan mengurangi potensi penyebaran virus COVID19. B. Sosialisasi Bela Negara Sosialisasi Bela Negara dilakukan untuk memberi wawasan bela negara melalui profesi dan kompetensi personal maupun lembaga masing-masing. Kegiatan ini juga iharapkan mamp membangkitkan moti asi dan semangat Teknik Mesin karena permasalahan lapangan yang diselesaikan pada kegiatan ini memerlukan waktu yang lebih panjang. Kegiatan penerapan pertama yaitu dengan melakukan perbaikan komputer seperti pada Gambar 4. Gambar tersebut menunjukkan aktivitas perbaikan komputer yang dilakukan oleh dosen Prodi Teknik Mesin Unhan RI. Komputer dapat diperbaiki sesuai dengan jangka waktu yang ditentukan. Permasalahan komputer yang kurang responsif telah diatasi dan komputer dapat beroperasi untuk pendataan pasien kembali dengan lebih baik. Tabel 1. Jadwal pelaksanaan PKM No. Kegiatan Januari 2021 Februari 2021 I II III IV I II III IV 1 Persiapan program PKM 2 Perbaikan komputer 3 Perbaikan dan pelatihan perawatan genset 4 Perbaikan alat RO 5 Pembuatan alat cuci tangan otomatis 6 Sosialisasi Bela Negara 7 Pembuatan laporan program Tabel 1. Jadwal pelaksanaan PKM No. Kegiatan Januari 2021 Februari 2021 I II III IV I II III IV 1 Persiapan program PKM 2 Perbaikan komputer 3 Perbaikan dan pelatihan perawatan genset 4 Perbaikan alat RO 5 Pembuatan alat cuci tangan otomatis 6 Sosialisasi Bela Negara 7 Pembuatan laporan program Teknik Mesin karena permasalahan lapangan yang diselesaikan pada kegiatan ini memerlukan waktu yang lebih panjang. Kegiatan penerapan pertama yaitu dengan melakukan perbaikan komputer seperti pada Gambar 4. Gambar tersebut menunjukkan aktivitas perbaikan komputer yang dilakukan oleh dosen Prodi Teknik Mesin Unhan RI. Komputer dapat diperbaiki sesuai dengan jangka waktu yang ditentukan. Permasalahan komputer yang kurang responsif telah diatasi dan komputer dapat beroperasi untuk pendataan pasien kembali dengan lebih baik. Teknik Mesin karena permasalahan lapangan yang diselesaikan pada kegiatan ini memerlukan waktu yang lebih panjang. Kegiatan penerapan pertama yaitu dengan melakukan perbaikan komputer seperti pada Gambar 4. Gambar tersebut menunjukkan aktivitas perbaikan komputer yang dilakukan oleh dosen Prodi Teknik Mesin Unhan RI. Komputer dapat diperbaiki sesuai dengan jangka waktu yang ditentukan. Permasalahan komputer yang kurang responsif telah diatasi dan komputer dapat beroperasi untuk pendataan pasien kembali dengan lebih baik. produk. B. Sosialisasi Bela Negara Sosialisasi Bela Negara dilakukan untuk memberi wawasan bela negara melalui profesi dan kompetensi personal maupun lembaga masing-masing. Kegiatan ini juga diharapkan mampu membangkitkan motivasi dan semangat berkarya pada bidangnya masing-masing sebagai tindakan bela negara dalam kehidupan berbangsa dan bernegara. Materi sosialisasi antara lain Dasar Negara Pancasila, Wawasan Nusantara, Bhinneka Tunggal Ika dan UUD 1945. Sosialisasi ini disampaikan dengan bahasa yang mudah dicerna dan menarik bagi para karyawan rumah sakit yang sebagian besar berlatar belakang SMA. Kegiatan ini merupakan puncak dari rangkaian PKM yang dilakukan oleh Prodi Teknik Mesin Unhan RI. Alokasi waktu untuk sosialisasi ini adalah dua jam yang dilangsungkan bersamaan dengan acara penutupan kegiatan pengabdian masyarakat di RSIA Melania Cibinong. Gambar 4. Aktivitas perbaikan komputer Gambar 4. Aktivitas perbaikan komputer Gambar 4. Aktivitas perbaikan komputer C. Jadwal PKM Gambar 5. Aktivitas perbaikan genset Kegiatan PKM Prodi Teknik Mesin Unhan RI dilaksanakan dalam rentang waktu dua bulan mulai dari Januari 2021 sampai dengan Februari 2021. Jadwal pelaksanaan kegiatan PKM tersebut dapat dilihat pada Tabel 1. Seperti dapat dilihat pada tabel, kegiatan ini diawali dengan tahap persiapan program sebagai koordinasi awal antara Prodi Teknik Mesin Unhan RI dengan RSIA Melania Cibinong. Program penerapan keahlian Teknik Mesin dimulai setelah tahap persiapan, sesuai dengan alokasi waktu yang ditetapkan sebelumnya. Puncak acara dari program ini adalah program sosialisasi Bela Negara yang dilakukan setelah berhasilnya pelaksanaan tahap penerapan keahlian. Program ini diakhiri dengan pembuatan laporan kegiatan. Gambar 5. Aktivitas perbaikan genset Kegiatan penerapan keahlian Teknik Mesin selanjutnya adalah perbaikan genset, seperti yang dapat dilihat pada Gambar 5. Gambar tersebut menunjukkan genset yang sedang diperbaiki oleh dosen dan teknisi Prodi Teknik Mesin Unhan RI. Perbaikan genset ini merupakan aktivitas yang memerlukan waktu paling lama dan sumber daya manusia yang paling banyak. Meskipun komponen utama mesin masih 1. Perbaikan komputer Dengan adanya mesin cuci tangan otomatis di lingkungan RSIA Melania Cibinong, diharapkan meningkatkan kesadaran karyawan dan pengunjung rumah sakit untuk tetap menjaga kebersihan dan mengurangi potensi penyebaran virus COVID19. 1. Perbaikan komputer Alokasi waktu untuk kegiatan perbaikan ini adalah lima minggu mulai dari tahap analisis kebutuhan peralatan dan tenaga kerja, pelaksanaan perbaikan sampai dengan persiapan metode perawatan. Perawatan dengan metode yang tepat sangat penting untuk menjaga keberlangsungan operasional genset [14, 15]. Oleh karena itu, pada kegiatan PKM ini juga dilakukan pelatihan perawatan genset kepada karyawan rumah sakit yang bertugas sebagai operator dari genset tersebut. Gambar 3. Alat RO pemurni air yang tidak berfungsi Alat RO pemurni air di RSIA Melania Cibinong seperti pada Gambar 3 merupakan jenis alat untuk skala pemakaian sedang. Kendala yang dihadapi adalah alat tersebut tidak berfungsi dengan baik karena pompa tidak menyala. Oleh karena pentingnya alat tersebut untuk operasional RSIA Melania Cibinong, maka perbaikan pemurni air ini dimasukkan dalam program PKM. Alokasi waktu untuk kegiatan perbaikan ini adalah tiga minggu mulai dari tahap analisis permasalahan, pelaksanaan perbaikan sampai dengan persiapan metode perawatan. Gambar 2. Mesin Genset RSIA Melania Cibinong 4. Pembuatan alat cuci tangan otomatis Mencuci tangan merupakan salah satu komponen dari Gerakan 3M yang disosialisasikan untuk mencegah penyebaran virus COVID19 [19, 20]. Sebagai bagian dari pengabdian masyarakat, maka Prodi Teknik Mesin Unhan RI mengupayakan tersedianya sarana cuci tangan yang efektif dan efisien serta nyaman bagi penggunanya. Oleh karena itu, dilakukan produksi terhadap mesin cuci tangan otomatis dengan sensor gerak inframerah yang telah dirancang pada kajian sebelumnya dengan berbasis karakteristik antropometri [21]. Alokasi waktu untuk kegiatan pembuatan ini adalah empat minggu mulai dari tahap pembuatan, pengiriman dan uji coba 4. Pembuatan alat cuci tangan otomatis Mencuci tangan merupakan salah satu komponen dari Gerakan 3M yang disosialisasikan untuk mencegah penyebaran virus COVID19 [19, 20]. Sebagai bagian dari pengabdian masyarakat, maka Prodi Teknik Mesin Unhan RI mengupayakan tersedianya sarana cuci tangan yang efektif dan efisien serta nyaman bagi penggunanya. Oleh karena itu, dilakukan produksi terhadap mesin cuci tangan otomatis dengan sensor gerak inframerah yang telah dirancang pada kajian sebelumnya dengan berbasis karakteristik antropometri [21]. Alokasi waktu untuk kegiatan pembuatan ini adalah empat minggu mulai dari tahap pembuatan, pengiriman dan uji coba Gambar 2. Mesin Genset RSIA Melania Cibinong DOI: 10.34148/komatika.v1i1.352 KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong Rukmono, L. Y. W. dan Jaya, A. III. PELAKSANAAN KEGIATAN Kegiatan PKM Prodi Teknik Mesin Unhan RI telah dilaksanakan di RSIA Melania Cibinong. Rangkaian kegiatan PKM diawali dengan berbagai kegiatan penerapan keahlian KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 DOI: 10.34148/komatika.v1i1.352 Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong 21 Gambar 7. Pemasangan dan pengujian mesin cuci tangan otomatis dengan sensor gerak inframerah dalam keadaan baik, pembiaran kerusakan yang terlalu lama memerlukan penggantian beberapa komponen, terutama pada bagian sambungan listrik, daerah pembakaran dan sumber daya pemantik. Tingkat kompleksitas mesin juga memerlukan tenaga teknisi yang berpengalaman supaya tidak menyebabkan kerusakan yang lebih parah. Namun demikian, genset telah berhasil diperbaiki dan dapat beroperasi kembali sebagai pembangkit listrik dalam keadaan pemadaman arus listrik. (a) (a) (b) Gambar 6. Aktivitas perbaikan pemurni air metode RO (a) Pemeriksaan komponen, (b) Pemasangan pemurni air yang telah diperbaiki. Gambar 7. Pemasangan dan pengujian mesin cuci tangan otomatis dengan sensor gerak inframerah Sebagai puncak dari kegiatan PKM Prodi Teknik Mesin Unhan RI adalah sosialisasi Bela Negara yang dilakukan selama satu hari. Gambar 8 menunjukkan aktivitas sosialisasi Bela Negara yang dilakukan di RSIA Melania Cibinong. Pimpinan dan staf rumah sakit mengikuti acara ini dengan antusias sehingga sosialisasi dapat berjalan dengan baik. Dalam kegiatan puncak ini juga dilakukan pelatihan perawatan genset oleh dosen Prodi Teknik Mesin kepada karyawan rumah sakit yang akan bertugas merawat genset yang telah dapat beroperasi kembali seperti terlihat pada Gambar 9. (a) (b) Gambar 8. Aktivitas sosialisasi Bela Negara (b) Gambar 6. Aktivitas perbaikan pemurni air metode RO (a) Pemeriksaan komponen, (b) Pemasangan pemurni air yang telah diperbaiki. Gambar 8. Aktivitas sosialisasi Bela Negara Selanjutnya, kegiatan yang melibatkan para dosen Prodi Teknik Mesin Unhan RI dalam perbaikan alat pemurni air dengan metode RO dapat dilihat pada Gambar 6. Dari pemeriksaan terhadap setiap komponen utama dari alat tersebut, seperti pada Gambar 6.(a), didapatkan bahwa terdapat kebocoran pada komponen sambungan aliran sehingga memerlukan penggantian komponen. Sambungan pada kabel listrik yang pernah diperbaiki sebelumnya juga diperbaiki lagi untuk mengurangi potensi kerusakan yang lebih fatal. Gambar 6.(b) menunjukkan aktivitas pemasangan kembali pemurni air yang telah diperbaiki. Alat pemurni air telah berhasil beroperasi kembali dengan baik di RSIA Melania Cibinong. Gambar 9. KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 III. PELAKSANAAN KEGIATAN Pelatihan perawatan genset Penerapan keahlian Prodi Teknik Mesin Unhan RI dalam PKM ini tidak hanya terkait proses perbaikan tetapi juga melibatkan proses desain dan produksi produk yang salah satunya adalah mesin cuci tangan otomatis dengan sensor gerak inframerah. Gambar 7 menunjukkan pemasangan hasil produksi dan pengujian performa kerja mesin cuci tangan otomatis yang ditempatkan di RSIA Melania Cibinong. Gambar 9. Pelatihan perawatan genset DOI: 10.34148/komatika.v1i1.352 KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong 22 Letkol (Cpl) Linus Yoseph Wawan Rukmono, S.T., M.T., Hadi Wahyudi, S.T., M.T., Ph.D., Dr. Edy Susanto, S.T., M.T., Dr. Arie Sukma Jaya, S.T., M.Eng., Dr. Arief Nur Pratomo S.T., M.T., Dr. Eng. Rando Tungga Dewa, S.T., M.Eng., Ariyo Nurrachman Satiya Permata, S.T., M.Eng., Aditia Aulia, S.T., M.Sc., Eka Irianto Bhiftime, S.T., M.T., Ridho Illahi Putra, S.H., Hanung Bayu Setiawan, S.T., M.Han. Pimpinan RSIA Melania Cibinong: Suster Maria Veronika SFS. Gambar 10. Foto bersama peserta sosialisasi Bela Negara Letkol (Cpl) Linus Yoseph Wawan Rukmono, S.T., M.T., Hadi Wahyudi, S.T., M.T., Ph.D., Dr. Edy Susanto, S.T., M.T., Dr. Arie Sukma Jaya, S.T., M.Eng., Dr. Arief Nur Pratomo S.T., M.T., Dr. Eng. Rando Tungga Dewa, S.T., M.Eng., Ariyo Nurrachman Satiya Permata, S.T., M.Eng., Aditia Aulia, S.T., M.Sc., Eka Irianto Bhiftime, S.T., M.T., Ridho Illahi Putra, S.H., Hanung Bayu Setiawan, S.T., M.Han. Pimpinan RSIA Melania Cibinong: Suster Maria Veronika SFS. REFERENSI [1] “Profil,” 2009. https://www.idu.ac.id/profil (diakses pada Mar. 29, 2021). [2] “Identitas,” 2009. https://www.idu.ac.id/profil/identitas (diakses pada Mar. 29, 2021). Gambar 10. Foto bersama peserta sosialisasi Bela Negara [3] H. R. P. Sinaga, “Pendidikan Bela Negara yang Diselenggarakan Pusdikif,” J. Prodi Peperangan Asimetris, vol. 3, no. 3, pp. 63–80, 2017. Kegiatan sosialisasi Bela Negara ditutup dengan melakukan foto bersama, pada Gambar 10, yang diikuti semua peserta sosialisasi dalam kegiatan PKM ini. Foto bersama ini masih tetap mengikuti protokol kesehatan dengan memakai masker dan menjaga jarak. Kegiatan PKM ini telah membuka kesempatan dan ruang baru untuk penerapan keahlian Teknik Mesin dalam mendukung berbagai misi kemanusiaan bagi tenaga medis. Sinergi yang saling mendukung antara semangat bela negara dan kemanusiaan antara dua institusi dalam kegiatan PKM ini tetap diharapkan terus terjalin baik. [4] J. Indrawan dan M. P. Aji, “Pendidikan Bela Negara Sebagai Mata Kuliah Di Perguruan Tinggi,” J. Pertahanan Bela Negara, vol. 8, no. 3, pp. 1–24, 2018, doi: 10.33172/jpbh.v8i3.437. [5] “PDDikti - Pangkalan Data Pendidikan Tinggi,” 2020. https://pddikti.kemdikbud.go.id/data_prodi/NUQ0RDY 2Q0MtRUQzOS00NkY1LUExMkItQzA4OENBNUUz NUI3 (diakses pada Mar. 29, 2021). [6] C. F. Suan, “Faktor-faktor yang berhubungan keputusan ibu primigravida menentukan tempat persalinan di RSIA Melania Cibinong”, Undergraduate thesis, STIK Sint Carolus, 2016. IV. KESIMPULAN Dengan terlaksananya kegiatan PKM Prodi Teknik Mesin Unhan di RSIA Melania Cibinong, maka dapat diperoleh beberapa simpulan yaitu [7] Stetoskoop, “Tentang Rumah Sakit Ibu dan Anak Melania - Cibinong,” 2021. http://stetoskoop.com/id/hospital/rumah-sakit-ibu-dan- anak-melania--cibinong (diakses pada Mar. 29, 2021). 1. Kegiatan PKM oleh masyarakat akademis di lingkungan Prodi Teknik Mesin Unhan RI telah dilakukan sebagai pemenuhan Tri Dharma perguruan tinggi. [8] SehatQ, “RSIA Melania Cibinong,” 2018. https://www.sehatq.com/faskes/rumah-sakit-melania- cibinong (diakses pada Mar. 29, 2021). 2. Terjadi sinergi yang saling menguatkan antara pelayanan kemanusiaan RSIA Melania Cibinong dengan pelayanan Bela Negara Prodi Teknik Mesin Unhan RI. 2. Terjadi sinergi yang saling menguatkan antara pelayanan kemanusiaan RSIA Melania Cibinong dengan pelayanan Bela Negara Prodi Teknik Mesin Unhan RI. [9] A. Alfirman, S. Sukamto, G. Sastria dan Z. Bahri, “Perakitan dan perbaikan komputer untuk meningkatan kualitas remaja di Desa Pandau Jaya Kecamatan Siak Hulu Kabupaten Kampar”, unricsce, vol. 1, pp. 243-254, Sep. 2019. 3. Seluruh pihak, baik dari Prodi Teknik Mesin Unhan RI maupun RSIA Melania Cibinong, terlibat aktif dalam pelaksanaan kegiatan PKM yang dilakukan. 4. Penerapan keahlian Teknik Mesin dapat mendukung keberlangsungan operasional rumah sakit dalam pelayanan kemanusiaan. 4. Penerapan keahlian Teknik Mesin dapat mendukung keberlangsungan operasional rumah sakit dalam pelayanan kemanusiaan. p [10] M. Y. T. Sulistyono, W. S. Sari dan I. Septriana, “Pemberdayaan Remaja Putus Sekolah dan Pengangguran Dalam Pembentukan Komunitas Wira Usaha Jasa Perbaikan Komputer dan Jaringan Komputer,” sens2. 2016. p y 5. Kegiatan sosialisasi Bela Negara bagi pimpinan dan karyawan rumah sakit mendukung sumber daya manusia yang memiliki jiwa ksatria dan bertanggung jawab tidak hanya untuk kepentingan kemanusiaan saat bertugas di rumah sakit tetapi juga untuk bangsa dan negara. 5. Kegiatan sosialisasi Bela Negara bagi pimpinan dan karyawan rumah sakit mendukung sumber daya manusia yang memiliki jiwa ksatria dan bertanggung jawab tidak hanya untuk kepentingan kemanusiaan saat bertugas di rumah sakit tetapi juga untuk bangsa dan negara. [11] A. Syaputra, “Pelatihan Maintenance Komputer Pada Himpunan Mahasiswa Teknik Informatika Kota Pagar Alam,” NGABDIMAS, Vol. 3, No. 2, pp. 75-81, 2020, https://doi.org/10.36050/ngabdimas.v3i2.274. p j g g g 6. Kegiatan PKM Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong menunjukkan sinergi bela negara dan kemanusiaan dalam rangka mendukung kehidupan berbangsa dan bernegara yang sehat dan humanis. [12] Y. Basongan dan M. Iswar. “IbM Perawatan dan Perbaikan Generator Set (Genset) di Desa Nisombalia,” Seminar Nasional Hasil Penelitian & Pengabdian Kepada Masyarakat (SNP2M), pp. 474– 478, 2017. Rukmono, L. Y. W. dan Jaya, A. S.: Sinergi Bela Negara dan Kemanusiaan dalam Kegiatan Pengabdian Kepada Masyarakat Prodi Teknik Mesin Unhan RI di RSIA Melania Cibinong KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 UCAPAN TERIMA KASIH Penulis menyampaikan terima kasih untuk dukungan teknis maupun operasional terhadap pelaksanaan PKM ini. Masyarakat akademis di lingkungan Prodi Teknik Mesin: [13] K. Purwanto, S. Suripto, R. O. Wiyagi, Y. Ardiyanto dan DOI: 10.34148/komatika.v1i1.352 KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341 23 M. Y. Mustar, “Implementasi Emergency Backup Pada Instalasi Listrik Di Masjid An Nuur, Gamping Tengah,” dalam Prosiding Seminar Nasional Program Pengabdian Masyarakat, 2020. M. Y. Mustar, “Implementasi Emergency Backup Pada Instalasi Listrik Di Masjid An Nuur, Gamping Tengah,” dalam Prosiding Seminar Nasional Program Pengabdian Masyarakat, 2020. Engagement and Services, Vol. 2, No. 2, pp. 1-6, 2019. [18] “Reverse Osmosis (RO) Fact Sheet,” 2019. [Online]. Available: https://www.wqa.org/Portals/0/Technical/Technical Fact Sheets/2019_RO.pdf. [14] D. Aribowo, Desmira, and D. A. Fauzan, “Sistem Perawatan Mesin Genset di PT (Persero) Pelabuhan Indonesia II,” Pros. Semin. Nas. Pendidik. FKIP, Vol. 3, No. 1, pp. 580–594, 2020. [19] T. H. Saputra, H. A. Pamasaria, B. Wiratmoko, R. Hermawan dan R. Supriyono, “Rancang Bangun Mesin Cuci Tangan Otomatis Portabel untuk Mengurangi Efek Pandemi Covid 19,” LOGISTA-Jurnal Ilmiah Pengabdian kepada Masyarakat, Vol. 4, No. 2, pp. 534- 540, 2020. [15] S. Teguh, “Uji Kelayakan Operasi Genset BRV20 RSG- GAS Setelah dilakukan Perbaikan,” dalam Prosiding Seminar Nasional Teknologi Pengelolaan Limbah XIV, pp. 268–277, 2016. [20] R. Romadhoni, B. Santoso dan M. Helmi, “Pembuatan Alat Cuci Tangan Mobile Dalam Pencegahan Corona Virus pada Tempat Ibadah,” INTEGRITAS: Jurnal Pengabdian, Vol. 4, No. 2, pp. 264-274, 2020. [16] L. K. Wardana, ”Pengolahan Air Mineral dalam Kemasan Rendah Kontaminasi,” Berdikari: Jurnal Inovasi dan Penerapan Ipteks, Vol. 6, No. 2, 162-172, 2018. [21] L. Y. W. Rukmono, A. S. Jaya, A. Aulia, and E. I. Bhiftime, “Rancang bangun mesin cuci tangan otomatis cutato-Unhan berbasis karakteristik anthropometri,” dalam Prosiding Seminar Nasional Ilmu Teknik dan Aplikasi Industri (SINTA), Vol. 3, 2020. [17] D. D. Djamaluddin, M. Anshar, Z. Muslimin, R.S. Sadrat, E. Palantei, M. Baharuddin, A. D. Achmad, Wardi dan S. Syarif, “Pelatihan Trouble Shooting Sistem RO di Kecamatan Marusu Kabupaten Maros,” JURNAL TEPAT: Applied Technology Journal for Community DOI: 10.34148/komatika.v1i1.352 KOMATIKA, Volume 1(1), Mei 2021, pp. 17-23 ISSN: 2774-5341
https://openalex.org/W4361221265	https://ejournal.stebisigm.ac.id/index.php/esha/article/download/702/321	Indonesian	null	Pengaruh Komitmen dan Kepuasan Kerja Terhadap Kinerja Pegawai Pada Dinas Kependudukan dan Pencatatan Sipil Kota Palembang	Economica sharia/Ekonomica Sharia	2,023	cc-by-sa	5,755	Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│295 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│295 Abstrack This study focuses on employees, especially the performance of employees of the Palembang City Population and Civil Registration Office. High employee performance is expected by the agency. The more employees who have high performance, the overall agency productivity will also increase. Employees are required to be able to complete their duties and responsibilities effectively and efficiently. Based on the calculation results, it can be concluded that the commitment variable has a partial effect on the Palembang Disdukcapil. The commitment variable has a significant effect on work performance at Disdukcapil Palembang at a significance level of 0.000 which is less than a significance level of 0.05, thus it can be stated that commitment has an effect on performance at Disdukcapil Palembang. Based on the calculation results, it can be concluded that the job satisfaction variable has no partial effect on work performance at Disdukcapil Palembang. The variable of job satisfaction in the Disdukcapil Palembang at 0.422 is greater than the significance level of 0.05. Thus there is no partial effect of job satisfaction on performance in the Disdukcapil Palembang Keywords: Commitment, Job Satisfaction and Performance PENGARUH KOMITMEN DAN KEPUASAN KERJA TERHADAP KINERJA PEGAWAI PADA DINAS KEPENDUDUKAN DAN PENCATATAN SIPIL KOTA PALEMBANG M. Kurniawan DP Universitas Indo Global Mandiri Palembang Email: Kurniawan@uigm.ac.id Kata Kunci : Komitmen, Kepuasan kerja dan Kinerja Abstrak Penelitian ini memfokuskan pada pegawai terutama Kinerja pegawai Dinas Kependudukan dan Pencatatan Sipil kota Palembang. Kinerja pegawai yang tinggi sangatlah diharapkan oleh instansi tersebut, Semakin banyak pegawai yang mempunyai kinerja yang tinggi, maka produktivitas instansi secara keseluruhan akan meningkat pula. Pegawai dituntut untuk mampu menyelesaikan tugas dan tanggung jawabnya secara efektif dan efisien. y g gg g j y f f f Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel komitmen memiliki pengaruh secara parsial terhadap disdukcapil Palembang. Variabel komitmen berpengaruh secara signifikan terhadap prestasi kerja di Disdukcapil Palembang dalam angka signifikansi 0,000 lebih kecil dari taraf signifikansi 0,05, dengan demikian dapat dinyatakan bahwa komitmen berpengaruh terhadap kinerja di disdukcapil Palembang. Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel kepuasan Kerja tidak memiliki pengaruh secara parsial terhadap prestasi kerja di Disdukcapil Palembang. Variabel kepuasan kerja pada disdukcapil Palembang dalam angka 0,422 lebih besar dari taraf signifikansi 0,05. Dengan demikian tidak adanya pengaruh secara parsial dari kepuasan kerja terhadap kinerja di disdukcapil Palembang Kata Kunci : Komitmen, Kepuasan kerja dan Kinerja Kata Kunci : Komitmen, Kepuasan kerja dan Kinerja 296│ 296│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… 96│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai…… 1. Latar Belakang Masalah Penelitian ini menggunakan variabel komitmen, komitmen adalah Menurut Mathis dan Jackson komitmen adalah derajat yang mana karyawan percaya dan menerima tujuan-tujuan organisasi dan akan tetap tinggal atau tidak akan meninggalkan organisasi. Sedangkan Mowdey menyebutkan komitmen merupakan dimensi perilaku penting yang dapat digunakan untuk menilai kecenderungan karyawan untuk bertahan sebagai anggota organisasi. Disamping itu Lincoln menyatakan komitmen itu mencakup kebanggaan anggota, kesetiaan anggota, dan kemauan anggota pada organisasi. Luthans menyatakan komitmen merupakan keinginan yang kuat untuk menjadi anggota dalam suatu kelompok, kemauan usaha yang tinggi untuk organisasi, serta suatu keyakinan tertentu dan penerimaan terhadap nilai- nilai dan tujuan-tujuan organisasi. Selain itu Mayer dan Allen mengidentifikasi tiga tema berbeda dalam mendefenisikan komitmen. Ketiga tema tersebut adalah komitmen sebagai ketertarikan pada organisasi (affective commitment), komitmen sebagai biaya yang harus ditanggung jika meninggalkan atau keluar organisasi (continuance commitment), dan komitmen sebagai kewajiban untuk tetap dalam organisasi (normative commitment). Tidak hanya komitmen yang memiliki pengaruh dalam kualitas kinerja karyawan, kepuasan kerja karyawan adalah salah satu hal terpenting dalam kualitas kinerja. Menurut Robins, kepuasan kerja atau job satisfaction diidentikkan dengan hal-hal yang bersifat individual. Karena itu, tingkat kepuasan setiap orang berbeda-beda dan hal ini terjadi apa bila beberapa faktor terpenuhi yaitu kebutuhan individu serta kaitannya dengan derajat kesukaan dan ketidaksukaan pekerja (Robins, 1999). Kepuasan dapat diartikan juga sebagai sekelompok faktor yang menyebabkan individu berperilaku dalam cara-cara tertentu. Kepuasan adalah pertimbangan yang penting bagi pemimpin karena kepuasan, bersama-sama kemampuan dan faktor-faktor lingkungan, sangat mempengaruhi kinerja individu. Proses kepuasan dimulai dengan terpenuhinya kebutuhan. Sebagai reaksinya, seorangpekerja mempertimbangkan alternatif-alternatif pemenuhan kebutuhan ini,seperti bekerja lebih keras agar gajinya dinaikkan atau mencari pekerjaan baru. Agar 297 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│297 perilaku seorang pekerja sesuai dengan tujuan organisasi, maka harus ada perpaduan antara kepuasan akan pemenuhan kebutuhan mereka sendiri dan permintaan organisasi. Berdasarkan penjelasan di atas bahwa dengan memberikan kepuasan yang tepat, pegawai akan terdorong untuk berbuat semaksimal mungkin dalam melaksanakan tugasnya, dan mereka meyakini bahwa dengan keberhasilan organisasi mencapai tujuan dan berbagai sasarannya, maka kepentingan- kepentingan pribadinya akan terpelihara pula. Agar dapat mewujudkan pegawai yang memiliki kepuasan dan kinerja yang tinggi diperlukan pula peran yang besar dari pimpinan organisasi, karena dalam perubahan organisasibaik yang terencana maupun tidak terencana, aspek yang terpenting adalah perubahan individu. Perubahan pada individu tidak mudah, tetapi harus melalui proses. Pemimpin sebagai panutan dalam organisasi, sehingga perubahan harus dimulai dari tingkat yang paling atas (pemimpin). 1. Latar Belakang Masalah Untuk itu organisasi memerlukan pemimpin yang reformis yang mampu menjadi motorpenggerak perubahan organisasi. Kemampuan kerja pegawai juga menjadi faktor penentu keberhasilan suatu organisasi karena sebagai motor penggerak organisasi kemampuan pegawai menjadi salah satu syarat mutlak apabila menginginkan pencapaian tujuan organisasi. Penelitian ini memfokuskan pada pegawai terutama Kinerja pegawai Dinas Kependudukan dan Pencatatan Sipil kota Palembang. Kinerja pegawai yang tinggi sangatlah diharapkan oleh instansi tersebut, Semakin banyak pegawai yang mempunyai kinerja yang tinggi, maka produktivitas instansi secara keseluruhan akan meningkat pula. Pegawai dituntut untuk mampu menyelesaikan tugas dan tanggung jawabnya secara efektif dan efisien. Kinerja pegawai Dinas Kependudukan dan Pencatatan Sipil kota Palembangdapat diukur melalui penyelesaian tugasnya secara efektif dan efisien serta melakukan peran dan fungsinya dan itu semua berhubungan linear dan berhubungan positif bagi keberhasilan instansi. Dihubungkan dengan keadaan sebenarnya dilapangan kinerja pegawai masih belum sesuai dengan keinginaninstansi hal tersebut dapat dilihat melalui hasil kerja yang dicapai oleh pegawaiyang belum sesuai dengan efektivitas dan efisiensi yang ingin dicapai oleh instansi. Pencapaian kinerja yang optimal pada Dinas kependudukan dan pencatatansipil kota Palembang diharapkan dapat dicapai oleh pegawai melalui pengetahuannya tentang 298 M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai…… komitmen affektif serta kualitas kinerja yang dibangun oleh pegawai bersama-sama dengan instansi, dengan kepemimpinanyang sejalan dengan tujuan instansi serta karakter pegawai yang mengacu padaperbaikan kemampuan kerja pegawai dalam melaksanakan tanggung jawabnyasehingga Dinas kependudukan dan pencatatan sipil kota Palembang mampu mencapai tujuan yang ditargetkan melalui optimalisasi kinerja pegawai yang belum tercapai pada kenyataannya. Fenomena yang terjadi di Dinas kependudukan dan pencatatan sipil kota Palembang, diantaranya adalah menurunnya keinginan pegawai untukmencapai prestasi kerja, ketepatan waktu dalam menyelesaikan pekerjaan hingga kedisiplinan, dan pengaruh yang berasal dari lingkungannya, teman kerja yang menurun semangatnya dan tidak ada contoh yang harus dijadikan acuan dalam pencapaian prestasi kerja yang baik. Sebagai instansi yang bergerak dibidang pelayanan publik yang menuntut segala sesuatunya termasuk target pekerjaan dapat terlaksana tepat waktu. Pada penelitian ini penulis lebih memfokuskan pada pengaruh Komitmen Affektif dan kepuasan kerja pegawai di dinas kependudukan dan pencatatan sipil kota Palembang, karena dilihat dari beberapa fenomena yang ada. Berdasarkan uraian diatas, penulis tertarik mengangkat masalah mengenai “Pengaruh Komitmen Affektif dan kepuasan kerja terhadap kinerja pegawai pada Dinas Kependudukan dan pencatatan sipil kota Palembang”. 4. Manfaat Penelitian Berdasarkan latar belakang dalam penelitian ini peneliti berharap dapat memberikan manfaat pada banyak pihak: Berdasarkan latar belakang dalam penelitian ini peneliti berharap dapat memberikan manfaat pada banyak pihak: a. Secara Teoritis 3. Tujuan Penelitian Berdasarkan uraian penelitian diatas, tujuan penelitian ini adalah untuk mengetahui: a. Pengaruh Komitmen Affektif terhadap kinerja kerja pada Dinas Kependudukan dan pencatatan sipil kota Palembang. b. Pengaruh kepuasaan kerja terhadap kinerja pada Dinas Kependudukan dan pencatatan sipil kota Palembang. c. Pengaruh Komitmen Affektif dan kepuasaan kerja terhadap kinerja pegawai pada Dinas Kependudukan dan pencatatan sipil kota Palembang. 2. Masalah Penelitian Berdasarkan tema yang diambil mengenai Pengaruh Komitmen Affektif dan kualitas kinerja terhadap kepuasaan kerja pegawai pada Dinas Kependudukan dan pencatatan sipil kota Palembang, maka dapat diidentifikasikan masalah dari penelitian ini adalah : a. Bagaimana Pengaruh Komitmen Affektif secara parsial terhadap kepuasaan kerja pegawai pada Dinas Kependudukan dan pencatatan sipil kota Palembang ? a. Bagaimana Pengaruh Komitmen Affektif secara parsial terhadap kepuasaan kerja pegawai pada Dinas Kependudukan dan pencatatan sipil kota Palembang ? b. Bagaimana Pengaruh kualitas kinerja secara parsial terhadap kepuasaan kerja pegawai pada Dinas Kependudukan dan pencatatan sipil kota Palembang c. Bagaimana Pengaruh Komitmen Affektif dan kualitas kinerja terhadap kepuasaan kerja pegawai pada Dinas Kependudukan dan pencatatan sipil kota Palembang ? 299 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│299 a. Definisi Kinerja a. Definisi Kinerja Kinerja apabila dikaitkan dengan performance sebagai kata benda (noun), maka pengertian performance atau kinerja adalah hasil kerja yang dapat dicapai oleh seseorang atau kelompok orang dalam suatu perusahaansesuai dengan wewenang dan tanggung jawab masing-masing dalam upaya pencapaian tujuan perusahaan secara ilegal, tidak melanggar hukumdan tidak bertentangan dengan moral dan etika (Rivai & Basri, 2004; Harsuko 2011). Dilihat dari sudut pandang ahli yang lain, kinerja adalah banyaknyaupaya yang dikeluarkan individu pada pekerjaannya (Robbins, 2001). Sementara itu menurut Bernandi & Russell 2001 (dalam Riani 2011) performansi adalah catatan yang dihasilkan dari fungsi suatu pekerjaan tertentu atau kegiatan selama periode waktu tertentu. b. Kriteria-Kriteria Kinerja TINJAUAN PUSTAKA 1. Kinerja M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… 300│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… a. Secara Teoritis Penulis berharap hasil penulisan ini dapat memberikan sumbangan bagi pengembangan ilmu pengetahuan tentang Komitmen affektif dan juga kepuasan kerja yang baik untuk mencapai kinerja yang baik . b. Secara Praktis 1) Bagi Instansi 1) Bagi Instansi Sebagai bahan informasi dan masukan bagi instansi dalam meningkatkan Kinerja yang maksimal pada dinas capil kota Palembang. Sebagai bahan informasi dan masukan bagi instansi dalam meningkatkan Kinerja yang maksimal pada dinas capil kota Palembang. 2) Bagi Pihak Lain 2) Bagi Pihak Lain Hasil penelitian ini diharapkan dapat dijadikan referensi bagi peneliti lain yang melakukan penelitian dengan topik yang sama Hasil penelitian ini diharapkan dapat dijadikan referensi bagi peneliti lain yang melakukan penelitian dengan topik yang sama b. Kriteria-Kriteria Kinerja Kriteria kinerja adalah dimensi-dimensi pengevaluasian kinerja seseorang pemegang jabatan, suatu tim, dan suatu unit kerja. Secara bersama-sama dimensi itu merupakan harapan kinerja yang berusaha dipenuhi individu dan tim guna mencapai strategi organisasi. Menurut Schuler dan Jackson 2004 (dalam Harsuko 2011) bahwa ada 3 jenis dasar kriteria kinerja yaitu: Menurut Schuler dan Jackson 2004 (dalam Harsuko 2011) bahwa ada 3 jenis dasar kriteria kinerja yaitu: 1. Kriteria berdasarkan sifat memusatkan diri pada karakteristik pribadi seseorang karyawan. Loyalitas, keandalan, kemampuan berkomunikasi, dan keterampilan memimpin merupakan sifat-sifat yang sering dinilai selama proses penilaian. Jenis kriteria ini memusatkan diri pada bagaimana seseorang, bukan apa yang dicapai atau tidak dicapai seseorang dalam pekerjaanya. 2. Kriteria berdasarkan perilaku terfokus pada bgaimana pekerjaan dilaksanakan. Kriteria semacam ini penting sekali bagi pekerjaan yang membutuhkan hubungan antar personal. Sebagai contoh apakah SDM- nyaramah atau menyenangkan. 3. Kriteria berdasarkan hasil, kriteria ini semakin populer dengan makin ditekanya produktivitas dan daya saing internasional. Kreteria ini berfokus pada apa yang telah 3. Kriteria berdasarkan hasil, kriteria ini semakin populer dengan makin ditekanya produktivitas dan daya saing internasional. Kreteria ini berfokus pada apa yang telah │301 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│301 dicapai atau dihasilkan ketimbang bagaimana sesuatu dicapai atau dihasilkan. Menurut Bernandin & Russell (2001 dalam Riani 2011) kriteria yang digunakan untuk menilai kinerja karyawan adalah sebagai berikut: 1) Quantity of Work (kuantitas kerja): jumlah kerja yang dilakukan dalam suatu periode yang ditentukan. 2) Quality of Work (kualitas kerja): kualitas kerja yang dicapai berdasarkan syarat- syarat kesesuaian dan ditentukan. 3) Job Knowledge (pengetahuan pekerjaan): luasnya pengetahuan mengenai pekerjaan dan keterampilannya. 4) Creativeness (kreativitas): keaslian gagasan-gagasan yang dimunculkan dan tindakan-tindakan untuk menyelesaikan persoalan yangtimbul. 5) Cooperation (kerja sama): kesedian untuk bekerjasama dengan orang lain atau sesama anggota organisasi. c. Faktor-Faktor yang Mepengaruhi Kinerja c. Faktor-Faktor yang Mepengaruhi Kinerja Menurut Steers (dalam Suharto & Cahyono 2005) faktor-faktor yang mempengaruhi kinerja adalah: 1. Kemampuan, kepribadian dan minat kerja. 1. Kemampuan, kepribadian dan minat kerja. 2. Kejelasan dan penerimaan atau kejelasan peran seseorang pekerja yang merupakan taraf pengertian dan penerimaan seseprang atas tugas yang diberikan kepadanya. 3. Tingkat motivasi pekerja yaitu daya energi yang mendorong, mengarahkan dan mempertahankan perilaku. b. Faktor-faktor Komitmen Semakin tinggi komitmen seorang pegawai maka akan semakin tinggi pula kinerja yang dicapainya. ketika komitmen diwujudkan dalam bentuk kesetiaan pengabdian pada organisasi. Namun dalam kenyataannya tidak semua pegawai melaksanakan komitmen seutuhnya. Ada komitmen kerja yang tinggi dan ada yang rendah. Faktor-faktor yang mempengaruhi komitmen adalah faktor intrinsik dan ekstrinsik dari pegawai yang bersangkutan. Adapun Faktor-faktor intrinsik karyawan meliputi aspek-aspek kondisi sosial ekonomi keluarga pegawai, usia pegawai, pendidikan pendidikan, pengalaman kerja, kestabilan kepribadian, dan gender. Sementara itu untuk faktor ekstrinsik yang dapat mendorong terjadinya komitmen tertentu antara lain adalah keteladanan pihak manajemen khususnya manajemen puncak dalam berkomitmen di berbagai aspek organisasi. c. Indikator komitmen kerja 02│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… tertentu,2). keinginan untuk berusaha keras sesuai keinginan organisasi 3). keyakinan tertentu, dan penerimaan nilai dan tujuan organisasi. tertentu,2). keinginan untuk berusaha keras sesuai keinginan organisasi 3). keyakinan tertentu, dan penerimaan nilai dan tujuan organisasi. b. Faktor-faktor Komitmen c. Indikator komitmen kerja Komitmen organisasi bersifat multidimensi, oleh karena itu Allen dan Meyer (dalam Luthans, 2006) membedakan bentuk komitmenorganisasi yang dibagi atas tiga komponen, yaitu: a. Affective commitment a. Affective commitment Merupakan hal yang berkaitan dengan keterikatan emosional atau emotional attachment, identifikasi, dan keterlibatan individu di dalam suatu organisasi. Individu yang memiliki komitmen afektif yang kuat akan terus bekerja dalam organisasi karena mereka memang ingin (want to) melakukan hal tersebut. b. Continuance commitment a. Pengertian Komitmen Organisasi Komitmen organisasi muncul sebagai topik penting dalam studi danperusahaan. Para peneliti memandang komitmen organisasi sebagai tantangan utama pada abad ke- 21. Individu yang loyal terhadap organisasi akan selalu bekerja dengan organisasi dan terus berusaha untuk mencapai tujuan organisasi. Sebaliknya, individu yang tidak berkomitmen tidak termotivasi untuk mencapai tujuan organisasi. Komitmen organisasi didefinisikan sebagai : 1). keinginan kuat untuk tetap sebagai anggota organisasi 302│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… tertentu,2). keinginan untuk berusaha keras sesuai keinginan organisasi 3). keyakinan tertentu, dan penerimaan nilai dan tujuan organisasi. 2│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… c. Normative commitment Komitmen normatif merupakan perasaan-perasaan individu tentangkewajiban yang harus ia berikan kepada organisasi, karena tindakantersebut merupakan hal benar yang harus dilakukan. Hal ini berarti individu dengan komitmen normatif yang tinggi akan merasa bahwamereka wajib (ought to) bertahan dalam organisasi dimana mereka bergabung. Kepuasan Kerja b. Continuance commitment Disebut juga komitmen berkelanjutan berkaitan denganpersepsi individu tentang kerugian yang akan dihadapinya jika ia meninggalkan organisasi. Jadi individu akan mempertimbangkan untung rugi apabila ingin tetap bergabung dengan organisasi atau justru meninggalkan organisasi. Hal ini mungkin karena hilangnya senioritas, promosi, atau benefit. Individu yang bekerja berdasarkankomitmen kontinuans ini akan bertahan 303 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│303 dalam organisasi karena memang mereka butuh (need to) melakukan hal tersebut karena tidak adanya pilihan lain. c. Normative commitment 304│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… 2) Faktor pekerjaan, yaitu jenis pekerjaan, struktur organisasi, pangkat (golongan), kedudukan, mutu pengawasan, jaminan finansial, kesempatan promosi jabatan, interaksi sosial, dan hubungan kerja. 2) Faktor pekerjaan, yaitu jenis pekerjaan, struktur organisasi, pangkat (golongan), kedudukan, mutu pengawasan, jaminan finansial, kesempatan promosi jabatan, interaksi sosial, dan hubungan kerja. b. Indikator-Indikator Kepuasan Kerja b. Indikator-Indikator Kepuasan Kerja Setiap karyawan memiliki tolak ukur kepuasan kerja yang berbeda standar kepuasannya.. Adapun indikator kepuasan kerja menurut Hasibuan (2013:203) antara lain: 1) Balas jasa yang adil dan layak, merupakan semua pendapatan yang berbentuk uang, barang langsung atau tidak langsung yang diterima karyawan sebagai imbalan atas jasa yang diberikan kepada perusahaan. 2) Penempatan yang sesuai dengan keahlian menempatkan posisi seseorang ke posisi pekerjaan yang tepat, seberapa baik seseorang karyawan cocok dengan pekerjaannya akan mempengaruhi jumlah dan kualitas pekerjaan. 3) Berat-ringannya pekerjaan, merupakan sekumpulan sejumlah kegiatan yang harus diselesaikan oleh seseorang atau sekelompok orang selama periode waktu tertentu sesuai dengan kemampuan pekerja. 4) Suasana dan lingkungan kerja, merupakan suasana dimana karyawan melakukan aktivitas kerjanya setiap hari dan lingkungan kerja merupakan segala sesuatu yang ada disekitar pekerja yang dapat mempengaruhi tugas yang dibebankan kepada pekerja. 5) Sikap pimpinan dalam kepemimpinannya, merupakan seseorang yang menggunakan wewenang formal untuk mengorganisasikan, mengarahkan, mengontrol, para bawahan yang bertanggung jawab, supaya semua pekerjaan dikoordinasi demi mencapai tujuan perusahaan. 5) Sikap pimpinan dalam kepemimpinannya, merupakan seseorang yang menggunakan wewenang formal untuk mengorganisasikan, mengarahkan, mengontrol, para bawahan yang bertanggung jawab, supaya semua pekerjaan dikoordinasi demi mencapai tujuan perusahaan. 3. Pengertian Kepuasan Kerja Kepuasan kerja merupakan salah satu faktor yang sangat penting untuk mendapatkan hasil kerja yang optimal. Ketika seseorang merasakan kepuasan dalam bekerja ia akan berupaya semaksimal mungkin dengan segenap kemampuan yang dimilikinya untuk menyelesaikan tugas pekerjaannya. Dengan demikian produktivitas dan hasil kerjanyan akan meningkat secara optimal. Menurut Robbins (2015:170) disebutkan bahwa kepuasan kerja adalah suatusikap umum terhadap pekerjaan seseorang sebagai perbedaan antara banyaknya ganjaran yang diterima pekerja dengan banyaknya ganjaran yang diyakini seharusnya diterima. Menurut Priansa (2014:291) kepuasan kerja merupakan perasaan pegawai terhadap pekerjaannya, apakah senang/ suka atau tidak senang/ tidak suka sebagai hasil interaksi pegawai dengan lingkungan pekerjaannya atau sebagai persepsi sikap mental, juga sebagai hasil penilaian pegawai terhadap pekerjaannya. Berdasarkan pengertian para ahli diatas dapat disimpulkan bahwa kepuasankerja adalah sikap positif atau keadaan emosional yang dimiliki seorang karyawan terhadap pekerjaan dan kondisi kerja yang terkait. a. Faktor-Faktor yang Mempengaruhi Kepuasan Kerja (2015) Faktor-faktor yang mempengaruhi kepuasan kerja, menurut Mangkunegara (2015) yaitu: 1) Faktor pegawai, yaitu kecerdasan (IQ), kecakapan khusus, umur, jenis kelamin, kondisi fisik, pendidikan, pengalam kerja, masa kerja, kepribadian, emosi, cara berfikir, persepsi, dan sikap kerja. 304│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… Gambar 2. Kerangka pikir penelitian Berdasarkan gambar 2 diatas, dapat dijelaskan bahwa variabel bebas (independent) dalam penelitian ini adalah Komitmen (X1), Kepuasan (X2), dan variabel terikat (dependent) Kinerja (Y). 4. Penelitian terdahulu Hasil penelitian dari Novita Dkk (2016) yang berjudul “Pengaruh Kepuasan Kerja Dan Komitmen Organisasional Terhadap Kinerja Karyawan” menyimpulkan bahwa . Hasil penelitian ini menunjukkan bahwa kepuasan kerja dan komitmen organisasional berpengaruh secara simultan terhadap kinerja karyawan dengan nilai sig.F 0,000 < α = 0,05. Kepuasan kerja berpengaruh secara parsial terhadap kinerja karyawan dengan nilai 305 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023 sig.t 0,000 < α = 0,05 namun, komitmen organisasional tidak berpengaruh secara parsial terhadap kinerja karyawan dengan nilai sig.t 0,344 > α = 0,05. Beberapa penelitian terdahulu yang dilakukan oleh para peneliti menemukan hasil yang berbeda mengenai hubungan komitmen organisasional dengan kinerja. Menurut hasil penelitian Keller (1997) hanya ditemukan hubungan yang lemah antara komitmen organisasional dan kinerja. Kemudian Suliman dan Iles (2000 b) dalam penelitiannya dikawasan Timur Tengah (Yordania) menemukan bahwa affective commitment, continuance commitment, serta normative commitment mempunyai hubungan yang positif dengan kinerja karyawan. 5. Kerangka penelitian Menurut Sugiyono (2014) mengemukakan bahwa kerangka berpikir merupakan model konseptual tentang bagaimana teori berhubungan denganberbagai faktor yang telah diidentifikasi sebagai masalah yang penting. Berikut ini adalah kerangka pemikiran yang digunakan dalam penelitian ini: Kepuasan Kerja Komitmen Kinerja Gambar 2. Kerangka pikir penelitian Kinerja Kepuasan Kerja Gambar 2. Kerangka pikir penelitian 306│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… H3 : Diduga Komitmen dan Kepuasan kerja berpengaruhterhadap Kinerja pegawai. H3 : Diduga Komitmen dan Kepuasan kerja berpengaruhterhadap Kinerja pegawai. METODE PENELITIAN Penelitian ini merupakan penelitian kuantitatif dengan menggunakan data primer dan data sekunder. Teknik pengumpulan data yang diambil oleh peneliti diperoleh dari kuisioner, observasi dan dokumentasi. Pendekatan yang digunakan dalam penelitian ini dengan menggunakan pendekatan kuantitatif di bantu dengan program SPSS, informasi di penelitian ini mengenai pengaruh komitmen dan kepuasan kerja terhadap kinerja pegawai di penelitian ini didapat dari informasi yang diberikan kepada karyawan melalui kuesioner untuk memperoleh data yang akurat. Sampel dalam penelitian ini meliputi 106 pegawai Dinas Kependudukan dan catatan sipil dan Teknik analisis menggunakan uji validitas, uji reliabilitas, uji normalitas, uji multikolinieritas, analisis deskriptif, analisis regresi linier berganda, uji parsial (uji T), uji simultan (uji F), koefisien korelasi (R), dan koefisien determinasi (R2). 6. Hipotesis Penelitian Hipotesis pada penelitian ini adalah sebagai berikut : H1 : Diduga Komitmen berpengaruh terhadap Kinerja pegawai. H2 : Diduga Kepuasan kerja berpengaruh terhadap Kinerja pegawai. 1. Uji Validitas Uji validitas dilakukan untuk mengukur tepat atau tidak indikator atau kuesioner dari masing-masing variabel. Pengujian dilakukan dengan membandingkan antara r- hitung dengan r-tabel menggunakan program SPSS. Pengujian validitas data dengan menggunakan uji dua sisi dengan taraf signifikan 5%. Pengujian validitas ini dilakukan terhadap 106 responden, maka r-tabel df = n-2 dengan taraf signifikan 5%, df = 106-2, maka r-tabel = 0,1606. Tingkat kevalidan indikator atau kuesioner dapat ditentukan, apabila r- hitung > r- tabel = valid dan r-hitung < r-tabel = tidak valid (Sugiyono 2017:176).Hasil uji validitas dapat dilihat dari tabel dibawah ini dengan menggunakan r- hitung dan r-tabel, sebagai berikut : Hasil Uji Validitas Komitmen Item Pertanyaan R Hitung R Tabel Keterangan Pertanyaan 1 0,446 0,279 Valid Pertanyaan 2 0,562 0,279 Valid 307 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│307 Pertanyaan 3 0,449 0,279 Valid Pertanyaan 4 0,533 0,279 Valid Pertanyaan 5 0,473 0,279 Valid Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│307 Pertanyaan 3 0,449 0,279 Valid Pertanyaan 4 0,533 0,279 Valid Pertanyaan 5 0,473 0,279 Valid Dari table diatas dapat dilihat bahwa dari keseluruhan item pertanyaan yang ada memiliki hasil uji validitas dengan nilai R Hitung lebih besar dari R Tabel, dengan demikian dapat dinyatakan bahwa seluruh data Komitmen adalah valid. Hasil Uji Validitas Kepuasan Kerja Item Pertanyaan R Hitung R Tabel Keterangan Pertanyaan 1 0,545 0,279 Valid Pertanyaan 2 0,391 0,279 Valid Pertanyaan 3 0,449 0,279 Valid Pertanyaan 4 0,529 0,279 Valid Pertanyaan 5 0,406 0,279 Valid Dari table diatas dapat dilihat bahwa dari keseluruhan item pertanyaan yang ada memiliki hasil uji validitas dengan nilai R Hitung lebih besar dari R Tabel, dengan demikin dapat dinyatakan bahwa seluruh data Kepuasan kerja adalah valid. Hasil Uji Validitas Kinerja Item Pertanyaan R Hitung R Tabel Keterangan Pertanyaan 1 0,502 0,279 Valid Pertanyaan 2 0,337 0,279 Valid Pertanyaan 3 0,616 0,279 Valid Pertanyaan 4 0,459 0,279 Valid Pertanyaan 5 0,530 0,279 Valid Dari table diatas dapat dilihat bahwa dari keseluruhan item pertanyaan yang ada memiliki hasil uji validitas dengan nilai R Hitung lebih besar dari R Tabel, dengan demikin dapat dinyatakan bahwa seluruh data Kinerja adalah valid. 2. Uji Reliabilitas Hasil Uji Reliabilitas Komitmen Hasil Uji Reliabilitas Komitmen Reliability Statistics Cronbach's Alpha N of Items .650 6 Sumber: SPSS 24.0 Dari table diatas dapat dilihat bahwa nilai Cronbach’s Alpha berada pada angka 0,650 yang mana angka ini lebih besar dari standar reliabilitas dengan nilai 0,60. Dengan demikian variable Komitmen dapat dinyatakan reliabel. 308│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… Hasil Uji Reliabilitas Kepuasan Kerja Reliability Statistics Cronbach's Alpha N of Items .649 6 Sumber: SPSS 24.0 308│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… Hasil Uji Reliabilitas Kepuasan Kerja Reliability Statistics Cronbach's Alpha N of It Hasil Uji Reliabilitas Kepuasan Kerja Reliability Statistics Cronbach's Alpha N of Items .649 er: SPSS 24.0 Dari table diatas dapat dilihat bahwa nilai Cronbach’s Alpha berada pada angka 0,649 yang mana angka ini lebih besar dari standar reliabilitas dengan nilai 0,60. Dengan demikian variable Kepuasan Kerja dapat dinyatakan reliabel. Hasil Uji Reliabilitas Kinerja Reliability Statistics Cronbach's Alpha N of Items .647 6 Sumber: SPSS 24.0 Hasil Uji Reliabilitas Kinerja Reliability Statistics Cronbach's Alpha N of Items .647 6 Sumber: SPSS 24.0 Dari table diatas dapat dilihat bahwa nilai Cronbach’s Alpha berada pada angka 0,647 yang mana angka ini lebih besar dari standar reliabilitas dengan nilai 0,60. Dengan demikian variable Kinerja dapat dinyatakan reliabel. 3. Uji Normalitas 3. Uji Normalitas Sumber: SPSS 24.0 Sumber: SPSS 24.0 Sumber: SPSS 24.0 Sumber: SPSS 24.0 Sumber: SPSS 24.0 Berdasar gambar diatas terlihat bahwa menyebar agak kekanan bagian kurva normal, sehingga dapat disimpulkan residual memenuhi asumsi normalitas. │309 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023 Sumber: SPSS 24 0 Gambar P-Plot Sumber: SPSS 24.0 Gambar P-Plot Berdasarkan gambar grafik p-p plot juga menunjukkan kesimpulan erupa dengan histrogram. Dari tampilan diatas terlihat bahwa ada data yang sedikit menyebar keluar garis diagonal, sehingga masih dapat dinyatakan normal. S b SPSS 13 0 G b S tt l t Sumber: SPSS 13.0 Gambar Scatterplot Sumber: SPSS 13.0 Gambar Scatterplot Berdasarkan gambar scatterplot juga menunjukkan kesimpulan serupa dengan histogram dan p-plot. Dari tampilan diatas dapat dilihat titik-titik menyebar dan tidak berkumpul pada satu titik, sehingga dapat dikatakan data masih normal. B = Koefisien Regresi Berdasarkan persamaan regresi diatas, maka peneliti membuat penjabaran perumusan penelitian sebagai berikut : Berdasarkan persamaan regresi diatas, maka peneliti membuat penjabaran perumusan penelitian sebagai berikut : Coefficientsa Unstandardized Coefficients Standardized Coefficients Beta t Sig. Model B Std. Error 1 (Constant) 11.254 2.836 3.968 .000 TOTAL.X1 .434 .107 .512 4.040 .000 TOTAL.X2 .073 .090 .103 .811 .422 a. Dependent Variable: TOTAL.Y Sumber: SPSS 24.0 Berdasarkan dari nilai-nilai koefisien diatas, dapat disusun persamaan regresi sebagai berikut :Y = α + β₁X₁ + β₂X₂ Y = 11,254 + 0,434X1 + 0,073X2 Y = 11,254 + 0,434X1 + 0,073X2 Y = 11,254 + 0,434X1 + 0,073X2 Dimana : Dimana : b₀ = 11,254 yang berarti angka tersebut menunjukkan tingkat Kinerja pegawai bila tingkat Kepuasan Kerja dan Motivasi Kerja diabaikan. b₀ = 11,254 yang berarti angka tersebut menunjukkan tingkat Kinerja pegawai bila tingkat Kepuasan Kerja dan Motivasi Kerja diabaikan. b₁ = 0,434 yang berarti setiap kenaikkan tingkat komitmen sebesar 1 persen, maka Kinerja akan mengalami peningkatan sebesar 0,434. b₂ = 0,073 yang berarti setiap kenaikkan tingkat kepuasan kerja sebesar 1 persen, maka kinerja mengalami peningkatan sebesar 0,073. 5. Uji Koefisien Determinasi (R2) Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate Durbin-Watson 1 .514a .265 .233 2.402 1.458 a. Predictors: (Constant), TOTAL.X2, TOTAL.X1 b. Dependent Variable: TOTAL.Y Sumber: SPSS 24.0 4. Analisis Regresi Linier Berganda Bentuk umum persamaan regresi yang digunakan adalah regresi berganda, yaitu sebagai berikut: Y = α + β₁X₁ + β₂X₂ 10│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… │ ; g p j p j g Dimana : Y = Kinerja X1 = Komitmen X2 = Kepuasan kerja B = Koefisien Regresi Dimana : Y = Kinerja X1 = Komitmen X2 = Kepuasan kerja 6. Hasil Uji F (Simultan) 6. Hasil Uji F (Simultan) ANOVAa Model Sum of Squares df Mean Square F Sig. 1 Regression 95.507 2 47.753 8.280 .001b Residual 265.310 46 5.768 Total 360.816 48 c. Dependent Variable: TOTAL.Y d. Predictors: (Constant), TOTAL.X2, TOTAL.X1 Sumber: SPSS24.0 Pada Tabel anova diperoleh nilai Fhitung sebesar 8,280 dan nilai sig 0,001 dimana nilai Ftabel untuk 50 sampel dengan probability 0,05 ialah sebesar 4,03 sehingga dapat disimpulkan bahwa 8,280 > 4,03, lalu sig 0,001 < 0,05 maka hasilnya komitmen dan kepuasan kerja secara bersama-sama (simultan) berpengaruh terhadap variabel kinerja pada kantor dinas disdukcapil Palembang walau dengan kontribusi yang berbeda-beda Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│3 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│3 Berdasarkan hasil perhitungan melalui program SPSS v.24 dapat diinterpretasikan sebagai berikut: Berdasarkan hasil perhitungan melalui program SPSS v.24 dapat diinterpretasikan sebagai berikut: a. Pada Model Summary diperoleh nilai R = 0,514 yang berarti bahwa hubungan variabel bebas yaitu komitmen dan kepuasan kerja dengan variabel terikat yaitu kinerja adalah kuat dan memiliki arah yang positif. b. Angka R Square sebesar 0,265 memberikan makna bahwa secara simultan atau serentak kedua variabel bebas yaitu komitmen dan kepuasan kerja mampu menjelaskan perubahan terhadap variabel kinerja sebesar 26,5% dan 73,5% lainnya dipengaruhi oleh variabel-variabel lain yang tidak termasuk dalam model regresi ini. 6. Hasil Uji F (Simultan) 6. Hasil Uji F (Simultan) 5. Uji Koefisien Determinasi (R2) 5. Uji Koefisien Determinasi (R2) │311 7. Hasil Uji t (Parsial) 7. Hasil Uji t (Parsial) Coefficientsa Unstandardized Coefficients Standardized Coefficients Beta t Sig. Model B Std. Error 1 (Constant) 11.254 2.836 3.968 .000 TOTAL.X1 .434 .107 .512 4.040 .000 TOTAL.X2 .073 .090 .103 .811 .422 a. Dependent Variable: TOTAL.Y Sumber: SPSS 24.0 7. Hasil Uji t (Parsial) Coefficientsa Unstandardized Coefficients Standardized Coefficients Beta t Sig. Model B Std. Error 1 (Constant) 11.254 2.836 3.968 .000 TOTAL.X1 .434 .107 .512 4.040 .000 TOTAL.X2 .073 .090 .103 .811 .422 a. Dependent Variable: TOTAL.Y Sumber: SPSS 24.0 1. Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel komitmen memiliki pengaruh secara parsial terhadap disdukcapil 1. Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel komitmen memiliki pengaruh secara parsial terhadap disdukcapil 1. Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel komitmen memiliki pengaruh secara parsial terhadap disdukcapil 312│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… Palembang. Variabel komitmen berpengaruh secara signifikan terhadap prestasi kerja di Disdukcapil Palembang dalam angka signifikansi 0,000 lebih kecil dari taraf signifikansi 0,05, dengan demikian dapat dinyatakan bahwa komitmen berpengaruh terhadap kinerja di disdukcapil Palembang. 312│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… Palembang. Variabel komitmen berpengaruh secara signifikan terhadap prestasi kerja di Disdukcapil Palembang dalam angka signifikansi 0,000 lebih kecil dari taraf signifikansi 0,05, dengan demikian dapat dinyatakan bahwa komitmen berpengaruh terhadap kinerja di disdukcapil Palembang. 312│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… Palembang. Variabel komitmen berpengaruh secara signifikan terhadap prestasi kerja di Disdukcapil Palembang dalam angka signifikansi 0,000 lebih kecil dari taraf signifikansi 0,05, dengan demikian dapat dinyatakan bahwa komitmen berpengaruh terhadap kinerja di disdukcapil Palembang. 2. Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel kepuasan Kerja tidak memiliki pengaruh secara parsial terhadap prestasi kerja di Disdukcapil Palembang. Variabel kepuasan kerja pada disdukcapil Palembang dalam angka 0,422 lebih besar dari taraf signifikansi 0,05. Dengan demikian tidak adanya pengaruh secara parsial dari kepuasan kerja terhadap kinerja di disdukcapil Palembang. 1. Simpulan Berdasarkan data yang diperoleh dan diolah kembali, maka hasil penelitian tentang Pengaruh Komitmen dan Kepuasan Kerja terhadap Motivasi Pegawai Dinas Kependudukan dan Pencatatan Sipil kota Palembang dapat disimpulkan bahwa: 1. Untuk uji F. komitmen dan kepuasan kerja secara bersama-sama (simultan) berpengaruh terhadap variabel kinerja pada kantor dinas disdukcapil Palembang walau dengan kontribusi yang berbeda-beda 2. Pada Model Summary diperoleh nilai R = 0,514 yang berarti bahwa hubungan variabel bebas yaitu komitmen dan kepuasan kerja dengan variabel terikat yaitu kinerja adalah kuat dan memiliki arah yang positif. 3. Angka R Square sebesar 0,265 memberikan makna bahwa secara simultan atau serentak kedua variabel bebas yaitu komitmen dan kepuasan kerja mampu menjelaskan perubahan terhadap variabel kinerja sebesar 26,5% dan 73,5% lainnya dipengaruhi oleh variabel lain yang tidak termasuk dalam model regresi ini. 4. Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel komitmen memiliki pengaruh secara parsial terhadap disdukcapil Palembang. Variabel komitmen berpengaruh secara signifikan terhadap │313 Ekonomica Sharia: Jurnal Pemikiran dan Pengembangan Ekonomi Syariah Volume 8 Nomor 2 Edisi Februari 2023│3 prestasi kerja di Disdukcapil Palembang dalam angka signifikansi 0,000 lebih kecil dari taraf signifikansi 0,05, dengan demikian dapat dinyatakan bahwa komitmen berpengaruh terhadap kinerja di disdukcapil Palembang. 5. Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel kepuasan Kerja tidak memiliki pengaruh secara parsial terhadap prestasi kerja di Disdukcapil Palembang. Variabel kepuasan kerja pada disdukcapil Palembang dalam angka 0,422 lebih besar dari taraf signifikansi 0,05. Dengan demikian tidak adanya pengaruh secara parsial dari kepuasan kerja terhadap kinerja di disdukcapil Palembang 5. Berdasarkan hasil perhitungan, dapat disimpulkan bahwa variabel kepuasan Kerja tidak memiliki pengaruh secara parsial terhadap prestasi kerja di Disdukcapil Palembang. Variabel kepuasan kerja pada disdukcapil Palembang dalam angka 0,422 lebih besar dari taraf signifikansi 0,05. Dengan demikian tidak adanya pengaruh secara parsial dari kepuasan kerja terhadap kinerja di disdukcapil Palembang 2. Saran Berdasarkan kesimpulan yang diperoleh dari hasil penelitian, maka terdapat beberapa saran yang penulis berikan yang berhubungan dengan Komitmen dan Kepuasan Terhadap Kinerja pegawai pada Dinas Kependudukandan Pencatatan Sipil Kota Palembang antara lain : a. Seluruh pegawai pada Dinas Kependudukan dan Pencatatan Sipil Kota Palembang agar dapat terus meningkatkan komitmennya dengan cara menjaga hubungan antar pegawai tetap harmonis agar aktivitas dalam bekerja bisa berjalan dengan baik. b. Seluruh pegawai pada Dinas Kependudukan dan Pencatatan Sipil Kota Palembang agar dapat terus menjaga dan meningkatkan kinerja yang baik dengan meningkatkan kepuasan kerja pada Dinas Kependudukan dan Pencatatan Sipil Kota Palembang, dan instansi perlu memperhatikan faktor faktor yang mempengaruhinya seperti lingkungan kerja dan komunikasi antar pegawai. Sehingga tercipta kepuasan kerja yang pada akhirnya tingkat kepedulian pegawai terhadap kinerjanya lebih tinggi. c. Untuk peneliti selanjutnya diharapkan untuk meneliti variabel-variabel lain yang tidak termasuk di penelitian ini seperti kepemimpinan, motivasi, pemberdayaan, lingkungan kerja, disiplin kerja, stress kerja, analisis jabatan maupun yang lainya. 314│ M. Kurniawan DP; Pengaruh Komitmen Dan Kepuasan Kerja Terhadap Kinerja Pegawai……… DAFTAR PUSTAKA Mathis, R.L., dan Jackson, J.H (2006), Human Resource Management (terj.), Jilid 1, Edisi Kesepuluh, Jakarta: Salemba Empat Setyaningdyah, E., Kertahadi, U.N., dan Thoyib, A (2013), “The Effects of Human Resource Competence, Organisational Commitment and Transactional Leadership on Work Dis- cipline, Job Satisfaction and Employee’s Performance”, Interdisciplinary Journal Of Contemporary Research InBusiness, 5 (4), page 9. Meyer. J. and Allen, N. (1997), Commitment in the Workplace: Theory, Research, andAp- plication, Sage Publications. Kusumastuti, A.F., dan Nurtjahjanti, H (2013), “Komitmen Afektif Organisasi Ditinjau dari Persepsi Terhadap Kepemimpinan Transaksional pada Pekerja Pelaksanadi Perusahaan Umum (Perum) XSemarang, Jurnal Studi Manajemendan Operasi, VOL 10 NO 1, JANUARI 2013, diakses http:// ejournal.undip.ac.id/index.php/smo/issue/ view/1165 Gautam, Thanswur, Rulf van Dick, Ulrich Wagner, Narottam Upadhyay, and Ann J. Davis. (2004). Organizational Citizenship Behavior and Organization on Commitment in Nepal. Aston Bussines School.Aston University. Birmingham. Meyer, J. & Allen, N. (1991). A Three-Component Conceptualization of Organizational Commit-Ment. Human Resource Management Review Windi Dwi Aprillianto, Sri Mintarti dan Irsan Tricahyadinata. (2019), “Pengaruh peran pemimpin dan komitmen afektif terhadap kepuasan kerja dan kinerja pegawai negeri sipil bagian umum dan kepegawaian sekretariat kabupaten kutai timur”, Jurnal Manajemen - Vol. 11 (1) 2019, 83-95 Novita, Bambang Swasto Sunuharjo dan Ika Ruhana. (2016), “Pengaruh Kepuasan Kerja Dan Komitmen Organisasional Terhadap Kinerja Karyawan ”, Jurnal Administrasi Bisnis (JAB)\|Vol. 34 No. 1 Mei 2016 Bernardin, H. John and Russel, E.A. (1993). Human Resource Management, An Experiential Approach. Singapore: Mc Graw Hill International Editions, Mac Graw Hill Book Co Schuler dan Jackson (1996) Tujuan Penilaian Kinerja diakses dari http://jurnalsdm.blogspot.com/ diakses pada tanggal 9 oktober 2011 (Rivai & Basri, (2004) Penilaian Kinerja. diakses dari http://jurnal- sdm.blogspot.com/ diakses pada tanggal 9 oktober 2011.
https://openalex.org/W4306638414	https://hal.science/hal-01263540/document	English	null	Modelling time evolving interactions in networks through a non stationary extension of stochastic block models	arXiv (Cornell University)	2,015	cc-by	1,734	Modelling time evolving interactions in networks through a non stationary extension of stochastic block models Marco Corneli, Pierre Latouche, Fabrice Rossi To cite this version: Marco Corneli, Pierre Latouche, Fabrice Rossi. Modelling time evolving interactions in networks through a non stationary extension of stochastic block models. International Conference on Ad- vances in Social Networks Analysis and Mining ASONAM 2015, IEEE/ACM, Aug 2015, Paris, France. pp.1590-1591, 10.1145/2808797.2809348. hal-01263540 HAL Id: hal-01263540 https://hal.science/hal-01263540v1 Submitted on 27 Jan 2016 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 - ShareAlike 4.0 International License Modelling time evolving interactions in networks through a non stationary extension of stochastic block models Fabrice Rossi SAMM laboratory Universit´e Paris 1 Pantheon-Sorbonne Paris, France 75634 Paris Cedex 13 Email: Fabrice.Rossi@univ-paris1.fr Pierre Latouche SAMM laboratory Universit´e Paris 1 Pantheon-Sorbonne Paris, France 75634 Paris Cedex 13 Email: pierre.latouche@univ-paris1.fr Marco Corneli SAMM laboratory Universit´e Paris 1 Pantheon-Sorbonne Paris, France 75634 Paris Cedex 13 Email: Marco.Corneli@malix.univ-paris1.fr over the interval [0, T] and a partition C1, . . . , CD of the same interval1. We introduce a random vector y = {yu}u≤U, not observed, such that: Abstract—The stochastic block model (SBM) [1] describes interactions between nodes of a network following a probabilistic approach. Nodes belong to hidden clusters and the probabilities of interactions only depend on these clusters. Interactions of time varying intensity are not taken into account. By partitioning the whole time horizon, in which interactions are observed, we develop a non stationary extension of the SBM, allowing us to simultaneously cluster the nodes of a network and the ﬁxed time intervals in which interactions take place. The number of clusters as well as memberships to clusters are ﬁnally obtained through the maximization of the complete-data integrated likelihood relying on a greedy search approach. Experiments are carried out in order to assess the proposed methodology. yu = d iff Iu :=]tu−1, tu] ∈Cd, ∀d ≤D. Iu :=]tu−1, tu] ∈Cd, ∀d ≤D. We attach to y a multinomial distribution: p(y\|β, D) = Y d≤D β\|Cd\| d , where \|Cd\| = #{Iu : Iu ∈Cd}. Assuming that labels c and y are independently distributed, their joint distribution is: p(c, y\|Φ, K, D) =  Y k≤K ω\|Ak\| k    Y d≤D β\|Cd\| d  , (1) I. A NON STATIONARY STOCHASTIC BLOCK MODEL (1) We consider a set of N nodes A = {a1, . . . , aN}. Directed links between these nodes are observed over the time interval [0, T] and self loops are not considered. Nodes in A belong to K disjoint clusters: where Φ = {ω, β}. I where Φ = {ω, β}. { } We now deﬁne N Iu ij as the number of observed connections from i to j, in the time interval Iu. The following crucial assumption is made: A = ∪k≤KAk, Al ∩Ag = ∅, ∀l ̸= g. p(N Iu ij \|ci = k, cj = g, yu = d) = p(N Iu ij \|λkgd), We introduce an hidden vector c = {c1, . . . , cN} such that: where: p(N Iu ij \|λkgd) = (∆uλkgd)N Iu ij N Iu ij ! e−∆uλkgd. (2) ci = k iff i ∈Ak, ∀k ≤K (2) and assume that c follows a multinomial distribution: We note Λ the set of all parameters λkgd. We note Λ the set of all parameters λkgd. P{ci = k} = ωk with X k≤K ωk = 1. g Remarks: For i and j ﬁxed and c known, the sequence: N I1 ij , . . . , N IU ij As a consequence, modulo an independence assumption, the joint density for vector c is: is independently but not identically distributed, therefore this model is a non-stationary extension of the SBM. Moreover, this is the sequence of increments of a non-homogeneous Poisson process, counting interactions from cluster ci to cluster cj p(c\|ω, K) = Y k≤K ω\|Ak\| k , cj. where \|Ak\| = #{ai : ai ∈Ak} is the number of nodes inside cluster Ak. j Notation: In the following, for simplicity, we will note: Y k,g,d := Y k≤K Y g≤K Y d≤D and Y ci=k := Y i:ci=k In order to introduce a temporal structure, consider a sequence of equally spaced, adjacent time steps: ∆u := tu −tu−1, u ∈{1, . . . , U} 1T and U are linked by the following relation: T = ∆uU. and similarly for Q cj=g and Q yu=d. The adjacency matrix, noted N ∆, has three dimensions (N × N × U): We focused on the ﬁrst conference day, namely the twenty four hours going from 8am of June 29th to 7.59am of June 30th. 2More informations can be found at: http://www.sociopatterns.org/datasets/hypertext-2009-dynamic-contact-network [ REFERENCES [1] P. Holland, K. Laskey, and S. Leinhardt, “Stochastic blockmodels: ﬁrst steps,” Social Networks, vol. 5, pp. 109–137, 1983. [1] P. Holland, K. Laskey, and S. Leinhardt, “Stochastic blockmodels: ﬁrst steps,” Social Networks, vol. 5, pp. 109–137, 1983. I. A NON STATIONARY STOCHASTIC BLOCK MODEL The day was partitioned in small time intervals of 20 seconds in the original data frame and face-to-face proximity interactions (less than 1.5 meters) were monitored by radio badges that conference attendees volunteered to wear. Further details can be found in [4]. We considered 15 minutes time aggregations, thus leading to a partition of the day made of 96 consecutive quarter-hours (U = 96 with previous notation). cj g yu d The adjacency matrix, noted N ∆, has three dimensions (N × N × U): N ∆= {N Iu ij }i≤N,j≤N,u≤U and its observed likelihood can be computed explicitly: p(N ∆\|Λ, c, y, K, D) = Y k,g,d Y ci=k Y cj=g Y yu=d p(N Iu ij \|λkgd) = Y k,g,d (∆λkgd)Skgd Pkgd e−∆λkgdRkgd, (3) (3) Aggregated Interactions (clustered) 0 200 400 600 800 1000 Aggregated Interactions (clustered) Time Interactions 8h 10h 12h 14h 16h 18h 20h 22h Fig. 1: Aggregated interactions for each time interval where we noted: Skgd := X ci=k X cj=g X yu=d N Iu ij , Pkgd = Y ci=k Y cj=g Y yu=d N Iu ij ! Rkgd := ( \|Ak\|\|Ag\|\|Cd\| if g ̸= k \|Ak\|\|Ak−1\|\|Cd\| if g = k Note that the subscript u was removed from ∆u to emphasize that time steps are equally spaced for every u. Note that the subscript u was removed from ∆u to emphasize that time steps are equally spaced for every u. II. EXACT ICL FOR NON STATIONARY SBM In order to estimate the label vectors c and y and the number of clusters K and D, we directly maximize an exact version of the Integrated Classiﬁcation Criterion (ICL) [2] by means of a greedy search [3]. The quantity we focus on is the complete data log-likelihood, integrated with respect to the model parameters Φ and Λ: Fig. 1: Aggregated interactions for each time interval The greedy search algorithm converged to a ﬁnal ICL of −53217.4, corresponding to 20 clusters for nodes (people) and 4 time clusters. In Figure 1, the total number of interactions for each quarter-hour can be observed. Different colors correspond to the time clusters found by the model, in particular time intervals corresponding to the highest number of interactions have been placed in cluster C4 (blue), those corresponding to an intermediate interaction intensity, in C2 (green) and C3 (yellow). Cluster C1 (magenta) contains those quarter-hours where the frequency of interactions is very low or null. It is interesting to note how the model closely recovers times of social gathering: ICL = log Z p(N ∆, c, y, Λ, Φ\|K, D)dΛdΦ . Introducing a prior distribution ν(Λ, Φ\|K, D) over the pair (Φ, Λ) and making an independence assumption, allow us to write the ICL as follows: ICL = log ν(N ∆\|c, y, K, D) + log (ν(c, y\|K, D)) . The choice of conjugated prior distributions is crucial to obtain an explicit form of the ICL. We impose a Gamma a priori over Λ: ν(λkgd\|a, b) = ba Γ(a)λa−1 kgd e−bλkgd • 9.00-10.30 - set-up time for posters and demos. • 13.00-15.00 - lunch break. and a factorizing Dirichlet a priori distribution to Φ: • 18.00-19.00 - wine and cheese reception. A complete program of the day can be found at: ν(Φ\|K, D) =DirK(ω; α, . . . , α) × DirD(β; γ, . . . , γ), ν(Φ\|K, D) =DirK(ω; α, . . . , α) × DirD(β; γ, . . . , γ), http://www.ht2009.org/program.php. where the parameters of each distribution have been set constant for simplicity. 2More informations can be found at: III. EXPERIMENTS [2] C. Biernacki, G. Celeux, and G. Govaert, “Assessing a mixture model for clustering with the integrated completed likelihood,” Pattern Analysis and Machine Intelligence, IEEE Transactions on, vol. 22, no. 7, pp. 719–725, 2000. [2] C. Biernacki, G. Celeux, and G. Govaert, “Assessing a mixture model for clustering with the integrated completed likelihood,” Pattern Analysis and Machine Intelligence, IEEE Transactions on, vol. 22, no. 7, pp. 719–725, 2000. The dataset we used was collected during the ACM Hyper- text conference held in Turin, June 29th - July 1st, 2009 and represents the dynamical network of face-to-face proximity interactions of 113 conference attendees over about 2.5 days2. [3] E. Cˆome and P. Latouche, “Model selection and clustering in stochastic block models based on the exact integrated complete data likelihood,” Statistical Modelling, 2015, to appear. [3] E. Cˆome and P. Latouche, “Model selection and clustering in stochastic block models based on the exact integrated complete data likelihood,” Statistical Modelling, 2015, to appear. k/. [4] L. Isella, J. Stehl, A. Barrat, C. Cattuto, J. Pinton, and W. Van den Broeck, “What’s in a crowd? analysis of face-to-face behavioral networks,” Journal of Theoretical Biology, vol. 271, no. 1, pp. 166–180, 2011. k/. [4] L. Isella, J. Stehl, A. Barrat, C. Cattuto, J. Pinton, and W. Van den Broeck, “What’s in a crowd? analysis of face-to-face behavioral networks,” Journal of Theoretical Biology, vol. 271, no. 1, pp. 166–180, 2011.
https://openalex.org/W3175436206	https://www.frontiersin.org/articles/10.3389/fpls.2021.777285/pdf	English	null	Genome-wide identification, expression and bioinformatic analyses of GRAS transcription factor genes in rice	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	13,333	ORIGINAL RESEARCH published: 26 November 2021 doi: 10.3389/fpls.2021.777285 ORIGINAL RESEARCH published: 26 November 2021 doi: 10.3389/fpls.2021.777285 Genome-Wide Identification, Transcript Profiling and Bioinformatic Analyses of GRAS Transcription Factor Genes in Rice 1 Department of Plant Sciences, University of Hyderabad, Hyderabad, India, 2 Department of Biotechnology, Indian Institute of Rice Research, Hyderabad, India, 3 Agri Biotech Foundation, PJTS Agricultural University Campus, Hyderabad, India Edited by: Edited by: Ramón Gerardo Guevara-Gonzalez, Universidad Autónoma de Querétaro, Mexico Edited by: Ramón Gerardo Guevara-Gonzalez, Universidad Autónoma de Querétaro, Mexico Our group has previously identified the activation of a GRAS transcription factor (TF) gene in the gain-of-function mutant population developed through activation tagging in rice (in an indica rice variety, BPT 5204) that was screened for water use efficiency. This family of GRAS transcription factors has been well known for their diverse roles in gibberellin signaling, light responses, root development, gametogenesis etc. Recent studies indicated their role in biotic and abiotic responses as well. Although this family of TFs received significant attention, not many genes were identified specifically for their roles in mediating stress tolerance in rice. Only OsGRAS23 (here named as OsGRAS22) was reported to code for a TF that induced drought tolerance in rice. In the present study, we have analyzed the expression patterns of rice GRAS TF genes under abiotic (NaCl and ABA treatments) and biotic (leaf samples infected with pathogens, Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight and Rhizoctonia solani that causes sheath blight) stress conditions. In addition, their expression patterns were also analyzed in 13 different developmental stages. We studied their spatio-temporal regulation and correlated them with the in-silico studies. Fully annotated genomic sequences available in rice database have enabled us to study the protein properties, ligand interactions, domain analysis and presence of cis-regulatory elements through the bioinformatic approach. Most of the genes were induced immediately after the onset of stress particularly in the roots of ABA treated plants. OsGRAS39 was found to be a highly expressive gene under sheath blight infection and both abiotic stress treatments while OsGRAS8, OsSHR1 and OsSLR1 were also responsive. Our earlier activation tagging based functional characterization followed by the genome-wide characterization of the GRAS gene family members in the present study clearly show that they are highly appropriate candidate genes for manipulating stress tolerance in rice and other crop plants. Reviewed by: Gerardo Acosta-Garcia, Technological Institute of Celaya, Mexico Ma. Cristina Vazquez Hernandez, Tecnologico Nacional de México en Roque, Mexico Reviewed by: Gerardo Acosta-Garcia, Technological Institute of Celaya, Mexico Ma. Keywords: GRAS genes, rice, stress tolerance, genome-wide analysis, transcription factor, transcript profiling Edited by: Cristina Vazquez Hernandez, Tecnologico Nacional de México en Roque, Mexico *Correspondence: Pulugurtha Bharadwaja Kirti pbkirti@uohyd.ac.in Mouboni Dutta moubonidutta@gmail.com orcid.org/0000-0002-1714-0435 Specialty section: This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science Received: 15 September 2021 Accepted: 26 October 2021 Published: 26 November 2021 Received: 15 September 2021 Accepted: 26 October 2021 Published: 26 November 2021 INTRODUCTION Among them, some important transcription factors include WRKY, MBS, MADS, ARF, AP2/EREBP, HB, SBP, bZIP, GRAS and others (Zhang et al., 2018; Shen et al., 2020; Sidhu et al., 2020).i g GRAS group of transcription factors are plant specific proteins that were first observed in bacteria and assigned to the Rossman fold methyl transferase superfamily (Zhang et al., 2012). Later, this group radiated towards the ancestors of bryophytes, lycophytes and other higher plants (Cenci and Rouard, 2017; Zhang et al., 2018). A large number of GRAS genes have been identified in various plant species including 34 in Arabidopsis, 60 in rice, 86 in maize, 106 in Populus trichocarpa and many others (Tian et al., 2004; Liu and Widmer, 2014; Guo et al., 2017). The higher number of genes in this gene family indicated that the expansion might have happened via segmental and tandem duplication events in evolution, followed by their retention (Tian et al., 2004; Huang et al., 2015). Till date, the GRAS family of TFs have been studied in 30 different plant species including Arabidopsis, rice, mustard, lotus, tomato, castor bean, poplar, pine, grapevine and others (Cenci and Rouard, 2017). This gene family has been divided into eight subfamilies in Arabidopsis and rice, while the number varied from eight to 13 in tomato, poplar and castor beans (Tian et al., 2004; Liu and Widmer, 2014; Huang et al., 2015; Xu et al., 2016). In our previous study (Moin et al., 2016b), we have generated a pool of gain-of-function mutants via activation tagging using tetrameric 35S enhancers and screening of some of these mutants for water use efficiency led to the identification of several genes that were associated with the target trait, the water use efficiency. These interesting gain of function mutants included RNA and DNA helicases (SEN1 and XPB2; Dutta et al., 2021), and genes for ribosome biogenesis (RPL6 and RPL23A), protein ubiquitination (cullin4) and transcription factors like WRKY 96 and GRAS (LOC_Os03g40080; Moin et al., 2016b). A GRAS gene was tagged in the mutant DEB.86 rice line, which showed a high quantum efficiency of 0.82 and a low Δ13C value of 18.06‰. Since high photosynthetic efficiency and low carbon isotope ratio are the proxies for high water use efficiency, DEB.86 was further analyzed for other phenotypic characters. INTRODUCTION growth regulatory cues and play significant roles in plant development. This family of genes is responsible for a variety of biological functions including gibberellic acid signaling (GAI and RGA of DELLA subfamily and SLR1 of rice; Pysh et al., 1999; Liu and Widmer, 2014; To et al., 2020), SHR and SCR genes for radial root patterning (Helariutta et al., 2000), SCL3 for root elongation (Huang et al., 2015), HAM for shoot meristem formation (Stuurman et al., 2002), PAT genes for phytochrome signaling (Bolle et al., 2000), NSP1 and NSP2 for nodulation signaling pathway (Huang et al., 2015) and some others for abiotic and biotic stress responses (Sun et al., 2012; Zhang et al., 2018; Zeng et al., 2019). In many higher angiosperms, several GRAS genes like ZmSCL7, AtRGA, AtGAI were shown to have roles in salt stress tolerance in maize and Arabidopsis (Zeng et al., 2019). PeSCL7 from Populus is associated with the modulation of drought and salt tolerance (Ma et al., 2010). OsGRAS23 (here named as OsGRAS22) was shown to induce drought stress tolerance in rice (Xu et al., 2015). The gene OsGRAS23 (LOC_Os04g50060) as mentioned by Xu et al. (2015) was based on the QTL found in chromosome 4. They followed the nomenclature of Tian et al. (2004). In this study we have considered the recent nomenclature of Liu and Widmer (2014), which is more inclusive and comprehensive. Based on the analysis by Liu and Widmer (2014), OsGRAS23 (as mentioned in Xu et al., 2015) bearing the locus number LOC_Os04g50060 is denoted as OsGRAS22. In the present investigation, OsGRAS23 is a different gene on chromosome 5 bearing the locus id LOC_Os05g31380. Therefore, it has been mentioned now that OsGRAS23 as reported by Xu et al. (2015) is named as OsGRAS22 in this study in order to avoid confusion. Identification and analysis of transcription factors (TFs) form the essential approaches in Functional Genomics research. TFs bind to DNA or protein sequences and regulate gene expression (Zhang et al., 2018). They play important roles in almost all cellular functions like growth, development, metabolism, signal transduction, resistance/tolerance to abiotic and biotic stress factors, among others. About 320 k TFs from 165 different plant species have been reported in literature. Citation: Dutta M, Saha A, Moin M and Kirti PB (2021) Genome-Wide Identification, Transcript Profiling and Bioinformatic Analyses of GRAS Transcription Factor Genes in Rice. Front. Plant Sci. 12:777285. doi: 10.3389/fpls.2021.777285 November 2021 \| Volume 12 \| Article 777285 1 Frontiers in Plant Science \| www.frontiersin.org Genome-Wide Identification of OsGRAS Genes Dutta et al. Frontiers in Plant Science \| www.frontiersin.org Genomic Distribution of GRAS Genes The coordinates of all 60 GRAS genes were obtained from RGAB-DB and were fed in the NCBI Genome Decoration Page. The outputs were combined and the genes were marked for understanding the genomic distribution of OsGRAS genes. MATERIALS AND METHODS The cis- acting elements in the promoter regions play a major role in the coordinated expression of the genes. Hence, it is crucial to identify these regulatory elements in order to correlate the expression data with the genetic components. We retrieved ≤1 kb upstream sequences of all 40 selected GRAS genes under study from the rice genome database and identified important regulatory elements responsible for biotic and abiotic stress responses in them. The identification of these elements was performed by subjecting the sequences in PlantCARE (Cis- Acting Regulatory Elements) database and manually mapping them on the chromosomes. INTRODUCTION This activation tagged line exhibited improved plant height with increased tillering and seed yield and had the ΨOsGRAS4 gene tagged for activation tagging (Moin et al., 2016b). A total of 60 GRAS genes have already been identified in rice (Liu and Widmer, 2014), out of which, OsGRAS23 has been reported to enhance tolerance to drought (Xu et al., 2015) and ΨOsGRAS4 has been identified to be associated with enhanced photosynthetic efficiency and water use efficiency with enhanced agronomic features (Moin et al., 2016b). These observations have prompted our group to analyze the GRAS gene family of rice in detail. GRAS proteins consist of 400–770 amino acid residues and derive the name from the first three identified members of this family viz. Gibberellin-Acid Insensitive (GAI), Repressor of GAI (RGA) and Scarecrow (SCR; Pysh et al., 1999; Bolle, 2004; Zhang et al., 2017). These genes have a conserved C-terminal region, which forms the GRAS domain and a variable N-terminal region. The conserved region or the GRAS domain comprises of five motifs in the following order; Leucine heptad repeat I (LHR I), VHIID motif, Leucine heptad repeat II (LHR II), PFYRE motif and the SAW motif (Pysh et al., 1999). The conserved C-terminal domain is responsible for the transcriptional regulation of the genes that exist under their control. The LHR region is required for protein dimerization and the VHIID is necessary for protein-DNA interactions. PFYRE and SAW are the other important regulatory domains that are present in GRAS TFs. The GRAS genes are mostly nuclear localized except PAT1, which is found in the cytoplasm (Pysh et al., 1999; Tian et al., 2004). The variable N-terminal region consists of intrinsically disordered regions (IDRs,) which are important for molecular recognition during plant development. Due to these IDRs, the GRAS transcription factors are functionally polymorphic (Sun et al., 2012). The members of this gene family integrate environmental and November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 2 Genome-Wide Identification of OsGRAS Genes Dutta et al. Motif Arrangements and Organization of GRAS Genes in Rice In the present study, we have shortlisted 40 genes, one gene representing each paralogous group and provided an experimental basis to identify the potential GRAS genes capable of imparting stress tolerance in rice. We have analyzed the genes selected in the GRAS family for their spatio- temporal and stress induced expression. The phylogenetic relationship among GRAS proteins, their genetic arrangements and structure, in-silico analysis of putative promoter elements and protein properties were also studied. This study helps us in the identification of important GRAS genes for stress tolerance, which aids in their further functional characterization. All the selected 40 GRAS genes were subjected to the MEME suite for conserved motif analysis using default parameters. The number of motif scan was set to 10. Based on the previous investigation of Pysh et al. (1999), the MEME-motifs were further classified into conserved GRAS motifs. The gene organization was studied by subjecting the genomic and coding sequences for analysis in the Gene Structure Display Server (GSDSv2), and the number of exons, introns, untranslated regions (UTRs) etc. were noted. Retrieval of GRAS Gene Sequences and Their Nomenclature Our previous work on gain of function mutants generated through activation tagging technology using the tetrameric 35S elements identified a GRAS gene as a potential player in enhancing water use efficiency in rice (Moin et al., 2016b). Also, Xu et al. (2015) suggested that OsGRAS23 is involved in inducing drought stress responses in rice. This has led us to undertake literature search in the present study and we observed that Tian et al. (2004) have identified 57 GRAS genes in rice. We searched the accession numbers of all 57 genes in NCBI and performed a BLASTN search in rice genome databases (RGAP-DB- http://rice.uga.edu, Orygenes DB- https:// orygenesdb.cirad.fr) and retrieved the locus numbers of 47 genes. Simultaneously, we performed a key word search for GRAS, DELLA, SCR, Monoculm, Chitin- Inducible gibberellin- responsive protein, Gibberellin response modulator protein, Nodulation signaling pathway and Short Root. Subsequently we combined the search results with the 47 genes retrieved from the literature search. Finally, we matched our list of 60 genes with that of Liu and Widmer (2014) and followed the same nomenclature. For more clarity, we had performed a protein database search for the GRAS domain in NCBI, Simple Modular Architecture Research Tool (SMART), Prosite and Pfam databases, and selected 40 genes, one from each of all the paralogous groups for our analyses. Biochemical Properties of GRAS Proteins The sequences of all the 40 shortlisted GRAS genes were subjected to the ExPASyProtParam tool to assess their encoded proteins for amino acid length, molecular weight and theoretical isoelectric points (pI). The three-dimensional structures of the proteins and their ligand interactions were studied using 3DLigandSite software (Wass et al., 2010). The structures were then subjected to Phyre2 (Protein Homology/Analogy Recognition Engine v2; Kelley et al., 2015) program for analysis of the protein secondary structure composition. This tool gives an idea of the percentage of secondary structures in a protein, i.e., the percentage of α-helix, β-sheets and the disordered regions in the proteins. The SMART online tool was used to analyse the protein domains and their low complexity regions (LCRs). ExpasyProtParam tool also indicates the GRAVY indices of the proteins, which provide information regarding the hydrophobicity of the subjected proteins. The localization and existence of transmembrane helices in the proteins coded by these genes were predicted using TargetP-2.0 and TMHMM software, respectively. Phylogenetic Relationships of Rice GRAS Genes For simulated abiotic stress experiments, BPT-5204 (Samba Mahsuri) rice seeds were surface sterilized using 70% ethanol for 50 s followed by 4% aqueous sodium hypochlorite solution for 15 min and five washes with sterile double distilled water, each of 1 min duration. The sterile seeds were germinated on Murashige and Skoog medium for 7 d under a 28 ± 2°C for In order to understand the evolutionary relationships between the rice GRAS genes, we aligned the amino acid sequences in MEGA7 software followed by the construction of an unrooted phylogenetic tree. The tree was constructed using the Neighbour Joining method with a bootstrap value of 1,000. November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 3 Genome-Wide Identification of OsGRAS Genes Dutta et al. 16 h/8 h photoperiodic cycle (Bakshi et al., 2017; Saha et al., 2017). The seedlings were then subjected to NaCl (250 μm) and ABA (100 μm) stress conditions for 60 h. The shoot and root samples were collected periodically at 0 h, 15 min, 3 h, 12 h, 24 h and 60 h after exposing the seedlings to the simulated stress treatments. The untreated samples were taken as controls for normalization of gene expression. of rice. Based on the literature and database search, we observed that chromosomes 8 and 9 did not carry any GRAS genes. The number of genes on a single chromosome ranged from a minimum of two on chromosome 10 to a maximum of 12 on chromosome 11. Among the rest, a total of nine genes were located on chromosome 3, while chromosomes 1, 7 and 12 carried six genes each, chromosomes 2, 4 and 5 exhibited five genes each while the chromosome 6 carried four genes (Figure 1). Out of the 60 genes located, we have shortlisted 40 genes for our study with one representative selected from each of the paralogous groups. For studying the native expression patterns of the GRAS genes, tissue samples from 13 regions in rice seedlings were collected following Moin et al. (2016a) and Saha et al. (2017). These included embryo and endosperm from 16 h soaked seeds, plumule and radicle from 3 d old germinating seeds, shoot and root tissues from 7 d old seedlings and shoot, root, root- shoot transition region, flower, spikes and grain samples from mature 20 d old plants post-transfer to the greenhouse. cDNA Preparation and qRT-PCRh p q The plant material collected was used to isolate of RNA using Tri-reagent following manufacturer’s protocol (Takara Bio, United Kingdom) and cDNA was prepared using 2 μg total RNA samples (Takara Bio, United Kingdom). The cDNA samples were diluted 10 times and an aliquot of 2 μl of each sample per reaction was used for quantitative real-time PCR (qRT-PCR). All the primers were designed using Primer3 software and 10 μm primer concentration was used per reaction. The PCR program included an initial denaturation step of 94°C for 2 min followed by 40 cycles of second denaturation of 30 s, annealing for 25 s and extension at 72°C for 30 s. The samples for the current study were taken in biological and technical triplicates, and the fold changes were calculated using the ΔΔCT method (Livak and Schmittgen, 2001). Rice actin and β-tubulin genes were used as two housekeeping genes for internal normalization. For abiotic and biotic expression studies, housekeeping genes and individual control samples were used for double normalization. In contrast, single normalization was performed using the CT value of housekeeping genes for native expression studies. The graphs were generated using MORPHEUS program and GraphPad Prism software. One way ANOVA was performed using SigmaPlot v.11 software for discerning the significance of statistical differences between samples. Phylogenetic Relationships of Rice GRAS Genes The schematic flowchart for obtaining the results reported in the investigation is provided in Supplementary Figure S1. Analysis of Evolutionary Relationship of OsGRAS Genes In order to understand the evolutionary relationship among the rice GRAS family of genes, we subjected the retrieved sequences to a phylogenetic analysis (Figure 2) in MEGA7 software. A total of 16 different clusters were observed. These clusters were divided into 14 subfamilies based on a previous report of Cenci and Rouard (2017). Members belonging to the same subfamily were found to cluster together except DLT and PAT subfamilies where some genes belonging to different orthologous groups (according to Cenci and Rouard, 2017) formed separate clusters. Each cluster has been colour coded in the Figure 2. The number of genes found in each subfamily included four in SCL3, three each in SCR, NSP2 and HAM, one in RAM, LS, SCL4/7and SCLA, two in DELLA, DLT, SHR and SCL32, six in PAT and nine in LISCL. LISCL was found to be the largest subfamily with the maximum number of genes getting clustered. ΨOsGRAS4 and ΨOsGRAS9 were placed close to LISCL family since these sequences were still unclassified. The highly expressed genes under biotic and abiotic stress conditions belonged to SCL3, SHR, DELLA, HAM and PAT subfamilies. In order to study the expression of GRAS genes under biotic stress conditions, leaf samples of 1 month old rice plants infected with Xanthomonas oryzae pv. oryzae [Xoo that causes Bacterial Leaf Blight (BLB)] and Rhizoctonia solani (that causes Sheath Blight, SB) were taken post 20 d and 25 d of infection, respectively. Samples from plants of the same age without the pathogen challenge were taken as the controls. The infection protocol was followed as per Saha et al. (2017). Analysis of GRAS Motifs and Gene Organizationh g The amino acid sequences of selected 40 genes were subjected to MEME analysis for identifying the conserved motifs in rice GRAS gene encoded proteins. A total of 10 motifs were identified, which corresponded to LHR I (motif 5, 9), VHIID (motif 2, 3, 10), LHR II (motif 8), PFYRE (motif 4, 7) and SAW (motif 1, 6) motifs (Figure 3 and Supplementary Figure S2). The C-terminal domain was found to contain the conserved GRAS motifs as reported earlier in literature. However, not all genes exhibited all the 10 MEME-motifs. PAT and LISCL subfamilies carried all the 10 domains, while others like SCR lacked motif 1. Proteins belonging to same subfamily had similar motif composition. The genomic and cDNA sequences of all the selected genes were subjected to GSDS server to observe the organization of different GRAS genes selected from each of the paralogous groups (Supplementary Figure S3). Based on the map that was generated by the server, it was observed that the genes varied in length and the distribution of exons, introns and UTRs. The majority of genes (31 out of 40 genes studied) Chromosomal Distribution of GRAS Genes in Rice Genome The number of intronic sequences among the genes varied between one (OsSCR1 and ΨOsGRAS10) to a maximum of seven (ΨOsGRAS4). All of them showed low (OsGRAS43), moderate (OsGRAS3, ΨOSGRAS4 and ΨOSGRAS8) and high (OsGRAS39, OsGRAS41, OsSCR1, ΨOsGRAS9 and ΨOsGRAS10) expression levels under abiotic and biotic stress conditions. Six out of nine genes (OsGRAS41, OsGRAS43, ΨOSGRAS4, ΨOsGRAS8, ΨOsGRAS9 and ΨOsGRAS10) did not exhibit any UTRs in their structure and were solely composed of introns and exons. The details of genetic organization of rice GRAS genes have been provided in the Supplementary Table S1. g OsGRAS39, the highly expressive gene under both biotic and abiotic stress conditions in the present study had three copies each of MYB binding factor sites and CGTCA-motif, five copies of STRE, two copies of ABRE and one copy each of DRE, TC-rich repeats and CCAAT-box justifying its expression under different stress treatments. Other responsive genes in both the stresses like OsGRAS8, OsSHR1 and OsSLR1 had combinations of MYB, STRE, ERE, WUN, TCA, CGTCA and MYC elements in their putative promoter regions. Apart from these, OsGRAS8 exhibited ABRE, LTR and W-box elements, OsSHR1 carried a DRE element and OsSLR1 had copies of TATC, WRE and TC- rich elements. ΨOsGRAS5, the only expressive gene in the shoot region had two copies each of MYB and MYC binding elements and three copies of ABRE. Other important abiotic stress responsive genes like ΨOsGRAS2 and OsSCR1 were also observed to have multiple copies (upto six) of ABRE, MYB and MYC elements, STRE elements and ERE, CGTCA, GARE and WRE motifs in their 5' upstream regions. OsCIGR1 that was found to be highly induced under biotic stress conditions carried 10 copies of ABRE, seven copies of STRE, five copies of CGTCA element and one copy each of CCAAT-box, DRE, MYB, MYC and WRE. Chromosomal Distribution of GRAS Genes in Rice Genome Liu and Widmer (2014) showed that there are 60 GRAS genes in the genome that are distributed on 10 out of 12 chromosomes November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 4 Dutta et al. Genome-Wide Identification of OsGRAS Genes FIGURE 1 \| Chromosomal distribution of GRAS genes in rice. Karyotypic representation of rice chromosomes obtained from NCBI Genome Decoration Page. Rice genome carries 60 GRAS genes, which are represented in the figure with red arrows indicating the position of each gene. The size of each chromosome and the number of genes present are provided below each chromosome in brackets. FIGURE 1 \| Chromosomal distribution of GRAS genes in rice. Karyotypic representation of rice chromosomes obtained from NCBI Genome Decoration Page. Rice genome carries 60 GRAS genes, which are represented in the figure with red arrows indicating the position of each gene. The size of each chromosome and the number of genes present are provided below each chromosome in brackets. box and MYB sites for binding of MYB transcription factors that are reported to be responsive to drought treatments, binding site for MYC transcription factors for defence responses, DRE or dehydration responsive elements, STRE or stress responsive elements, TC-rich repeats for defence and stress responses, and the LTR or low temperature responsive element. Several phytohormones and wound responsive elements were also observed in their upstream regions, which included TCA-element for salicylic acid responses, CGTCA-motif or TGACG-motif as a methyl jasmonate responsive element, GARE-motif, TATC-box and P-box for gibberellin responses, ERE as ethylene responsive elements, TGA-element or AuxRR core or AuxRE for auxin responses, WUN-motif and WRE for responses against wounding, box-S for wounding and pathogen elicitation, and the W-box for binding of WRKY transcription factors. lacked introns in their gene structure and were only composed of exonic sequences and UTRs. OsGRAS11 exon is flanked by a long stretch of UTR at its 5' and 3' ends. It completely lacked introns and is the longest gene in this study (6.7Kb). Ten genes were observed to contain only coding sequences in their structure without any introns and UTRs. Among them, ΨOsGRAS3 had the smallest sequence of only 414 bp. The nine genes carrying introns only in their structure were OsGRAS3, OsGRAS39, OsGRAS41, OsGRAS43, OsSCR1, ΨOSGRAS4, ΨOsGRAS8, ΨOsGRAS9 and ΨOsGRAS10. Putative Promoter Analysis of GRAS Genes and the Search for cis-Regulatory Elementsf The functions of each of these stress responsive elements has been provided in Table 1 and the physical mapping of the important stress responsive elements on the putative promoter regions of the genes is provided in Figure 4. theoretical pI through ExPASyProtParam program. It was observed that the proteins had a molecular weight ranging from 15 kDa (ΨOsGRAS3) to 94 kDa (OsGRAS39). ΨOsGRAS3 showed a minimum amino acid (aa) length of 137 aa while OsGRAS39 had a maximum length of 854 aa. The pI of the proteins ranged from acidic to basic (4.5–10.1) with only eight proteins having a pI of more than 7. The majority of the proteins fall under the pI range of 4–7. Likewise, the remaining 32 proteins were found to be in the acidic range, i.e., pI < 7. This is because of the observation that the proteins carried more negatively charged (acidic) amino acid residues like Aspartic and Glutamic acids in their composition as compared to basic amino acid residues. Only OsGRAS39 was found to Putative Promoter Analysis of GRAS Genes and the Search for cis-Regulatory Elementsf Since diverse expression patterns were observed for different GRAS genes under abiotic and biotic stress conditions, we tried to correlate their expression patterns with the putative regulatory sequences observed in their upstream regions. In order to achieve this correlation, we retrieved 1 kb sequences from 5' upstream region of each gene under study from the rice genome database and subjected them to an in-silico analysis for the identification of the putative cis-regulatory elements observed in them. A total of 18 stress responsive elements were observed in the upstream putative promoter regions of the GRAS genes. These included ABRE or ABA responsive elements, CCAAT November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 5 Genome-Wide Identification of OsGRAS Genes Dutta et al. FIGURE 2 \| Phylogenetic analysis of OsGRAS genes. An unrooted phylogenetic tree showing the evolutionary relationship of OsGRAS genes. The tree was constructed using the Neighbour Joining method in MEGA7 software with a bootstrap value of 1,000. The number at each node represents the percentage bootstrap values. Based on the previous literature, the genes have been divided into 14 subfamilies (mentioned in boxes) and each subfamily has been colour coded. FIGURE 2 \| Phylogenetic analysis of OsGRAS genes. An unrooted phylogenetic tree showing the evolutionary relationship of OsGRAS genes. The tree was constructed using the Neighbour Joining method in MEGA7 software with a bootstrap value of 1,000. The number at each node represents the percentage bootstrap values. Based on the previous literature, the genes have been divided into 14 subfamilies (mentioned in boxes) and each subfamily has been colour coded Other expressive genes under biotic stress conditions included OsGRAS2, ΨOsGRAS3, OsGRAS19, OsGRAS20 and OsGRAS23, which had combinations of TCA-elements, W-box, WRE, ERE, AuxRE, CGTCA-box, box-S and WUN elements apart from other stress responsive elements. The functions of each of these stress responsive elements has been provided in Table 1 and the physical mapping of the important stress responsive elements on the putative promoter regions of the genes is provided in Figure 4. Other expressive genes under biotic stress conditions included OsGRAS2, ΨOsGRAS3, OsGRAS19, OsGRAS20 and OsGRAS23, which had combinations of TCA-elements, W-box, WRE, ERE, AuxRE, CGTCA-box, box-S and WUN elements apart from other stress responsive elements. Frontiers in Plant Science \| www.frontiersin.org Properties of GRAS Proteins, Their Ligand Interactions and Domain Analysis y We studied the properties of 40 shortlisted GRAS proteins like amino acid length (aa), molecular weight (kDa) and November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 6 Dutta et al. Genome-Wide Identification of OsGRAS Genes FIGURE 3 \| MEME-motif analysis of OsGRAS genes. Figure showing the identified MEME-motifs of OsGRAS genes. The conserved GRAS-motifs are provided at the top. A search for 10 MEME-motifs was done and each of them has been assigned to the corresponding GRAS-motifs. Each coloured box represents one motif and the legend has been provided below. The genes were organized based on their subfamilies. FIGURE 3 \| MEME-motif analysis of OsGRAS genes. Figure showing the identified MEME-motifs of OsGRAS genes. The conserved GRAS-motifs are provided at the top. A search for 10 MEME-motifs was done and each of them has been assigned to the corresponding GRAS-motifs. Each coloured box represents one motif and the legend has been provided below. The genes were organized based on their subfamilies. have an equal number of acidic and basic residues in its composition. According to TargetP-2.0 server, OsGRAS39 was predicted to be localized to the chloroplast while no signal peptides for chloroplast or mitochondria could be specified by the tool for the rest of the proteins. (14%) extent of β-sheets were noticed in the secondary structure of OsGRAS32. No β-sheets were present in ΨOsGRAS2 and ΨOsGRAS3. Several metallic and non-metallic ligands were also observed to be interacting with the GRAS proteins, which included Mg+2, Ca+2, SAM, SAH, NAP, NAD, ATP, Zn+2 and Ni+2. The three dimensional structures of the proteins along with their interacting ligands have been provided in the Supplementary Figure S4. We have also analyzed the proteins for their three dimensional structures and ligand binding residues in the 3DLigand site and the structures were submitted to the Phyre2 program to analyze their secondary structures like the percentage of disordered regions, α-helix and β-sheets. ΨOsGRAS3 showed a maximum of 71% and OsGRAS8 had a minimum of 31% of α-helical structure. Similarly, maximum Low complexity region are repetitive amino acid sequences found abundantly in the eukaryotic proteins. These play essential roles in protein-protein and protein-nucleic acid interactions (Toll-Riera et al., 2012). It was noted that the number of LCRs November 2021 \| Volume 12 \| Article 777285 7 Genome-Wide Identification of OsGRAS Genes Dutta et al. Expression Analysis of GRAS Genes Under Simulated Abiotic Stress Conditionsi We have identified a GRAS transcription factor as a potential stress tolerance gene by screening a pool of gain-of-function mutants in rice in our previous study (Moin et al., 2016b). Another report by Xu et al. (2015) suggested the role of OsGRAS23 (reported as OsGRAS22 in this study) in drought tolerance in rice. These observations have prompted us to analyze the differential expression pattern of GRAS family of transcription factors under the influence of biotic and simulated abiotic stress conditions in the present study. We have analyzed the expression patterns of 40 selected genes separately in shoot and root tissues at six different time points for two abiotic (NaCl and ABA) and two biotic (BLB and SB) stresses. The native expression patterns of these genes in 13 different tissues were also studied. Based on the pattern of expression, we have divided the genes as immediate early (IE), early (E) and late (L) responsive genes. Some genes were expressed up to 100 folds after the incidence of stress. Thus, the genes were also categorized as expressive (2–10 fold), moderately expressive (10–30 fold) and highly expressive (≥30 fold) types. Genes showing an upregulation of ≥2 folds were considered as expressive.h Wound responsive element (Whitbred and Schuler, 2000) The majority of the genes got upregulated in the root (Figures 5A,B) compared to the shoot (Figures 5C,D). As indicated in the pie chart, about 55–60% of the total genes showed IE type expression under both NaCl and ABA treatments. NaCl, however, induced more early (12.5%) responsive genes than the late genes (2.5%) whereas ABA induced more late genes (12.5%) than early ones (2.5%). The list of the expressed genes has been provided in Figure 5. More than half of the IE genes continued their expression till 60 h of treatment, while some others became downregulated or showed no expression at all later during the experimental timeline. Under ABA treatment, all highly upregulated genes, i.e., ΨOsGRAS2, OsSHR1, OsSCR1 and OsGRAS39 were IE type and their expression persisted till the last time point of treatment, i.e., 60 h. Other IE type genes showed a split before increasing their expression at subsequent time points. Only OsGRAS39 was highly expressive under both ABA and NaCl treatments (100 fold and 65 fold, respectively). Properties of GRAS Proteins, Their Ligand Interactions and Domain Analysis TABLE 1 \| List of cis-regulatory elements and their functions. Name of cis-element Function ABRE ABA responsive element (Choi et al., 2000) MYB/MBS MYB binding site for drought inducibility (Ambawat et al., 2013) DRE Dehydration responsive element (Narusaka et al., 2003) MYC Transcription factor for stress responses, helps in dehydration induced expression of genes (Tran et al., 2004) STRE Stress responsive element (Hwang et al., 2010) TCA element Element for salicylic acid responsiveness (Wei et al., 2013) CGTCA-motif/TGACG-motif Methyl-Jasmonate responsive element (Wang et al., 2011) TC-rich motifs Responsible for defense and stress, transcription regulation (Bernard et al., 2010; Liu et al., 2017) Box S Responsive to wounding and pathogen elicitation (Yin et al., 2017); Stress responsiveness (Ding et al., 2019) GARE-motif/TATC-box Gibberellin responsive element (Bastian et al., 2010) ERE Element for ethylene responses (Oñate- Sánchez and Singh, 2002) TGA-element/AuxRR core/AuxRE Element for auxin response (Sakai et al., 1996) WUN motif Wound responsive element for biotic stress (Xu et al., 2011) LTR Low temperature responsive element (Zhang et al., 2020) W box Binding sites for WRKY transcription factors (Dhatterwal et al., 2019) CCAAT box Binding site for MYB transcription factors P-box Gibberellin responsiveness (Zhang et al., 2020) WRE Wound responsive element (Whitbred and Schuler, 2000) TABLE 1 \| List of cis-regulatory elements and their functions. in addition to the GRAS domain were found in OsSLR1 and OsGRAS18, respectively. DELLA proteins are transcriptional regulators, which function in gibberellic acid signaling by binding with GA receptor, GID1 followed by proteasomal degradation of DELLA domain (Murase et al., 2008). OsGRAS41 had a transmembrane region, OsGRAS43 and OsGRAS53 had two RPT1 domains (internal repeats) along with single GRAS domains. The detailed list of the domains and the LCRs with their sequences have been provided in the Supplementary Table S3. The presence of transmembrane domain in OsGRAS41 was further confirmed through TMHMM software. Frontiers in Plant Science \| www.frontiersin.org Expression Analysis of GRAS Genes Under Simulated Abiotic Stress Conditionsi OsGRAS2, ΨOsGRAS2, OsGRAS25, OsGRAS35 and OsSCR1 under NaCl and OsGRAS22 under ABA were early (E) expressed genes respectively, while ΨOsGRAS9, OsGRAS3, OsGRAS11 and in each of the proteins varied from none to a maximum of eight in OsGRAS20 and OsGRAS43, respectively. g p y Grand average of hydropathicity index or GRAVY index indicates the hydrophobicity of a protein taking into consideration its charge and the size. Usually GRAVY values range from −2 to +2 with more positive values indicating hydrophobicity and more negative values indicating hydrophilicity (Morel et al., 2006). Seven proteins had a positive GRAVY value while the rest 33 proteins had a values lesser than zero, which indicated that the majority of the GRAS proteins are hydrophilic. The list of all the observations have been provided in the Supplementary Table S2. In order to study the domains present in the genes, we utilized the SMART online tool and observed that all the proteins had at least one GRAS domain with ΨOsGRAS4, ΨOsGRAS8, OsGRAS39, ΨOsGRAS10 exhibiting two GRAS domains. Among them, ΨOsGRAS4 and ΨOsGRAS10 had two internal repeats designated as RPT1 along with two GRAS domains. One DELLA domain and one SCOP domain November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 8 Genome-Wide Identification of OsGRAS Genes Dutta et al. FIGURE 4 \| In-silico analysis of putative promoter regions of GRAS genes. The selected GRAS genes were subjected to in silico analysis for cis-regulatory elements in their putative promoter regions (sequence retrieved from about ≤1 kb upstream region). This was performed in PlantCARE database and the figu was prepared by mapping the stress regulatory elements in the each of the sequences. The index for each element along with its functions are mentioned b the figure. FIGURE 4 \| In-silico analysis of putative promoter regions of GRAS genes. The selected GRAS genes were subjected to in silico analysis for cis-regulatory elements in their putative promoter regions (sequence retrieved from about ≤1 kb upstream region). This was performed in PlantCARE database and the figure was prepared by mapping the stress regulatory elements in the each of the sequences. The index for each element along with its functions are mentioned below FIGURE 4 \| In-silico analysis of putative promoter regions of GRAS genes. Expression Analysis of GRAS Genes Under Simulated Abiotic Stress Conditionsi The selected GRAS genes were subjected to in silico analysis for cis-regulatory elements in their putative promoter regions (sequence retrieved from about ≤1 kb upstream region). This was performed in PlantCARE database and the figure was prepared by mapping the stress regulatory elements in the each of the sequences. The index for each element along with its functions are mentioned below the figure. OsGRAS26 under ABA and ΨOsGRAS9 became upregulated under NaCl treatment with late (L) expression. were either downregulated or showed no change in the level of expression. Among them, OsGRAS7, OsGRAS23, OsGRAS28 and ΨOsGRAS8 were downregulated under both the treatments. ΨOsGRAS3, ΨOsGRAS4, OsCIGR1 and OsGRAS32 under ABA treatment and OsCIGR1 under NaCl treatment showed an immediate expression, but were either downregulated or showed no expression at subsequent time points. p Twelve and 13 genes (30 and 32%) were mild to moderately expressed, respectively under the ABA treatment, whereas seven genes (17%) were moderately expressive under NaCl treatment and rest 19 genes (47%) exhibited mild expression. Nine genes (22%) under ABA and 13 (32%) genes under NaCl treatment November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 9 Genome-Wide Identification of OsGRAS Genes Dutta et al. The shoot region did not evidence major GRAS gene expression compared to the root region. However, ΨOsGRAS5 is the only gene that showed moderate expression (25–30 fold) under both ABA and NaCl treatments in the shoot This gene was an IE type maintaining its expression till 60 h under NaCl, but showed a split before reaching a peak under the ABA treatment. On the contrary, it showed low expression (2–3 fold) in root tissues under ABA and NaCl treatments A B C D FIGURE 5 \| Expression analysis of GRAS genes under abiotic stress. Heat map representation of temporal expression pattern of GRAS genes developed using MORPHEUS program. 7 d old seedlings were subjected to NaCl (250 μm) and ABA (100 μm) treatments and the obtained quantitative real-time values were double normalized using rice actin and tubulin as the internal reference genes and that of the unstressed samples using the ΔΔCT method. The experiment was conducted separately for root (A,B) and shoot (C,D) tissues. Percentage of genes upregulated under NaCl and ABA treatments is represented in the form of a pie chart beside their corresponding heat maps. DISCUSSION We have studied the expression of the selected GRAS transcription factor genes in the leaf samples of rice infected with Xoo and R. solani pathogens that cause BLB and Sheath Blight (SB) diseases, respectively (Figure 6). Six genes were upregulated in BLB of which five (OsGRAS1, OsGRAS18, OsCIGR2, ΨOsGRAS9, OsGRAS53) showed low expression while one gene (OsCIGR1) was highly upregulated up to 57 folds. More genes were upregulated in SB infected leaves compared to the BLB treated ones. Out of the 30 expressed genes in SB infected leaves, only 12 showed high expression levels while the rest of the genes exhibited low to moderate expression. OsGRAS2, ΨOsGRAS3, OsGRAS19, OsGRAS20, OsGRAS23 and OsSHR1 were expressed by ≥100 folds under the SB treatment. A total of 22 genes in BLB and three in SB treated samples were downregulated. Those that were downregulated in SB treated samples (OsSHR2, OsGRAS24 and OsGRAS43) were also downregulated in BLB treated leaves. Twelve genes under BLB and seven under SB did not show any changes in their expression levels. Being sessile, plants cannot escape the onslaught from environmental stresses like cold, heat, drought etc., nor can they avoid harmful interactions with microorganisms like fungi and bacteria (Lin et al., 2017). Such adversities impose a threat to agricultural productivity and sustainability. In order to support the burgeoning global population, the development of stress tolerant crops is of utmost importance. Characterization of insertional mutants is an important functional genomics based method of identifying novel genes responsible for inducing stress tolerance in crop plants (Cushman and Bohnert, 2000). TF genes are of particular importance in this context as they act upstream in the pathway(s) and control the expression of several genes working under their control. Because of this, the manipulation undertaken using TF genes as ‘Master’ genes would render the plant more accommodative towards the particular stress under consideration. Our previous studies have identified several key players for stress tolerance in an indica rice variety via enhancer based activation tagging method and a GRAS transcription factor gene, ΨOsGRAS4 was one of the important genes that was identified in the study along with others for enhanced water use efficiency associated with enhanced photosynthetic efficiency (Moin et al., 2016b). Expression Analysis of GRAS Genes Under Simulated Abiotic Stress Conditionsi The genes were separated based on their time point(s) of expression and annotated as immediate early (IE), early (E) and late (L) expressive genes. The names of the genes is provided in the list below. The experiment was conducted using biological and technical triplicates (n = 3), and the mean value was used to plot the heat map in the MORPHEUS software. B A B B A D D C C D C FIGURE 5 \| Expression analysis of GRAS genes under abiotic stress. Heat map representation of temporal expression pattern of GRAS genes developed using MORPHEUS program. 7 d old seedlings were subjected to NaCl (250 μm) and ABA (100 μm) treatments and the obtained quantitative real-time values were double normalized using rice actin and tubulin as the internal reference genes and that of the unstressed samples using the ΔΔCT method. The experiment was conducted separately for root (A,B) and shoot (C,D) tissues. Percentage of genes upregulated under NaCl and ABA treatments is represented in the form of a pie chart beside their corresponding heat maps. The genes were separated based on their time point(s) of expression and annotated as immediate early (IE), early (E) and late (L) expressive genes. The names of the genes is provided in the list below. The experiment was conducted using biological and technical triplicates (n = 3), and the mean value was used to plot the heat map in the MORPHEUS software. The shoot region did not evidence major GRAS gene expression compared to the root region. However, ΨOsGRAS5 is the only gene that showed moderate expression (25–30 fold) under both ABA and NaCl treatments in the shoot. This gene was an IE type maintaining its expression till 60 h under NaCl, but showed a split before reaching a peak under the ABA treatment. On the contrary, it showed low expression (2–3 fold) in root tissues under ABA and NaCl treatments. November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 10 Genome-Wide Identification of OsGRAS Genes Dutta et al. only seven were expressed in mature vegetative and reproductive tissues. OsGRAS2 and OsGRAS3 were upregulated only in mature leaves, OsGRAS28 in 20 d root, flower and spike, OsCIGR1 and OsCIGR2 in root-shoot transition and leaves, OsGRAS39 in 20 d root and OsGRAS47 in 20 d root and flower. Native Expression Analysis of GRAS Genes in Various Tissues at Specific Developmental Stages in Rice In order to study the native expression patterns of GRAS transcription factors in different tissues of the rice plant, we performed qRT-PCR analysis of 13 different tissues, which included shoot, root, root-shoot transition, flag leaves, flower, spikes and grain of mature Expression Analysis of GRAS Genes Under Simulated Abiotic Stress Conditionsi Out of these seven genes, five were upregulated either in the roots or in the root-shoot transition region indicating the preference of GRAS genes towards expression in the root tissue. This is also in accordance with the expression analysis under abiotic stress condition, where the genes were highly expressive in roots rather than in the shoot tissue. Three out of seven mildly expressive genes were upregulated in flower and spike of 20 d old plants while none of them expressed in the grain. OsGRAS39 that was upregulated in root tissues under native conditions is highly expressive in roots under abiotic stress conditions also responding immediately after the application of stress treatment. This might be an indication of its tissue specificity and its potential as a stress tolerance transcription factor gene for genetic manipulation in rice. Among the other genes that were mildly expressive in both root and shoots were ΨOsGRAS2, OsGRAS12, OsGRAS19, OsGRAS24, OsGRAS25 and OsSCR1. The rest of the genes were mainly downregulated or did not show any change in expression in shoot tissues under both the stress treatments. The expression level of all the genes studied has been provided in Supplementary Table S4. Among the genes studied, some were observed to be expressed only under NaCl or ABA treatments at certain time points, whereas some were found to be expressive under both the treatments. Such overlaps have been depicted in the form of Venn diagrams generated through InteractiVenn software (Heberle et al., 2015) in Supplementary Figure S5. The corresponding list of genes clearly demonstrates that several GRAS genes were up/down-regulated simultaneously under both ABA and NaCl treatments at certain time points. In roots, the expression of 37.5% of the genes (IE type) overlapped under both stress treatments, while ΨOsGRAS5 (IE) was expressive in shoots only. Frontiers in Plant Science \| www.frontiersin.org Evolutionary Relationships, Gene Organization and Protein Properties of Rice GRAS Genes Plants use certain acclimation and adaptive measures to cope up with the impending stress, which is mostly modulated through the action of hormones and regulator genes (Lin et al., 2017). Thus, understanding the expression patterns of GRAS family of genes, which play a key role in gibberellin signaling and their spatio-temporal regulation help us identify the candidate genes for improving the endogenous defence ability of plants, particularly rice in the present context. In this study, we have 20 d old plants (after shifting to the greenhouse), shoot and root of 7 d old seedlings, 3 d old plumule and radicle, embryo and endosperm of 16 h germinating seeds (Figure 7). The mean values were used to plot the heat maps (Figure 8).h The expression analysis showed a conspicuous downregulation of all genes in most of the tissues particularly in plumule, radicle, embryo and the endosperm. Out of 40 selected genes, November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 11 Dutta et al. Genome-Wide Identification of OsGRAS Genes shortlisted important GRAS genes responsible for abiotic and In our analysis we have classified the genes into 14 subfamilies A B FIGURE 6 \| Quantitative real-time expression analysis of GRAS genes under biotic stress. Expression analysis of GRAS genes under the infection of Xanthomonas oryzae pv. oryzae causing bacterial leaf blight (A) and Rhizoctonia solani causing sheath blight (B) were studied. The genes were double normalized using rice actin and tubulin as internal reference genes and the CT values of untreated samples by ΔΔCT method. The experiment was conducted using biological and technical triplicates (n = 3) and a one way ANOVA was performed on the data using SigmaPlot v. 11 to gauge their statistical significane. a represents p < 0.05, b represents p < 0.025 and c represents p < 0.001. The data represent mean ± SE. A B B FIGURE 6 \| Quantitative real-time expression analysis of GRAS genes under biotic stress. Expression analysis of GRAS genes under the infection of Xanthomonas oryzae pv. oryzae causing bacterial leaf blight (A) and Rhizoctonia solani causing sheath blight (B) were studied. The genes were double normalized using rice actin and tubulin as internal reference genes and the CT values of untreated samples by ΔΔCT method. Frontiers in Plant Science \| www.frontiersin.org Evolutionary Relationships, Gene Organization and Protein Properties of Rice GRAS Genes The data was single normalized using rice actin as the internal reference gene. retroposition of intronless genes (Huang et al., 2015). Our genomic organization study revealed 31 OsGRAS genes out of 40 selected, to be intronless and this observation is in line with previous studies. Several interacting metallic and non-metallic ligands associated with GRAS genes along with their hydrophilic nature (as indicated by the GRAVY index) indicated their involvement in cell signaling, catalysis and protein-protein interactions (Ulucan et al., 2014; Jing et al., 2017).h with low pI tend to minimize the chances of non-specific interactions with nucleic acids and other acidic proteins (Takakura et al., 2015). All GRAS genes have at least one GRAS domain, but some were found to have two or possess a DELLA domain, which is known to have important role in gibberellic acid signaling (Urbanova and Leubner-Metzger, 2018). Evolutionary Relationships, Gene Organization and Protein Properties of Rice GRAS Genes Genome-Wide Identification of OsGRAS Genes FIGURE 7 \| Spatial regulation of OsGRAS genes. The native expression pattern of GRAS genes was studied in 13 different developmental tissues of rice plant. Majority of the genes were downregulated with some of them getting upregulated in mature vegetative and reproductive tissues. The list of the genes expressed in each tissue is mentioned in the boxes beside them. The figure has been adopted from Saha et al. (2017). FIGURE 8 \| Native expression analysis of GRAS genes. Heat map representing the spatial expression pattern of GRAS genes under 13 different developmental stages of rice. The experiment was conducted using biological and technical triplicates (n = 3) and the mean expression values were used to generate the map using the MORPHEUS program. The data was single normalized using rice actin as the internal reference gene. FIGURE 7 \| Spatial regulation of OsGRAS genes. The native expression pattern of GRAS genes was studied in 13 different developmental tissues of rice plant. Majority of the genes were downregulated with some of them getting upregulated in mature vegetative and reproductive tissues. The list of the genes expressed in each tissue is mentioned in the boxes beside them. The figure has been adopted from Saha et al. (2017). FIGURE 7 \| Spatial regulation of OsGRAS genes. The native expression pattern of GRAS genes was studied in 13 different developmental tissues of rice plant. Majority of the genes were downregulated with some of them getting upregulated in mature vegetative and reproductive tissues. The list of the genes expressed in each tissue is mentioned in the boxes beside them. The figure has been adopted from Saha et al. (2017). FIGURE 8 \| Native expression analysis of GRAS genes. Heat map representing the spatial expression pattern of GRAS genes under 13 different developmental stages of rice. The experiment was conducted using biological and technical triplicates (n = 3) and the mean expression values were used to generate the map using the MORPHEUS program. The data was single normalized using rice actin as the internal reference gene. FIGURE 8 \| Native expression analysis of GRAS genes. Heat map representing the spatial expression pattern of GRAS genes under 13 different developmental stages of rice. The experiment was conducted using biological and technical triplicates (n = 3) and the mean expression values were used to generate the map using the MORPHEUS program. Frontiers in Plant Science \| www.frontiersin.org Evolutionary Relationships, Gene Organization and Protein Properties of Rice GRAS Genes The experiment was conducted using biological and technical triplicates (n = 3) and a one way ANOVA was performed on the data using SigmaPlot v. 11 to gauge their statistical significane. a represents p < 0.05, b represents p < 0.025 and c represents p < 0.001. The data represent mean ± SE. In our analysis, we have classified the genes into 14 subfamilies (Cenci and Rouard, 2017) of which LISCL constituted the maximum number of genes. However, most expressive genes belonged to SCL3, SHR1, DELLA, HAM and PAT subfamilies. The 10 MEME-identified motifs were categorized into five conserved C-terminal GRAS motifs. Genes belonging to the same subfamily exhibited similar motif arrangements, but this varied within the subfamilies, which might be due to the diverse biological functions of GRAS genes. This group of proteins were reported to have originated in bacteria, which later expanded into eukaryotic genomes via horizontal gene transfer and repeated duplication events with the possible In our analysis, we have classified the genes into 14 subfamilies (Cenci and Rouard, 2017) of which LISCL constituted the maximum number of genes. However, most expressive genes belonged to SCL3, SHR1, DELLA, HAM and PAT subfamilies.hii shortlisted important GRAS genes responsible for abiotic and biotic stress tolerance. We have also studied the in-silico properties of these genes and have correlated them with our expression data. According to the published evidence that is available (Liu and Widmer, 2014), 60 GRAS genes were reported in the rice genome, which are distributed on all the 12 chromosomes except chromosome numbers 8 and 9. Highest number of GRAS gene density was observed on chromosome 11. We have selected 40 genes for our study, drawing one member representing each paralogous group. The availability of high quality genomic sequences enabled us to get an insight into the phylogenetic, genomic and protein properties of the GRAS genes in rice. The 10 MEME-identified motifs were categorized into five conserved C-terminal GRAS motifs. Genes belonging to the same subfamily exhibited similar motif arrangements, but this varied within the subfamilies, which might be due to the diverse biological functions of GRAS genes. This group of proteins were reported to have originated in bacteria, which later expanded into eukaryotic genomes via horizontal gene transfer and repeated duplication events with the possible November 2021 \| Volume 12 \| Article 777285 12 Dutta et al. Differential Expression Patterns of OsGRAS Genes and Their Spatio-Temporal Regulation Also, previous studies indicated that it is probable that the role of these genes in pattern formation and signal transduction enables them to be more expressive in roots (Pysh et al., 1999). Out of 13 different developmental tissues, only seven genes were induced in mature vegetative and reproductive tissues. Proteins belonging to DELLA subfamily of GRAS transcription factors are known to be negative regulators of seed germination as bioactive gibberellic acid causes proteasomal degradation of such proteins for gibberellin signaling to occur (Urbanova and Leubner-Metzger, 2018). This explains the downregulation of all genes in plumule, radicle, embryo and endosperm. Thus, OsGRAS genes might be associated with developmental regulation in mature rice plants. The induction of OsGRAS28 belonging to HAM subfamily and OsGRAS47 belonging to LISCL subfamily in reproductive tissues indicates their role in floral development. This can be correlated with the expression of PhHAM genes of Petunia hybrida (Stuurman et al., 2002), AtSCL6 and AtSCL27 genes of Arabidopsis (Fan et al., 2017) and PtGRAS67 of Populus (Liu and Widmer, 2014) flowers. All these genes belonging to HAM subfamily were reported to be involved in floral differentiation. LISCL subfamily gene from lily plants were also reported to be associated with miscrosporogenesis (Bolle, 2004). GRAS proteins are involved in growth signaling and plant developmental regulation. OsGRAS39 was expressive in roots under native as well as abiotic stress conditions indicating its tissue specificity. This gene belongs to SCL3 subfamily, which is known for modulating GA signaling in roots via protein–protein interactions (Weng et al., 2020). This group of proteins are involved in maintaining gibberellic acid homeostasis in roots, which in turn help them in controlling root development (Heo et al., 2011). During salt or ABA stress, roots are the primary organs to perceive the stress signals and initiate a signaling cascade towards the shoot. Therefore, OsGRAS39 (belonging to subfamily SCL3) along with other notable candidate genes like OsSHR1 (belonging to SHR subfamily) and OsSLR1 (belonging to SCR subfamily) might have an impact on the SCL3-DELLA interaction and SHR/SCR pathway in the root tissue leading to stress tolerance. Hence, high expression pattern of the above mentioned genes can be further exploited for their potential role in stress tolerance. A schematic diagram has been provided in the Supplementary Figure S6. p ( y ) Rice productivity is severely hampered by BLB and SB diseases. Differential Expression Patterns of OsGRAS Genes and Their Spatio-Temporal Regulation BLB infection during tillering stage can cause a yield decline of 20–40%, while it can reduce crop productivity by 50% at a younger stage. Upto 45% yield losses in rice are caused by SB infections (Chukwu et al., 2019; Singh et al., 2019). Thus, identification of key genes and understanding their expression patterns are important for developing tolerant varieties of rice for these diseases. Under BLB treatment, only six genes (15%) were expressive compared to 30 (75%) expressive genes under SB infection. Quite a number of genes were expressive under SB infection and both abiotic stress conditions. Noteworthy among them are OsGRAS39, OsGRAS8, OsSHR1 and OsSLR1. Thus, these genes can be considered to be quite important as they are expressive under both stress conditions with important roles in disease resistance. Majority of the genes were highly expressive in ABA treated roots and SB infection. The presence of multiple stress responsive elements in their putative promoter regions are corroborated by our expression data and these observations indicate their probable roles in improving plant defence against biotic and abiotic stress. GRAS gene family has been studied extensively in many plant species, but we have successfully provided a backdrop based on which future exploration on rice GRAS genes can be done. The differential expression patterns of these genes indicates their importance in stress remediation. Our study provides an insight into the role of GRAS genes in stress tolerance along with their spatio-temporal regulation. Based on this report, it would be possible to pick up important genes that can be further manipulated for developing stress tolerant varieties in rice and other related crops. Differential Expression Patterns of OsGRAS Genes and Their Spatio-Temporal Regulation Such preferential expression of GRAS genes in roots over shoots indicates their important role in stress responses (Janiak et al., 2016). Also, previous studies indicated that it is probable that the role of these genes in pattern formation and signal transduction enables them to be more expressive in roots (Pysh et al., 1999). transcripts and proteins in the cells for their adjustment to the environmental changes. Most of the GRAS genes were observed to be upregulated in roots with 55–60% of them showing IE type of gene expression. In plants, stress responses can be divided broadly into early and late response types. Early responsive genes are expressed within minutes of stress induction and this provides protection and repair from the initial stress. Such response “alarms” the plant to prepare for further stress tolerance or avoidance. On the other hand, late responsive genes are mostly involved in protein synthesis that regulates downstream genes, thereby responding to the “adaptation” part of stress mediation (Bahrami and Drabløs, 2016; Lin et al., 2017). ΨOsGRAS9 was observed to be late expressive under both treatments in root indicating that it might have an important role in subsequent steps of stress amelioration. Interestingly, 50–60% of IE expressed genes continued to express till the end point of the treatment. Among them, OsGRAS39 was the only gene that was observed to be highly expressive under both NaCl and ABA treatments in roots. Apart from this, ΨOsGRAS2, OsSHR1 and OsSCR1 continued their expression till 60 h under ABA treatment. This probably indicates that these constitute an important set of genes required by the plant throughout for stress remediation. Others like ΨOsGRAS3, ΨOsGRAS4, OsCIGR1 and OsGRAS32 under ABA and OsCIGR1 under NaCl were the genes induced initially (IE type), which either stopped expressing or got downregulated at subsequent time points. These genes are probably required for initial stress responses whose function is later on taken up by the other downstream genes in the signaling cascade. ΨOsGRAS5 (IE type) was found to be the only moderately expressive gene in shoot under both stress conditions. Since root is the first organ to perceive the stress signal, it induces a signaling cascade that extends towards the shoot. Such preferential expression of GRAS genes in roots over shoots indicates their important role in stress responses (Janiak et al., 2016). Differential Expression Patterns of OsGRAS Genes and Their Spatio-Temporal Regulation The majority of the genes were observed to have a pI less than seven and were found to be rich in negatively charged amino acids like Glutamic acid and Aspartic acid. This makes the interactions of GRAS proteins very specific as proteins Based on external cues, spatio-temporal regulation of gene transcription is required to control the concentration of particular November 2021 \| Volume 12 \| Article 777285 13 Genome-Wide Identification of OsGRAS Genes Dutta et al. transcripts and proteins in the cells for their adjustment to the environmental changes. Most of the GRAS genes were observed to be upregulated in roots with 55–60% of them showing IE type of gene expression. In plants, stress responses can be divided broadly into early and late response types. Early responsive genes are expressed within minutes of stress induction and this provides protection and repair from the initial stress. Such response “alarms” the plant to prepare for further stress tolerance or avoidance. On the other hand, late responsive genes are mostly involved in protein synthesis that regulates downstream genes, thereby responding to the “adaptation” part of stress mediation (Bahrami and Drabløs, 2016; Lin et al., 2017). ΨOsGRAS9 was observed to be late expressive under both treatments in root indicating that it might have an important role in subsequent steps of stress amelioration. Interestingly, 50–60% of IE expressed genes continued to express till the end point of the treatment. Among them, OsGRAS39 was the only gene that was observed to be highly expressive under both NaCl and ABA treatments in roots. Apart from this, ΨOsGRAS2, OsSHR1 and OsSCR1 continued their expression till 60 h under ABA treatment. This probably indicates that these constitute an important set of genes required by the plant throughout for stress remediation. Others like ΨOsGRAS3, ΨOsGRAS4, OsCIGR1 and OsGRAS32 under ABA and OsCIGR1 under NaCl were the genes induced initially (IE type), which either stopped expressing or got downregulated at subsequent time points. These genes are probably required for initial stress responses whose function is later on taken up by the other downstream genes in the signaling cascade. ΨOsGRAS5 (IE type) was found to be the only moderately expressive gene in shoot under both stress conditions. Since root is the first organ to perceive the stress signal, it induces a signaling cascade that extends towards the shoot. Frontiers in Plant Science \| www.frontiersin.org REFERENCES in rice. Theor. Appl. Genet. 134, 2253–2272. doi: 10.1007/s00122-021-03823-0 Fan, S., Zhang, D., Gao, C., Zhao, M., Wu, H., Li, Y., et al. (2017). Identification, classification, and expression analysis of GRAS gene family in Malus domestica. Front. 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Genome wide analysis of W-box element in Arabidopsis thaliana reveals TGAC motif with genes down regulated by heat and salinity. Sci. Rep. 9:1681. doi: 10.1038/ s41598-019-38757-7 Liu, X., and Widmer, A. (2014). Genome-wide comparative analysis of the GRAS gene family in Populus, Arabidopsis and Rice. Plant Mol. Biol. Rep. AUTHOR CONTRIBUTIONS grateful to M. S. Madhav, Department of Biotechnology, Indian Institute of Rice Research, Hyderabad, India, for providing the infected rice samples and wild type BPT-5204 seeds, her friend Indu for her help in creating phylogenetic tree and her sister Shinjini for her help in the digital art. PK acknowledges the National Academy of Sciences-India for the award of NASI- Platinum Jubilee Senior Scientist position. MM acknowledges the INSPIRE Faculty Fellowship received from the Department of Science and Technology (DST), Government of India. The authors are grateful to Prof. M. Uday Kumar for his help in Δ13C analysis. PK and MD designed the experiments and prepared the manuscript. MD performed the experiments. AS helped in the qRT-PCR experiments and analysis. MM identified GRAS gene from activation tagged lines and conceived the initial idea. PK supervised the work. All authors contributed to the article and approved the submitted version. PK and MD designed the experiments and prepared the manuscript. MD performed the experiments. AS helped in the qRT-PCR experiments and analysis. MM identified GRAS gene from activation tagged lines and conceived the initial idea. PK supervised the work. All authors contributed to the article and approved the submitted version. 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. November 2021 \| Volume 12 \| Article 777285 14 Genome-Wide Identification of OsGRAS Genes Dutta et al. SUPPLEMENTARY MATERIAL MD is grateful to UoH BBL and UGC for research fellowship and contingency and to DBT for funding the rice activation tagging project. She is also grateful to S. Dayananda, Dean School of Life Sciences, UoH for his help. MD and AS are grateful to DBT for providing research fellowship and contingency. MD is The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021.777285/ full#supplementary-material REFERENCES 3DLigandSite: predicting ligand-binding sites using similar structures. Nucleic Acids Res. 38, W469–W473. doi: 10.1093/nar/gkq406i Morel, J., Clavero, S., Mongrand, S., Furt, F., Fromentin, J., Bessoule, J. J., et al. (2006). Proteomics of plant detergent-resistant membranes. Mol. Cell. Proteomics 5, 1396–1411. doi: 10.1074/mcp.M600044-MCP200 Wei, Q., Cao, H., Li, Z., Kuai, B., and Ding, Y. (2013). 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Role of low-complexity sequences in the formation of novel protein coding sequences. Mol. Biol. Evol. 29, 883–886. doi: 10.1093/molbev/msr263 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 Tran, L. S. P., Nakashima, K., Sakuma, Y., Simpson, S. D., Fujita, Y., Maruyama, K., et al. (2004). Isolation and functional analysis of arabidopsis stress-inducible November 2021 \| Volume 12 \| Article 777285 Frontiers in Plant Science \| www.frontiersin.org 16 Dutta et al. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. 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https://openalex.org/W3115506618	https://europepmc.org/articles/pmc7781710?pdf=render	English	null	Development of an Immunoassay for the Detection of Amyloid Beta 1-42 and Its Application in Urine Samples	Journal of immunology research	2,020	cc-by	7,248	Anurak Wongta ,1,2 Surat Hongsibsong ,1,2,3 Somporn Chantara ,4 Mookda Pattarawarapan ,5 Ratana Sapbamrer ,6 Korawan Sringarm ,3,7 Zhen-Lin Xu ,8 and Hong Wang8 Anurak Wongta ,1,2 Surat Hongsibsong ,1,2,3 Somporn Chantara ,4 Mookda Pattarawarapan ,5 Ratana Sapbamrer ,6 Korawan Sringarm ,3,7 Zhen-Lin Xu ,8 and Hong Wang8 1Environmental Science Ph.D. Program, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand 2School of Health Science Research, Research Institute for Health Science, Chiang Mai University, Chiang Mai 50200, Thailand 3Cluster of Research and Development of Pharmaceutical and Natural Products Innovation for Human or Animal, Chiang Mai University, Chiang Mai 50200, Thailand g y g 4Environmental Chemistry Research Laboratory, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand g y g 4Environmental Chemistry Research Laboratory, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand 5Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand 6Department of Community Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 7Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand 8Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China artment of Community Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 6Department of Community Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 7Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand 8Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China 7Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand 8Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China Correspondence should be addressed to Surat Hongsibsong; surat.hongsibsong@cmu.ac.th eceived 9 September 2020; Revised 8 December 2020; Accepted 11 December 2020; Published 28 December 202 Academic Editor: Xiao Feng Yang Academic Editor: Xiao Feng Yang Copyright © 2020 Anurak Wongta 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. Amyloid beta peptides (Aβ1-42) have been found to be associated with the cause of Alzheimer’s disease (AD) and dementia. Currently, methods for detecting Aβ1-42 are complicated and expensive. Hindawi Journal of Immunology Research Volume 2020, Article ID 8821181, 9 pages https://doi.org/10.1155/2020/8821181 Hindawi Journal of Immunology Research Volume 2020, Article ID 8821181, 9 pages https://doi.org/10.1155/2020/8821181 Anurak Wongta ,1,2 Surat Hongsibsong ,1,2,3 Somporn Chantara ,4 Mookda Pattarawarapan ,5 Ratana Sapbamrer ,6 Korawan Sringarm ,3,7 Zhen-Lin Xu ,8 and Hong Wang8 The present study is aimed at developing an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect Aβ1-42 by using a polyclonal antibody from alpaca, an application used in urine samples. The serum was collected from the alpaca after immunizing it with Aβ1-42 at 500 μg/injection 5 times. The ic-ELISA was developed and showed a half-maximal inhibitory concentration (IC50) of 103.20 ng/ml. The limit of detection (LOD) was 0.39 ng/100 μl. The cross-reactivity was tested with Aβ1-40 and 8 synthesized peptides that had sequence similarities to parts of Aβ1-42. The cross-reactivity of Aβ1-40 and peptide 1 (DAEFRHDSGYE) was 55% and 69.4%, respectively. The ic-ELISA was applied to analyze Aβ1-42 in the urine and precipitated protein urine samples. This method can be used for detecting a normal level of total soluble Aβ (approximately 1 ng in 5 mg of precipitated urine protein) and can be used for detecting the early stages of AD. It is considered to be an easy and inexpensive method for monitoring and diagnosing AD. 2. Materials and Methods After wash- ing, the plate was blocked with 200 μl/well of 1% skim milk in PBS as a blocking solution and incubated for 1 hour at 25°C. Then, the blocking solution was discarded, and the plate was dried by patting with paper towels. Alpaca antiserum, which was prepared with 8 dilutions from 1 : 1,000 to 1 : 32,000, was added to the wells and incubated for 1 hour at 25°C. Then, the plate was washed as in the previous washing procedure. In the next step, 100 μl of goat anti-llama IgG labeled with horseradish peroxidase (1: 5,000 in PBST) (Abcam, USA) was added to each well and incubated for 1 hour at 25°C. Then, the washing procedure was repeated. For determina- tion, 100 μl of TMB substrate (12.5 ml citrate buﬀer pH 4, 200 μl stock TMB, and 50μl of 1% H2O2) was added and rested in the dark at 25°C for 10min. The reaction was stopped with 2 M H2SO4. The optical density (OD) was mea- sured at 450 nm in a multiscan spectrophotometer (Tecan, Austria) to evaluate the optimal dilution of the alpaca serum and the concentration of coating [24]. Brain imaging techniques, which are crucial in the assessment of patients with AD, include ﬂuoro-D-glucose integrated with positron emission tomography (FDG-PET) and amyloid positron emission tomography (amyloid PET) imaging [16, 17]. However, there are still many limi- tations of these methods, including cost, potential compli- cations, the need for a specialist, and the requirement for high-performance equipment. While these conventional methods for identifying AD by detecting Aβ1-42 are still required and useful, they cannot detect the early stages of AD. The immunoassay is a simple, economical technique with high sensitivity and speciﬁcity [18, 19] that can be developed and applied to biological samples. In the present day, immu- noassays for human Aβ have been developed and can be pur- chased from pharmaceutical companies throughout the world. The most commercial enzyme-linked immunosorbent assay (ELISA) test kits are developed based on sandwich- ELISA, which requires two antibodies to detect the target antigen. They are used for serum, plasma, tissue homoge- nates, and cerebrospinal ﬂuid (CSF). The sensitivity of com- mercial kits is a LOD between 4.73 and 1,000 pg/ml and a detection range between 7.8 and 480,000 pg/ml for all Aβ. Aβ1-42 can be present at a lower concentration, where the LOD is between 4.73 and 9.38 pg/ml. 2. Materials and Methods 2.1. Preparation of the Antibody. A single alpaca was raised at Nanchang (Jiangxi Province, People’s Republic of China) and immunized subcutaneously 5 times at 2-week intervals with a 500 μg/injection of Aβ1-42 to produce antibodies against Aβ1-42. The immunogen was prepared with Aβ1- 42, which was purchased from Thermo Fisher (USA), dis- solved in phosphate-buﬀered saline (PBS) pH 7.2, and gently mixed with an equal volume of Freund’s Complete Adjuvant (Sigma-Aldrich, USA) to make an emulsion for the ﬁrst immunization and Freund’s Incomplete Adjuvant (Sigma- Aldrich, USA) to make an emulsion for the 2nd, 3rd, 4th, and 5th immunization. The blood was collected from the jugular vein of the alpaca before the 4th and 5th injection and 1 week after the last immunization. The 5th serum was used to develop the method in this study. The protocol of the experiment was approved by the animal ethical commit- tee of South China Agricultural University, Guangdong, Guangzhou, China, and the study has been conducted in accordance with the guidelines for the animal screening of antiserum. Several genetic, cell biology, biochemical, and animal studies support the concept that Aβ plays a central role in the development of AD pathology [9, 10]. The most common isoforms are Aβ1-40 and Aβ1-42 [11]. Aβ1-42 and Aβ1-43 are the major constituents of senile plaques and neuroﬁbril- lary tangles that occur in the hippocampus, neocortex, and amygdala of patients with AD [12]. Aβ1-42 is a good marker for the early stages of AD and is often detectable in several biological samples such as CSF, saliva, and urine in normal stages before the patient shows symptoms of AD, suggesting it is involved in the cause of AD [13–15]. 2.2. Indirect ELISA for the Titration of the Alpaca Antiserum. An indirect ELISA was used to determine the optimal dilution factor of the antibodies that were later used in ic- ELISA procedure [22, 23]. Standard Aβ1-42 was serially diluted into coating buﬀer (carbonate buﬀer, pH 9.6), pro- ducing 11 concentrations of Aβ1-42, and coated on Maxi- sorp microwell plates (Thermo Fisher Scientiﬁc, Denmark) at 4°C for 16 hours overnight. After incubation, the plate was washed 4 times with 400 μl/well PBST (PBS with 0.05% Tween) by a microplate washer (Thermo Fisher Scientiﬁc, Finland) and dried by patting with paper towels. 1. Introduction social costs. AD can be caused by several reasons, such as genetic causes, aging, malnutrition, immune system dysfunc- tion, infectious agents, severe brain damage, congenital dis- eases (i.e., hypertension and diabetes), and the environment (i.e., exposure to pesticides or aluminum) [2, 3]. Alzheimer’s disease (AD) is an age-related chronic degenera- tive disease that damages the nervous system. It is mostly found in the elderly as a progressive neurodegenerative dis- ease that predominantly aﬀects cognitive functions [1]. The increasing number of AD patients is becoming an issue of serious concern due to the concomitant rise in medical and Amyloid beta peptides (Aβ) are neurotrophic and neuro- toxic proteins that are used as a biomarker for detecting AD. Extracellular Aβ can lead to synapse loss and oxidative stress. Journal of Immunology Research 2 component, and the monomeric Aβ level is related to the clinical dementia rating score (CDR) [21]. Intracellular Aβ can increase the production of free radical species (ROS), causing increased levels of neurotoxicity and the death of neurons [4, 5]. Aβ is formed after sequential cleavage of the amyloid precursor protein (APP) by β-secre- tase, which removes the soluble APPβ (sAPPβ) and leaves a 99 amino acid C-terminal APP fragment (C99) in the cell membrane. Then, C99 is serial cleaved by γ-secretase at the ε-, ζ-, and γ-sites in the transmembrane domain (TMD), which releases the APP intracellular domain (AICD) into the cytosol and the 37-43 amino acid Aβ species into the extracellular space [6]. Aβ molecules can aggregate to form ﬂexible soluble oligomers that may exist in several forms [7]. Aβ is the major constituent of the ﬁbrils deposited into senile plaques and cerebral blood vessels of patients with AD. For Aβ levels under normal conditions, the daily excre- tion of intact soluble amyloid beta in the urine represents less than 1% of the circulating pool [8]. component, and the monomeric Aβ level is related to the clinical dementia rating score (CDR) [21]. 2. Materials and Methods 31.77 g of ammonium sulfate added to 100 ml urine (to reach 50% saturation) and well-mixed by a magnetic stirrer for 1 hour on ice. Then, the mixture was centrifuged 4,000 rpm for 1 hour at 4°C. The supernatant was discarded; then, the protein pellet was resuspended in 1 ml PBS buﬀer (pH 7.2) and kept at 4°C. The speciﬁcity and sensitivity of this method were mea- sured with alpaca antiserum (1 : 1,000 in PBST), and the competition between the free standard Aβ1-42 with the coated antigen (Aβ1-42) was observed. The microwell plate was coated at 4°C for 16 hours overnight with 100μl/well of 2 μg/ml Aβ1-42 in coating buﬀer. Twelve concentrations of competitive Aβ1-42 were prepared (0-4,000 ng/ml, twofold dilution), and ic-ELISA was performed with 1 : 1,000 (v/v) dilution of alpaca antiserum, as described above. 2.6. Assessment of the Matrix Eﬀect on Aβ1-42 ic-ELISA Calibration Graph. The eﬀects of urine and precipitated urine protein on the performance of the ic-ELISA were assessed. Twelve dilutions of each urine sample (1: 1-1 : 5,120 in PBS) and precipitated urine protein (0.001-4.56 mg/ml) were determined by ic-ELISA, as described above. The nonmatrix eﬀect was determined by the same value OD of the sample dilution and the OD of the PBS sample. The calibration curve was obtained and calculated as IC50 . The limit of detection (LOD) was calculated by using the formula [25] as follows: LOD = B0 – 3SD, ð1Þ ð1Þ where B0 is the optical density (OD) of blank (no Aβ1‐42) and SD is the standard deviation of B0. Calibration curves of the Aβ1-42 in urine and precipi- tated urine protein were obtained. Twelve concentrations of Aβ1-42 (0–8,000 ng/ml) were spiked into 1 : 160 urine in PBS and 0.456 mg/ml precipitated urine protein, and then, ic-ELISA was performed as described above. LODs were cal- culated as described above. 2.4. Assessment of the Cross-Reactivity of Aβ1-42 ic-ELISA. Standard Aβ1-40, Aβ1-42, and 8 synthesized Aβ peptides (Aβ1-8, Aβ7-15, Aβ14-21, Aβ20-27, Aβ26-33, Aβ32-39, Aβ35-42, and Aβ36-43) were tested in the ic-ELISA assay as described above. Calibration curves were obtained. The cross-reactivity (CR) values were calculated by using the formula [26] as follows: The correlation curves were obtained by comparing the concentration of spiked Aβ1-42 samples and the detected concentration in the calibration curves. 3. Results Cross‐reactivity rate % ð Þ ð2Þ = IC50 of Aβ1 −42 IC of the structural related protein ð Þ × 100 , ð2Þ 3.1. Development of ic-ELISA. To develop the ic-ELISA for the detection of Aβ1-42, alpaca antiserum was tested using indirect ELISA in the ﬁrst step to determine the optimal con- centration of coating antigen and the optimal dilution of antiserum. Six concentrations of Aβ1-42 in coating buﬀer were coated on the wells, and six serial dilution ratios from 1 : 1,000 to 1 : 32,000 of alpaca antiserum were investigated. The 1 : 1,000 dilution showed the highest binding sensitivity among all coating antigen concentrations (Table 1). Then, sensitivity of the indirect ELISA was tested with 15 concen- trations of Aβ1-42. The half-maximal eﬀective concentration (EC50) of a 1 : 1,000 (v/v) dilution of alpaca antiserum was 928.40 ng/ml as shown in Figure 1(a). = IC50 of the structural‐related protein ð Þ × 100 , = IC50 of the structural‐related protein ð Þ × 100 2. Materials and Methods Conventional antibod- ies such as polyclonal antibodies (pAb) and monoclonal anti- bodies (mAb) from rodents are used in the test kits. The price of one test kit is in the range of 395-875 dollars for 96 tests [20], which is far too expensive to be widely used. 2.3. Indirect Competitive ELISA for Quantiﬁcation of Aβ1-42. The ic-ELISA assay was performed with alpaca antiserum (1 : 1,000 in PBST), and the competition between the free This study was conducted to develop ELISA for detecting Aβ1-42 in urine samples in which Aβ is found as a normal Journal of Immunology Research 3 standard Aβ1-42 and the coated antigen (Aβ1-42) was observed in order to determine the concentration of the free standard. The microwell plate was coated at 4°C for 16 hours overnight with 100μl/well of 3 concentrations of coating antigen (Aβ-42, 2-4 μg/ml in coating buﬀer). The plate was then washed and blocked as described above. An equal vol- ume of standard Aβ-42 was added to the alpaca antiserum in a u-shaped plate at 25°C for 1 hour. After the blocking solution was discarded and dried, the mixture of standard Aβ1-42 and alpaca antiserum was added in triplicate to the wells of each coating antigen. After the plate was incubated for 1 hour at 25°C and washed, anti-llama IgG labeled with horseradish peroxidase (1 : 5,000 in PBST) and TMB sub- strate were added following the ELISA protocol, as described above. Table 1: Results of the ELISA checker-board titration test of the binding of 6 concentrations of Aβ1-42 and 6 serially diluted antiserums by the indirect ELISA method. Antiserum (1 : X) OD at 450 nm Concentration of coated Aβ1-42 (ng/ml) 4000 2000 1000 500 250 125 1,000 1.053 0.938 0.852 0.74 0.644 0.525 2,000 0.929 0.799 0.686 0.56 0.443 0.368 4,000 0.821 0.622 0.475 0.379 0.272 0.217 8,000 0.659 0.441 0.342 0.235 0.178 0.146 16,000 0.456 0.278 0.203 0.149 0.113 0.099 32,000 0.293 0.171 0.13 0.098 0.081 0.073 31.77 g of ammonium sulfate added to 100 ml urine (to reach 50% saturation) and well-mixed by a magnetic stirrer for 1 hour on ice. Then, the mixture was centrifuged 4,000 rpm for 1 hour at 4°C. The supernatant was discarded; then, the protein pellet was resuspended in 1 ml PBS buﬀer (pH 7.2) and kept at 4°C. where IC50 is the half-maximal inhibitory concentration. where IC50 is the half-maximal inhibitory concentration. 2.5. Preparation of the Urine Protein. A pool of urine was prepared from 10 healthy donors (5 male and 5 female) and used in this study. Due to the small amount of Aβ1-42 in urine, the concentration of the urine proteins was increased by a precipitation technique. Urine protein was prepared by the salting out technique, which is the most common method used to precipitate proteins. Ammonium sulfate ((NH4)2SO4) was used to compress the solvation layer and increase the protein–protein interactions. The increasing salt concentration caused the protein to separate and fall to the bottom of the sample solution by aggregation and precip- itation activities [27]. The precipitation was performed with As the second step, the ic-ELISA was used to optimize the concentration of coating antigen (Aβ1-42). Three concentra- tions of Aβ1-42, prepared at 2,000, 3,000, and 4,000 ng/ml in the coating buﬀer, were used as the coating antigen, and Journal of Immunology Research Absorbance at 450 nm 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1 2 Log concentration A𝛽1-42 (ng/ml) 3 4 5 6 EC50 = 928.50 ng/ml (a) %Binding Log concentration A𝛽1-42 (ng/ml) 3.50 3.25 3.00 2.75 2.50 2.25 2.00 1.75 1.50 –5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 IC50 = 532.00 ng/ml IC50 = 533.90 ng/ml IC50 = 517.10 ng/ml (b) –1 0 25 50 75 100 %Binding 0 1 2 Log concentration A𝛽1-42 (ng/ml) 3 4 IC50 = 103.20 ng/ml (c) Concentration A𝛽1-42 (ng/ml) 70 60 50 40 30 20 10 0 Absorbance at 450 nm 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 R2 = 0.9923 Y = –0.0086X+1.621 (d) Figure 1: Development of Aβ1-42 detection. (a) Indirect ELISA calibration curves for Aβ1-42 using 1 : 1,000 (v/v) dilution of alpaca antiserum. (b) Optimization of coating antigen using indirect competitive ELISA. The percent binding showed a competition percentage of 6 concentrations of competitive Aβ1-42 (62, 125, 250, 500, 1,000, and 2,000 ng/ml) to a 1 : 1,000 (v/v) dilution of alpaca antiserum at 3 concentrations of coating antigen (■= 2,000, ▲= 3,000, and ▼= 4,000 ng/ml of Aβ1-42 in coating buﬀer). (c) ic-competitive ELISA calibration curves for Aβ1-42 using a 1 : 1000 (v/v) dilution of alpaca antiserum. The calibration curve was obtained using analyte standards prepared in PBS buﬀer. where IC50 is the half-maximal inhibitory concentration. (d) ic-competitive ELISA calibration curves obtained by OD. 4 Journal of Immunology Research Absorbance at 450 nm 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1 2 Log concentration A𝛽1-42 (ng/ml) 3 4 5 6 %Binding Log concentration A𝛽1-42 (ng/ml) 3.50 3.25 3.00 2.75 2.50 2.25 2.00 1.75 1.50 –5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 (a) –1 0 25 50 75 100 %Binding 0 1 2 Log concentration A𝛽1-42 (ng/ml) 3 4 (b) Concentration A𝛽1-42 (ng/ml) 70 60 50 40 30 20 10 0 Absorbance at 450 nm 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 R2 = 0.9923 Absorbance at 450 nm %Binding (c) (d) Figure 1: Development of Aβ1-42 detection. (a) Indirect ELISA calibration curves for Aβ1-42 using 1 : 1,000 (v/v) dilution of alpaca antiserum. (b) Optimization of coating antigen using indirect competitive ELISA. The percent binding showed a competition percentage of 6 concentrations of competitive Aβ1-42 (62, 125, 250, 500, 1,000, and 2,000 ng/ml) to a 1 : 1,000 (v/v) dilution of alpaca antiserum at 3 concentrations of coating antigen (■= 2,000, ▲= 3,000, and ▼= 4,000 ng/ml of Aβ1-42 in coating buﬀer). (c) ic-competitive ELISA calibration curves for Aβ1-42 using a 1 : 1000 (v/v) dilution of alpaca antiserum. The calibration curve was obtained using analyte standards prepared in PBS buﬀer. (d) ic-competitive ELISA calibration curves obtained by OD. ic-ELISA was performed with a 1 : 1,000 (v/v) dilution of alpaca antiserum, as described above. The coated concen- tration of 2,000 ng/ml Aβ1-42 was selected since it showed the same half-maximal inhibitory concentration (IC50) as the above concentrations (Figure 1(b)). the test (the sequence of peptides is shown in Table 2). The data within a range of 12 concentrations of the Aβ1-42 stan- dard (0–4,000 ng/ml, twofold dilution in PBST) were used for the calculation of cross-reactivity. Aβ7-15, Aβ14-21, Aβ20- 27, Aβ26-33, Aβ32-39, Aβ35-42, and Aβ36-43 showed no detectable cross-reactivity. However, Aβ1-8 showed 69.40% and Aβ1-40 showed 55.00% cross-reactivity as shown in Table 2, suggesting both can have some contribution to the measurement of the Aβ1-42 level with this method. For the third step, the sensitivity of this method was determined. The IC50 of ic-ELISA was 103.20 ng/ml as shown in Figure 1(c). where IC50 is the half-maximal inhibitory concentration. The results had no matrix eﬀect OD at 1 : 160 for the pooled urine, as shown in Figure 2(a), and 0.456 mg/ml for the urine protein, as shown in Figure 2(b). The calibration curve was obtained using analyte standard Aβ1-42 prepared in 1 : 160 diluted pool urine and 0.456 mg/ml precipitated urine protein. The results showed an IC50 = 115:90 ng/ml for 1 : 160 pooled urine, as shown in Figure 3(a), and 579.00 ng/ml for precipitated urine protein, as shown in Figure 3(b). The LOD of this method was 16.83 ng/ml for the pooled urine sample and 139.60 ng/ml for the precipi- tated urine protein. The correlation of the detected and spiked concentrations of Aβ1-42 in the samples by using the ic-ELISA calibration curve showed R2 = 0:9973 for pooled urine (in the range 7.80-1000.00 ng/ml Aβ1-42) as shown in Figure 3(c) and R2 = 0:9918 for the precipitated urine protein (in the range 15.63-1000.00 ng/ml Aβ1-42) as shown in Figure 3(d). However, our method showed 69.4% and 55% cross- reaction to the synthesized Aβ1-8 and Aβ1-40, respectively, as expected, since Aβ1-40 has a sequence close to Aβ1-42. Synthesized Aβ1-8 also has a sequence present at the begin- ning of every Aβ. The LOD of this method cannot detect Aβ1-42 in urine samples. However, due to the high cross- reactivity, the method can be developed to detect total Aβ in urine and could be used to detect total Aβ in other biolog- ical samples. p In urine samples, the LOD was 16.83 ng/ml and 139.60 ng/ml for precipitated urine protein. Since urine con- tains inorganic ions (K +, Na +, Cl −, and Ca2 +), organic molecules such as creatinine, urea, and uric acid, and more than 1,500 proteins, mainly extracellular, membranes, cell membranes, and cell debris, the higher LOD may come from the matrix in urine, which can interrupt the binding of anti- gens with antibodies used in the ELISA assays [30]. Another limitation of commercial ELISA kits is the price; most are expensive and cannot be routinely used for early detection in people. The yields of pAb from rodents are limited due to the size of the animal. In this study, the antibody was pro- duced in an alpaca, whose blood can be collected in larger quantities, which allows our method to be developed at a lower cost. where IC50 is the half-maximal inhibitory concentration. The LOD of this method was 0.39 ng/100 μl, calculated by the formula and OD calibration curve shown in Figure 1(d). 3.3. Matrix Eﬀect on the Aβ1-42 ic-ELISA. The developed ic- ELISA was performed by using a 1 : 1,000 (v/v) dilution of alpaca antiserum in 12 diluted samples (1 : 1–1 : 5120 in PBS) of pooled urine and 12 diluted samples of precipitated urine protein (urine protein = 0:001‐4:56mg/ml) that were 3.2. Cross-Reactivity of the Assay. The speciﬁcity of the devel- oped ic-ELISA was assessed by measuring the cross-reactivity toward the same structural proteins as Aβ1-42, using Aβ1- 40, and 8 synthesized Aβ peptides (GenScript, China) in Journal of Immunology Research 5 Table 2: Cross-reactivity of synthesized peptides and two beta amyloid proteins. Table 2: Cross reactivity of synthesized peptides and two beta amyloid proteins. Sample Sequence IC50 (ng/ml) % cross-reaction Aβ1-42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA 103.20 100.00 Aβ1-8 DAEFRHDS 148.80 69.40 Aβ1-40 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV 187.70 55.00 Aβ7-15 DSGYEVHHQ >4000 - Aβ14-21 HQKLVFFA >4000 - Aβ20-27 FAEDVGSN >4000 - Aβ26-33 SNKGAIIG >4000 - Aβ32-39 IGLMVGGV >4000 - Aβ35-42 MVGGVVIA >4000 - Aβ36-43 VGGVVIAT >4000 - used as the matrix antigen of competition with coated Aβ1- 42. The results had no matrix eﬀect OD at 1 : 160 for the pooled urine, as shown in Figure 2(a), and 0.456 mg/ml for the urine protein, as shown in Figure 2(b). The calibration curve was obtained using analyte standard Aβ1-42 prepared in 1 : 160 diluted pool urine and 0.456 mg/ml precipitated urine protein. The results showed an IC50 = 115:90 ng/ml for 1 : 160 pooled urine, as shown in Figure 3(a), and 579.00 ng/ml for precipitated urine protein, as shown in Figure 3(b). The LOD of this method was 16.83 ng/ml for the pooled urine sample and 139.60 ng/ml for the precipi- tated urine protein. The correlation of the detected and spiked concentrations of Aβ1-42 in the samples by using the ic-ELISA calibration curve showed R2 = 0:9973 for pooled urine (in the range 7.80-1000.00 ng/ml Aβ1-42) as shown in Figure 3(c) and R2 = 0:9918 for the precipitated urine protein (in the range 15.63-1000.00 ng/ml Aβ1-42) as shown in Figure 3(d). used as the matrix antigen of competition with coated Aβ1- 42. 4. Discussion donors (proteinuria < 5 mg/dl), the excretion of soluble Aβ was calculated at 0:81 ± 0:26 ng/5 mg of urinary proteins (13 ± 4 ng/24 h) [8]. Therefore, at this Aβ level, our method could measure the level of monomer Aβ in the urine. By applying urine sample preparation methods, such as precip- itation techniques to increase the concentration of Aβ in the urine sample, we can use 1 dl of urine to prepare 5 mg urine protein, which can reach approximately 1 ng of Aβ in normal donors. In addition, from the urine matrix tests, our method found an eﬀect of the pooled urine at under 1 : 160 dilution (urine protein < 32:25 ng in 100 μl sample). This means that it is not possible to use this method to deter- mine Aβ in urine samples without a sample pretreatment process. However, since precipitated urine protein has shown nonmatrix eﬀect results at 0.456 mg/ml (urine protein = 0:05 mg in 100 μl sample) and unidentiﬁed inhibition results of 4.56 and 0.91 mg/ml (urine protein = 0:46 and 0:09mg in donors (proteinuria < 5 mg/dl), the excretion of soluble Aβ was calculated at 0:81 ± 0:26 ng/5 mg of urinary proteins (13 ± 4 ng/24 h) [8]. Therefore, at this Aβ level, our method could measure the level of monomer Aβ in the urine. By applying urine sample preparation methods, such as precip- itation techniques to increase the concentration of Aβ in the urine sample, we can use 1 dl of urine to prepare 5 mg urine protein, which can reach approximately 1 ng of Aβ in normal donors. In addition, from the urine matrix tests, our method found an eﬀect of the pooled urine at under 1 : 160 dilution (urine protein < 32:25 ng in 100 μl sample). This means that it is not possible to use this method to deter- mine Aβ in urine samples without a sample pretreatment process. However, since precipitated urine protein has shown nonmatrix eﬀect results at 0.456 mg/ml (urine protein = 0:05 mg in 100 μl sample) and unidentiﬁed inhibition results of 4.56 and 0.91 mg/ml (urine protein = 0:46 and 0:09mg in The serum and CSF sample collection requires invasive methods and can be harmful to participants, while urine samples can be easily and not harmfully collected. 4. Discussion We propose a new alternative method for the detection of Aβ in human biological samples, an ic-ELISA for the detection of Aβ1-42 in urine samples that we have developed. Most com- mercial ELISA test kits for Aβ in humans are developed based on sandwich-ELISA, which requires two antibodies for detection, and only a few commercial test kits can detect Aβ in urine. One advantage of our method is that it only requires one speciﬁc antibody and can be detected in urine samples. Another advantage of using a single antibody is reducing nonspeciﬁc bounding since other substances may aﬀect each antibody. The commercial kits have a LOD between 4.73 and 1,000 pg/ml, and their detection range is between 7.8 and 480,000 pg/ml for all Aβ. Aβ1-42 shows a lower concentration, where LOD is usually between 4.73 and 9.38 pg/ml. Our method shows that the concentration of the LOD for Aβ1-42 is higher than the commercial kit at 3.90 ng/ml (390 pg/well), based on the levels of Aβ1-42 in human biological samples that are regularly monitored at relatively low levels: 750 pg/ml for CSF samples [28] and 17.65 pg/ml for serum samples [29]. So, we are not able to use the commercial methods to detect Aβ1-42 in these samples. A previous study reported that Aβ is removed from cir- culation by the capillary beds of the kidneys, liver, and gas- trointestinal tract, as well as through the skin and excreted in the urine [31]. According to studies from Takata et al., Aβ has a correlation with the clinical dementia rate (CDR) of people with AD and mild cognitive impairment (MCI), with an increasing value of Aβ in mild AD (CDR 0.5 to 1) and a decreasing value in more severe AD (CDR 2 to 3), since aggregated Aβ plaques might not be able to be eliminated via normal clearance routes in severe cases. The detection sensi- tivity of their method was 40pg/ml, which still cannot be quantiﬁed [21]. Since our new ic-ELISA method can detect this amount of Aβ in urine samples, it can be used as a pre- liminary assessment for AD. y Aβ peptides are a normal component of human urine as identiﬁed in the study of Ghiso et al. [8], who conducted immunoprecipitation experiments with anti-Aβ antibodies followed by detection and identiﬁcation by immunoblots and MALDI mass spectrometry. 4. Discussion For Aβ levels in normal 0.6 0.8 1 1.2 1.4 1.6 1.8 1 5 10 20 40 80 160 320 640 1280 2560 5120 Absorbance at 450 nm Dilution of urine (1:X) Urine PBS (a) 0.6 4.56 0.912 0.456 0.228 0.114 0.057 0.0285 0.01425 0.007125 0.0035625 0.00178125 0.000890625 0.8 1 1.2 1.4 1.6 1.8 Absorbance at 450 nm Concentration of precipitated urine protein (mg/ml) Precipitated urine protein PBS (b) Figure 2: Observation of the urine matrix eﬀect using ic-ELISA. (a) The curve was obtained using analyte 12 dilutions of pooled urine prepared in PBS buﬀer (1 : 1–1 : 5120). (b) The curve was obtained using analyte 12 concentrations of precipitated urine protein prepared in PBS buﬀer (0.001-4.56 mg/ml). 6 Journal of Immunology Research Journal of Immunology Research 0.6 0.8 1 1.2 1.4 1.6 1.8 1 5 10 20 40 80 160 320 640 1280 2560 5120 Absorbance at 450 nm Dilution of urine (1:X) Urine PBS (a) 0.6 4.56 0.912 0.456 0.228 0.114 0.057 0.0285 0.01425 0.007125 0.0035625 0.00178125 0.000890625 0.8 1 1.2 1.4 1.6 1.8 Absorbance at 450 nm Concentration of precipitated urine protein (mg/ml) Precipitated urine protein PBS (b) igure 2: Observation of the urine matrix eﬀect using ic-ELISA. (a) The curve was obtained using analyte 12 dilutions of poo repared in PBS buﬀer (1 : 1–1 : 5120). (b) The curve was obtained using analyte 12 concentrations of precipitated urine protein n PBS buﬀer (0.001-4.56 mg/ml). Journal of Immunology R 6 0.6 0.8 1 1.2 1.4 1.6 1.8 1 5 10 20 40 80 160 320 640 1280 2560 5120 Absorbance at 450 nm Dilution of urine (1:X) Dilution of urine (1:X) Figure 2: Observation of the urine matrix eﬀect using ic-ELISA. (a) The curve was obtained using analyte 12 dilutions of pooled urine prepared in PBS buﬀer (1 : 1–1 : 5120). (b) The curve was obtained using analyte 12 concentrations of precipitated urine protein prepared in PBS buﬀer (0.001-4.56 mg/ml). Figure 2: Observation of the urine matrix eﬀect using ic-ELISA. (a) The curve was obtained using analyte 12 dilutions of pooled urine prepared in PBS buﬀer (1 : 1–1 : 5120). (b) The curve was obtained using analyte 12 concentrations of precipitated urine protein prepared in PBS buﬀer (0.001-4.56 mg/ml). 100 μl sample), it is possible to develop this method to detect amyloid in urine samples in combination with appropriate urine protein sample preparation. 4. Discussion The results can be corrected referencing the concentration of Aβ against urine creatinine and reported as microgram per milliliter urine or microgram per gram creatinine, as reported in sev- eral previous studies. Most of the immunoassay methods were developed based on conventional antibodies and have some limitations, i.e., the lack of speciﬁcity of pAb and complicated production of mAb [32]. However, the production of a single-domain antibody (sdAb) from a phage display technique could pro- vide more sensitivity, speciﬁcity, and stability of the antibody. In addition, a large number of antibodies can be produced by Journal of Immunology Research 7 Log concentration A𝛽1-42 (ng/ml) 5 4 3 2 1 0 %Binding 0 10 20 30 40 50 60 70 80 90 IC50 = 115.09 ng/ml (a) %Binding Log concentration A𝛽1-42 (ng/ml) 0 10 20 30 40 50 60 70 80 90 100 IC50 = 579.00 ng/ml 5 6 4 3 2 1 0 (b) Spiked concentration (ng/ml) Detected concentration (ng/ml) –50 0 250 500 750 1000 1250 200 450 700 950 1200 R2 = 0.9973 (c) Spiked concentration (ng/ml) Detected concentration (ng/ml) 0 0 250 250 500 500 750 750 1000 1000 1250 R2 = 0.9918 (d) Figure 3: The ic-ELISA calibration curves for Aβ1-42 using a 1 : 1000 (v/v) dilution of alpaca antiserum were obtained using analyte standards prepared in 0.456 mg/ml precipitated urine. (c) The correlation curve of the detected and spiked concentrations of Aβ1-42 in 1 : 160 diluted urine. (d) The correlation curve of the detected and spiked concentrations of Aβ1-42 in 0.456 mg/ml precipitated urine. 7 Journal of Immunology Research %Binding Log concentration A𝛽1-42 (ng/ml) 0 10 20 30 40 50 60 70 80 90 100 5 6 4 3 2 1 0 Log concentration A𝛽1-42 (ng/ml) 5 4 3 2 1 0 %Binding 0 10 20 30 40 50 60 70 80 90 IC50 = 115.09 ng/ml 50 g (b) Spiked concentration (ng/ml) Detected concentration (ng/ml) 0 0 250 250 500 500 750 750 1000 1000 1250 R2 = 0.9918 (d) Detected concentration (ng/ml) Detected concentration (ng/ml) (d) (c) Figure 3: The ic-ELISA calibration curves for Aβ1-42 using a 1 : 1000 (v/v) dilution of alpaca antiserum were obtained using analyte standards prepared in 0.456 mg/ml precipitated urine. (c) The correlation curve of the detected and spiked concentrations of Aβ1-42 in 1 : 160 diluted urine. 4. Discussion (d) The correlation curve of the detected and spiked concentrations of Aβ1-42 in 0.456 mg/ml precipitated urine. these methods [33]. An sdAb is an antibody fragment con- sisting of a single monomeric variable antibody domain, unlike a whole antibody, which can selectively bind to a very speciﬁc antigen. The sdAb could be produced from both con- ventional antibodies [34] and heavy chain antibodies from camelids [35]. reactivity to other synthesized Aβ peptides, which have the same sequence as Aβ1-42 from diﬀerent segments of the sequence. We can presume that the binding site of the anti- body has the same sequence as Aβ1-8 (DAEFRHD). The assay, as described in this study, has great potential to detect Aβ1-42 protein in cases of high-risk and early stages of AD, with an easy and rapid method when applied to a urine sam- ple. Moreover, this assay can be widely used at a low cost and applied to other types of biological samples. In our study, an alpaca was immunized with Aβ1-42 to produce antibodies. For our novel ic-ELISA for Aβ, pAb was applied due to its convenience in the development of this method of examination, but sdAb for Aβ can be developed in the future. All data were shown in the manuscript. All data were shown in the manuscript. The developed ic-ELISA indicated sensitivity for Aβ1-42 and peptides having a similar structure. Aβ1-40 showed 55% cross-reactivity, and the synthesized Aβ1-8, which consists of the same beginning sequence for all Aβ, showed 69.4% cross-reactivity. In addition, the method showed no cross- References [18] S. D. Gan and K. R. Patel, “Enzyme immunoassay and enzyme-linked immunosorbent assay,” Journal of Investigative Dermatology, vol. 133, no. 9, pp. 1–3, 2013. [1] A. S. Schachter and K. L. Davis, “Alzheimer’s disease,” Dia- logues in Clinical Neuroscience, vol. 2, no. 2, pp. 91–100, 2000. [19] S. D. Schmidt, R. A. Nixon, and P. M. 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https://openalex.org/W4224046913	https://discovery.ucl.ac.uk/id/eprint/10147588/1/Hepatology%20Communications%20-%202022%20-%20Engelmann%20-%20Body%20fat%20composition%20determines%20outcomes%20before%20and%20after%20liver.pdf	English	null	Body fat composition determines outcomes before and after liver transplantation in patients with cirrhosis	Hepatology communications	2,022	cc-by	8,152	Correspondence Cornelius Engelmann, Department of Hepatology and Gastroenterology, Campus Virchow-Klinikum, Charité– Universitaetsmedizin Berlin, Berlin, Germany Email: cornelius.engelmann@charite.de O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Body fat composition determines outcomes before and after liver transplantation in patients with cirrhosis Body fat composition determines outcomes before and after liver transplantation in patients with cirrhosis Cornelius Engelmann1,2,3,4 \| Niklas F. Aehling1 \| Stefan Schob5 \| Ines Nonnenmacher1 \| Luise Handmann1 \| Jane Macnaughtan2 \| Adam Herber1 \| Alexey Surov6 \| Thorsten Kaiser7 \| Timm Denecke6 \| Rajiv Jalan2 \| Daniel Seehofer8 \| Michael Moche6,9 \| Thomas Berg1 Cornelius Engelmann1,2,3,4 \| Niklas F. Aehling1 \| Stefan Schob5 \| Ines Nonnenmacher1 \| Luise Handmann1 \| Jane Macnaughtan2 \| Adam Herber1 \| Alexey Surov6 \| Thorsten Kaiser7 \| Timm Denecke6 \| Rajiv Jalan2 \| Daniel Seehofer8 \| Michael Moche6,9 \| Thomas Berg1 1Division of Hepatology, Department of Medicine II, Leipzig University Medical Center, Leipzig, Germany 2Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, UK 3Department of Hepatology and Gastroenterology, Campus Virchow- Klinikum, Charité–Universitaetsmedizin Berlin, Berlin, Germany 4Berlin Institute of Health, Berlin, Germany 5Department for Neuroradiology, University Hospital Leipzig, Leipzig, Germany 6Department of Diagnostic and Interventional Radiology, University Hospital Leipzig, Leipzig, Germany 7Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany 8Department of Visceral, Vascular, Thoracic and Transplant Surgery, University Hospital Leipzig, Leipzig, Germany 9Diagnostic and Interventional Radiology, Park Hospital Leipzig, Leipzig, Germany Correspondence Cornelius Engelmann, Department of Hepatology and Gastroenterology, Campus Virchow-Klinikum, Charité– Universitaetsmedizin Berlin, Berlin, Germany Email: cornelius.engelmann@charite.de 1Division of Hepatology, Department of Medicine II, Leipzig University Medical Center, Leipzig, Germany Abstract Cachexia occurs in late stages of liver cirrhosis, and a low-fat mass is poten- tially associated with poor outcome. This study compared different computed tomography (CT)–derived fat parameters with respect to its prognostic im- pact on the development of complications and death before and after liver transplantation. Between 2001 and 2014, 612 patients with liver cirrhosis without hepatocellular carcinoma listed for liver transplantation met the in- clusion criteria, including abdominal CT scan (±200 days to listing). A total of 109 patients without cirrhosis served as controls. The subcutaneous fat index (SCFI), the paraspinal muscle fat index, and the visceral fat index were assessed at L3/L4 level and normalized to the height (cm2/m2). Data were col- lected and analyzed retrospectively. Low SCFI was associated with a higher rate of ascites and increased C-reactive protein levels (p < 0.001). In addition, multivariate Cox regression analysis adjusting for sex, age, body mass index (BMI), and Model for End-Stage Liver Disease showed that decreasing SCFI was also associated with an increased risk of cirrhosis-related complications (p = 0.003) and death on the transplant wait list (p = 0.013). Increased par- aspinal and visceral fat were not only positively correlated with creatinine levels (p < 0.001), BMI, and metabolic comorbidities (all p < 0.001) before transplantation, but also predictive for 1-year mortality after transplantation. Conclusion: The distribution of body fat is a major determinant for complica- tions and outcome in cirrhosis before and after liver transplantation. 2Liver Failure Group, Institute for Liver and Digestive Health, University College London, Royal Free Campus, London, UK 3Department of Hepatology and Gastroenterology, Campus Virchow- Klinikum, Charité–Universitaetsmedizin Berlin, Berlin, Germany \| 1 wileyonlinelibrary.com/journal/hep4 Received: 8 October 2021 \| Accepted: 17 February 2022 DOI: 10.1002/hep4.1946 Received: 8 October 2021 \| Accepted: 17 February 2022 DOI: 10.1002/hep4.1946 Hepatology Communications. 2022;00:1–12. INTRODUCTION Europe.[1–3] The treatment of underlying liver diseases effectively prevents the development of complications in a proportion of patients, but some progress to later disease stages in which liver transplantation is the only Liver cirrhosis is a major chronic disease that ranges among the 10 most important causes of death in Hepatology Communications. 2022;00:1–12. \| 1 wileyonlinelibrary.com/journal/hep4 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases Cornelius Engelmann and Niklas F. Aehling contributed equally to this work. Hepatology Communications. 2022;00:1–12. This is an open access article under the terms of the Creative Commons Attribu any medium, provided the original work is properly cited, the use is non-comme © 2022 The Authors. Hepatology Communications published by Wiley Periodica Cornelius Engelmann and Niklas F. Aehling contributed equally to this work. BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT 2 2 therapeutic option.[4] To reduce the number of deaths and to tailor individual management strategies, it is es- sential to identify patients at high risk for fatal outcomes and disease-related complications among a cohort with very heterogeneous clinical phenotype, ranging from well-compensated mild cirrhosis to severely de- compensated patients with additional organ failures, so-called acute-on-chronic liver failure (ACLF). transplantation,[17] and making high demands on body fat composition as a prognostic marker in this cohort. Therefore, we performed a retrospective study as- sessing the nutritional status of patients listed for liver transplantation by measuring three CT-derived fat pa- rameters to evaluate its impact on clinical outcomes before and after liver transplantation. Herein, we could show that a decreased subcutaneous fat is associated with the development of disease-related complications and death in patients with liver cirrhosis before trans- plantation, whereas an increased muscle fat mass and visceral fat mass enhances the likelihood of death after transplantation. ( ) The indication and urgency for liver transplantation depends on multiple aspects, of which the severity of liver disease is one major criterion. Study design and population Between March 2001 and September 2014, a total of 1326 patients were evaluated for liver transplantation in the University Hospital Leipzig. Of those, 612 pa- tients with cirrhosis and without hepatocellular car- cinoma who received an abdominal CT scan at the time of transplant assessment ± 200 days were in- cluded. Patients younger than 18 years, with previ- ous liver transplantation and those with insufficient data were excluded (Figure S1). A total of 109 pa- tients without liver diseases with CT scan as part of their diagnostic work-up after polytrauma served as a control cohort. Clinical data including age, gender, causes of cirrhosis, history of cirrhosis–associated complications and co-morbidities at baseline, as well as the development of cirrhosis-associated complica- tions, biochemistry data and patients’ survival status during follow-up, were retrieved retrospectively from patients’ records. Chronic liver disease and cirrhosis are associated with alterations in body composition and cachexia. Muscle wasting is a well-known phenomenon associ- ated with late stages of liver disease,[8] but the body fat distribution might also be a marker for disease sever- ity and outcome in patients with liver cirrhosis. Indirect impedance-based techniques revealed that wasting and loss of body fat is a typical feature of liver cirrho- sis.[9,10] Computed tomography (CT) or magnetic reso- nance imaging–based techniques have been allocating a high risk of liver-related complications to those with low muscle fat or subcutaneous fat mass, especially in female patients.[11,12] However, the link between body fat composition and occurrence of individual complica- tions tends to be more controversial, as individual fat compartments might impact differently on the develop- ment of disease-related complications.[11,13,14] INTRODUCTION In late stages of cirrhosis, liver dysfunction develops with high Model for End-Stage Liver Disease (MELD) score and other disease-related complications such as hepatorenal syndrome (HRS), ascites or hepatic encephalopathy (HE), indicating an elevated likelihood of death.[5,6] However, the current MELD-based allocation system does not reflect the true survival probability in some subgroups such as patients with refractory ascites or ACLF, in whom the MELD score is less predictive.[7] The second aspect that needs to be considered during assessment for liver transplantation strongly relates to the likelihood of success after transplantation, which is majorly defined by the risk of posttransplant complica- tion.[4] However, its prediction remains inaccurate also because its genesis is less well defined. Therefore, new prognostic markers might help to prioritize patients on the wait list and to avoid fatal complications before and after liver transplantation. Patients’ baseline characteristics The mean age of patients with liver cirrhosis was 52 years, which was significantly younger than the con- trol group (61.7 years; p < 0.001). Of 612 patients with liver cirrhosis, 63.6% had alcohol-associated liver dis- ease and 66.7% were male (Tables 1 and 2). There was no significant difference in terms of gender or BMI between patients with cirrhosis and the control group. Patients with cirrhosis were primarily Child Pugh B (51.2%), the mean MELD score was 17 ± 7.1. Further baseline clinical and biochemical data are provided in Table 2. Out of 612 patients, 264 patients received liver transplantation. In total, 67% of patients suffered from ascites, 80.8% from gastric and/or esophageal varices, and 27.7% had a history of variceal bleeding. Transjugular intrahepatic portosystemic shunt (TIPS) was implanted in 6.7%. At least one episode of HE occurred in 83.2% in their past medical history; 18.6% were at least once treated for spontaneous bacterial Assessment of body fat content by CT The body fat content was determined using CT scans. Two trained observers (interrater agreement: Cohen’s Kappa coefficient of 0.89; p < 0.001) analyzed the CT images. SliceOmatic V 5.0 software (Tomovision, Montréal, Canada) was used to analyze an axial section of the abdomen at the level of the spinal segment L3/ L4. SliceOmatic uses previously reported Hounsfield unit (HU) thresholds to quantify different tissue com- partments in cross-sectional images. The subcutane- ous fat index (SCFI), paraspinal intramuscular fat index (PSFI), and visceral fat index (VFI) was identified and quantified using HU thresholds of −150 to −30. The SliceOmatic V4.3 software automatically calculated the cross-sectional areas (cm²). Normalization to body height was performed by dividing the fat area by body height in square meters. Alterations in body fat composition might also have a persisting impact on patients’ disease course even beyond the time point of liver transplantation. Metabolic complications become more prevalent during mainte- nance after transplantation,[15] and, indeed, it has been shown that obesity and a metabolic syndrome is a neg- ative predictor for complications and outcome after liver transplantation.[16] These observations emphasize the complexity of mechanisms linking body fat composition with dis- ease severity in liver cirrhosis and success after liver HEPATOLOGY COMMUNICATIONS \| 3 3 TA B L E 1 Demographics for patients with cirrhosis (derivation cohort) on the wait list for transplantation and for patients after polytraumatic accidents (group comparison with Mann-Whitney U test) Statistical analysis Statistical analysis was performed using SPSS 22 soft- ware (SPSS Inc., Chicago, IL). Categorical variables were displayed as frequency (%), continuous variables as mean ± SD, or median (range), as appropriate. A two- sided p-value of p ≤ 0.05 implicated statistical signifi- cance. Group comparisons for categorical variables were performed using the χ²-test and for metric variables using the Mann-Whitney U test. For more than two groups, data were analyzed by one-way analysis of variance followed by a Dunnett’s post hoc analysis for unequal variances. About one third of patients was transplanted within 1 year after listing. We used Cox regression analysis adjusting for age, body mass index [BMI], and MELD score to as- sess factors modifying the cause-specific hazard func- tions, censoring time-to-event endpoints at the time of transplant, including transplantation itself. Parameter Cirrhosis n = 612 Polytrauma n = 109 Level of significance Age (years) 52 ± 9.1 61.7 ±18 p < 0.001 Gender ♂ 408 (66.7%) ♂ 75 (68.8%) p = 0.661 ♀ 204 (33.3%) ♀ 34 (31.2%) Body height (cm) 171.7 ± 9.1 171.8 ± 9.1 p = 0.998 Body weight (kg) 78 ± 16.8 79.2 ± 15.4 p = 0.535 BMI (kg/m2) 26.3 ± 4.8 26.9 ± 4.5 p = 0.226 peritonitis (SBP); and 20% had a history of bacterial infections. To define patients with high-fat and low-fat mass indices, the cohort was divided based on tertiles. As calculated by Cox regression modeling based on the metric variable, a decreased SCFI was associated with an increased risk for complications and death before transplantation; therefore the lower tertile was chosen as the cutoff. VFI and PSFI showed higher values as- sociated with worsened outcome after liver transplan- tation. Therefore, the upper tertile was chosen as the cutoff. Taking into account that there are gender-related fat mass differences, we defined cutoffs individually for men and women as follows: PSFI male, 4.04 cm²/m²; PSFI female, 4.93 cm²/m²; SCFI male, 38.44 cm²/m²; SCFI female, 39.58 cm²/m²; VFI male, 58.61 cm²/m²; and VFI female, 49.97 cm²/m². RESULTS Patients’ baseline characteristics Fat indices and severity of liver disease PSFI, SCFI, and VFI were significantly lower in pa- tients with cirrhosis compared to controls (PSFI: 3.9 ± 2.3 cm²/m² vs. 5.3 ± 3.5 cm²/m² [p = 0.002]; SCFI: 56.7 ± 32.2 cm²/m² vs. 69.2 ± 35.4 cm²/m² [p = 0.002]; and VFI: 47.6 ± 24 cm²/m² vs. 61.3 ± 33.8 cm²/m² [p < 0.001]) (Figure 1; Table S1). Patients with liver cirrhosis who suffered from refractory ascites or who had a his- tory of SBP showed lower SCFI values (refractory as- cites: 45 ± 29.3 cm²/m² vs. no ascites 67.2 ± 32.3 cm²/ m² [p < 0.001]; SBP: 49.3 ± 30.7 cm²/m² vs. no SBP 58.7 ± 31.2 cm²/m² [p = 0.008]), whereas the PSFI and VFI were no different. In contrast, patients with Child-Pugh score C had a higher PSFI than patients with Child Pugh Score A (3.3 ± 1.8 cm²/m² vs. 4.3 ± 2.6 cm²/m² [p = 0.028]). There was neither a significant difference between Child Pugh A and B nor between Child Pugh B and C. Neither a history of HE nor TIPS was associated with alterations of fat indi- ces. However, there were slight differences of fat indices between genders (Figures S2–S4, Table S1A–C). Fat indices and development of complications or death before liver transplantation TA B L E 2 (Continued) 4 TA B L E 2 Baseline characteristics of patients with liver cirrhosis in this study Etiology of liver disease (n = 612) • ALD 389 (63.6%) • Viral (hepatitis B and C) 50 (8.2%) • NASH 30 (4.9%) • Others 143 (23.4%) MELD score (n = 595) 17 ± 7.1 • 6–11 points 136 (22.9.0%) • 12–24 points 375 (63%) • 25–40 points 84 (14.1%) Child-Pugh classification (n = 410) • Class A 52 (12.7%) • Class B 210 (51.2%) • Class C 148 (36.1%) Bilirubin (µmol/l) 48.9 (4.6–887.8) Creatinine (µmol/l) 82 (32–868) GFR (ml/min) (MDRD-Formel) 82.4 (6.4–251.6) INR 1.45 (0.9–5.1) Albumin (g/l) 32.9 (12.6–52.8) WBC (exp9/l) 6.1 (1.2–45.2) CRP (mg/dl) 9.7 (0.2–218.5) Platelet count (exp9/l) 102.5 (12–1180) Ascites (n = 555) • No ascites 183 (33%) • Mild to moderate ascites 144 (25.9%) • Massive 228 (41.1%) TIPS at evaluation Yes/No 41 (6.7%)/571 (93.3%) History of bacterial Infections (n = 643) Yes/No 99 (20%)/395 (80%) History of SBP (n = 643) Yes/No 92 (18.6%)/403 (81.4%) History of HE (n = 333) Yes/No 252 (83.2%)/51 (16.8%) History of hepatorenal syndrome (n = 650) Yes/No 109 (17.8%)/393 (78.3%) Varices (n = 724) Yes/No 449 (80.8%)/107 (19.2%) History of variceal bleeding (n = 664) Yes/No 142 (27.7%)/371 (72.3%) Diabetes mellitus (n = 556) Yes/No 187 (33.6%)/369 (66.4%) Arterial hypertension (n = 536) Yes/No 219 (40.9%)/317 (59.1%) Obesity (n = 524) Yes/No 107 (20.4%)/417 (79.6%) TA B L E 2 Baseline characteristics of patients with liver cirrhosis in this study S2; Figure 2). This result was also confirmed after ex- clusion of patients with nonalcoholic steatohepatitis (NASH) cirrhosis (n = 30) or patients with TIPS (n = 41) (Table S2). After including the paraspinal muscle index (as published by Engelmann et al.[8]) into the multivari- ate regression model, only the paraspinal muscle index remained as an independent predictor of 1-year mortal- ity, whereas the SCFI cost its significance (Figure S5). Furthermore, the association between fat indices and occurrence of cirrhosis-associated complica- tions within 1 year after listing for liver transplantation was calculated by univariate Cox regression analysis. Fat indices and development of complications or death before liver transplantation Univariate Cox regression analysis showed a de- creased risk of death within 1 year after listing with a higher SCFI (hazard ratio [HR] = 0.992 [0.985–1.000], p = 0.043), whereas an increased PSFI was associ- ated with higher risk (HR = 1.081 [1.001–1.167], p = 0.046) (Table S2). Multivariate Cox regression analysis adjusted to age, BMI, MELD score, white blood count, and gender revealed that the SCFI remained as an in- dependent predictor of death before liver transplanta- tion with a HR of 0.984 (0.971–0.997; p = 0.013) (Table BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT 4 BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT Etiology of liver disease (n = 612) Coronary heart disease (n = 537) Yes/No 31 (5.8%)/506 (94.2%) Note: Values are displayed as median (range) or number (%), respectively. The numbers in brackets represent the number of patients providing information. Abbreviations: ALD, alcohol-associated liver disease; CRP, C-reactive protein; GFR, glomerular filtration rate; HE, hepatic encephalopathy; INR, international normalized ratio; NASH, nonalcoholic steatohepatitis. Fat indices and development of complications or death before liver transplantation Accordingly, higher SCFI values were associated with a reduced risk of developing any complication (HR = 0.993 [0.989–0.998], p = 0.005), bacterial infection (HR = 0.989 [0.983–0.995], p < 0.001), SBP (HR = 0.988 [0.979–0.998], p = 0.015), and variceal bleedings (HR = 0.983 [0.970–0.995], p = 0.007) but not with the de- velopment of HRS or HE episodes. The PSFI showed an opposite risk correlation with complications being higher with increased PSFI values for any complication (HR = 1.080 [1.024–1.140], p = 0.005) and HE (HR = 1.117 [1.043–1.197], p = 0.002). After multivariate analy- sis adjusted to age, BMI, MELD score and gender, only the SCFI remained as independent predictor for any complication (HR = 0.989 [0.981–0.996], p = 0.003), bacterial infection (0.984 [0.974–0.994], p = 0.001), and SBP (0.970 [0.955–0.984], p < 0.001) (Table S3; Figure 2). To define patients with low-fat and high-fat mass in- dices, the cohort was divided based on index’s tertiles. A decreasing SCFI was associated with an increasing risk of complication and death before liver transplanta- tion. Therefore, the lower SCFI tertile was chosen as the cutoff (male: 38.44 cm²/m²; female: 39.58 cm²/m²). Patients with a low SCFI had an increased risk of death on the wait list (HR = 1.490 [0.966, 2.300], p = 0.072) and an increased risk of developing complications be- fore liver transplantation (HR = 1.403 [1.057, 1.862], p = 0.019) (Figure 3). They suffered more often from ascites (80.4% vs. 62% [p < 0.001]) and had higher C-reactive protein (CRP) levels (14 vs. 7.2 mg/dl [p < 0.001]). The subgroup of patients with a high SCFI suffered more often from diabetes mellitus (35.3% vs. 22%; p < 0.001) and had a higher BMI (26.7 vs. 22.4 kg/m²; Table 3). \| 5 5 HEPATOLOGY COMMUNICATIONS I G U R E 1 According to Table S1C, different computed tomography (CT)–derived fat parameters were obtained at baseline. Groups ere compared by Mann-Whitney U test or one-way analysis of variance and post hoc Dunnett’s test (unequal variances), depending on e number of groups according to subcutaneous fat index (SCFI) (A), paraspinal muscle fat index (PSFI) (B), and visceral fat index (VFI) C). BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT 6 6 F I G U R E 2 Multivariate Cox regression analysis of complications and 1-year-mortality before liver transplantation. (A–C) After multivariate Cox regression analysis, adjusting fat parameters to age, body mass index (BMI), MELD and gender, the SCFI remained as an independent predictor for complications, particularly bacterial infections and SBP. All other parameters were not associated with the development of cirrhosis-related complications before liver transplantation. (D) After multivariate Cox regression analysis, adjusting fat parameters to age, BMI, MELD, white blood count (WBC) and gender, the SCFI remained as independent predictor for death within 1 year before liver transplantation (Table S2). HR, hazard ratio F I G U R E 2 Multivariate Cox regression analysis of complications and 1-year-mortality before liver transplantation. (A–C) After multivariate Cox regression analysis, adjusting fat parameters to age, body mass index (BMI), MELD and gender, the SCFI remained as an independent predictor for complications, particularly bacterial infections and SBP. All other parameters were not associated with the development of cirrhosis-related complications before liver transplantation. (D) After multivariate Cox regression analysis, adjusting fat parameters to age, BMI, MELD, white blood count (WBC) and gender, the SCFI remained as independent predictor for death within 1 year before liver transplantation (Table S2). HR, hazard ratio F I G U R E 3 One-year mortality and development of complications before liver transplantation of patients with a high or low SCFI. Patients were divided into a cohort of low-fat and high-fat mass according to the gender-dependent, individual lower tertile (SCFI male: 38.44; SCFI female: 39.58). A lower SCFI was associated with a higher risk for death (A) and particularly complication (B) before live transplantation F I G U R E 3 One-year mortality and development of complications before liver transplantation of patients with a high or low SCFI. Patients were divided into a cohort of low-fat and high-fat mass according to the gender-dependent, individual lower tertile (SCFI male: 38.44; SCFI female: 39.58). A lower SCFI was associated with a higher risk for death (A) and particularly complication (B) before live transplantation Fat indices and development of complications or death before liver transplantation CPT, Child-Pugh Turcotte; HE, hepatic encephalopathy; HRS, hepatorenal syndrome; MELD: Model for End-Stage Liver Disease; S pontaneous bacterial peritonitis; TIPS, transjugular intrahepatic portosystemic shunt F I G U R E 1 According to Table S1C, different computed tomography (CT)–derived fat parameters were obtained at baseline. Groups were compared by Mann-Whitney U test or one-way analysis of variance and post hoc Dunnett’s test (unequal variances), depending on the number of groups according to subcutaneous fat index (SCFI) (A), paraspinal muscle fat index (PSFI) (B), and visceral fat index (VFI) (C). CPT, Child-Pugh Turcotte; HE, hepatic encephalopathy; HRS, hepatorenal syndrome; MELD: Model for End-Stage Liver Disease; SBP, spontaneous bacterial peritonitis; TIPS, transjugular intrahepatic portosystemic shunt Fat indices and the risk of death after liver transplantation [1.001–1.020], p = 0.031). Multivariate Cox regres- sion adjusting to age, BMI, MELD score, and gender also showed that a higher PSFI or VFI was associated with an increased risk of death after liver transplanta- tion (PSFI HR = 1.188 [1.032–1.368], p = 0.017; VFI HR = 1.014 [1.001–1.027], p = 0.037) (Figure 4). Fat indices and the risk of death after liver transplantation After Univariate Cox regression model showed a higher risk of death within 1 year with an increased PSFI (HR = 1.141 [1.029–1.264], p = 0.012) and VFI (HR = 1.011 \| 7 HEPATOLOGY COMMUNICATIONS TA B L E 3 Patients were divided into a cohort of low-fat and high-fat mass according to the gender-dependent, individual high-risk tertile (PSFI male: 4.04 cm²/m²; PSFI female: 4.93 cm²/ m²; SCFI male: 38.44 cm²/m²; SCFI female: 39.58 cm²/m²; VFI male: 58.61 cm²/m²; VFI female: 49.97 cm²/m²) Baseline SCFI PSFI VFI Subgroup Low High p Low High p Low High p MELD 15 (6–40) 15 (6–40) 0.232 15 (6–40) 17 (5–39) 0.101 15 (6–40) 16.5 (7–40) 0.128 Ascites 119 (80.4%) 184 (62%) <0.001 253 (68.9%) 133 (66.5%) 0.552 254 (68.3%) 129 (67.2%) 0.792 HE 33 (24.1%) 84 (29.9%) 0.215 100 (29.3%) 64 (35%) 0.184 96 (28.3%) 66 (36.3%) 0.062 Varices 108 (75.5%) 242 (82.6%) 0.082 293 (80.5%) 154 (81.1%) 0.875 288 (78.9%) 156 (93.9%) 0.163 Bilirubin (µmol/l) 44.2 (4.6–649.5) 45.3 (5.5–887.8) 0.146 48 (4.6–887.8) 52.8 (4.6–688.8) 0.759 50.7 (4.6–887.8) 46.8 (5.5–688.8) 0.665 Creatinine (µmol/l) 84.5 (34–509) 79 (32–868) 0.563 76 (32–868) 96 (45–577) <0.001 76 (32–868) 93.5 (41–739) <0.001 INR 1.4 (0.9–3.86) 1.4 (0.93–5.09) 0.227 1.4 (0.93–5.09) 1.38 (0.9–3.5) 0.615 1.4 (0.9–5.09) 1.36 (0.93–3.5) 0.554 WBC (×109/l) 6.1 (1.2–20.3) 5.8 (1.3–36) 0.330 5.8 (1.2–36) 6.4 (1.3–25.5) 0.023 5.8 (1.2–20.8) 6.5 (1.3–36) 0.007 CRP (mg/l) 14 (0.72–218.5) 7.2 (0.15–144-6) <0.001 9.2 (0.15–218.5) 9.6 (0.47–127.8) 0.096 9.24 (0.15–218.5) 8.4 (0.47–127.8) 0.122 Diabetes mellitus 31 (22%) 103 (35.3%) 0.005 101 (28.1%) 84 (43.3%) <0.001 97 (27%) 89 (46.6%) <0.001 AHT 47 (34.1%) 112 (39.3%) 0.297 122 (34.9%) 96 (51.9%) <0.001 118 (34%) 100 (54.3%) <0.001 CKD 41 (28.5%) 100 (33.4%) 0.293 105 (28.4%) 81 (41.5%) 0.002 104 (28.3%) 81 (42%) 0.001 BMI (kg/m²) 22.4 (15.4–33.8) 26.7 (17.4–49) <0.001 24.4 (15.4–37) 27.7 (20.9–49) <0.001 24.4 (15.4–41.7) 27.8 (21.2–49) <0.001 Age (years) 50 (21–71) 54 (27–71) 0.001 50 (21–70) 57 (27–71) <0.001 51 (21–71) 54.5 (27–71) <0.001 Note: Groups were compared by Mann-Whitney U test or one-way analysis of variance and post hoc Dunnett’s test (unequal variances), depending on the number of groups. p-Values are displayed if significant in comparison to the control group. BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT 8 8 F I G U R E 4 Mortality after liver transplantation according to different CT-derived fat parameters. After multivariate Cox regression analysis, adjusting fat parameters to age, BMI, MELD and gender, the VFI and PSFI could be identified as an independent predictor for the occurrence of death within 1 year after liver transplantation (Table S2) F I G U R E 4 Mortality after liver transplantation according to different CT-derived fat parameters. After multivariate Cox regression analysis, adjusting fat parameters to age, BMI, MELD and gender, the VFI and PSFI could be identified as an independent predictor for the occurrence of death within 1 year after liver transplantation (Table S2) F I G U R E 5 One-year mortality of patients with a high or low PSFI and VFI. Patients were divided into a cohort of low-fat and high-fat mass according to the gender-dependent, individual upper tertile (PSFI male: 4.04; PSFI female: 4.93; VFI male: 58.61; FRI female: 49.97). The PSFI (A) and VFI (B) inversely correlated with the posttransplant 1-year-survival F I G U R E 5 One-year mortality of patients with a high or low PSFI and VFI. Patients were divided into a cohort of low-fat and high-fat mass according to the gender-dependent, individual upper tertile (PSFI male: 4.04; PSFI female: 4.93; VFI male: 58.61; FRI female: 49.97). The PSFI (A) and VFI (B) inversely correlated with the posttransplant 1-year-survival exclusion of patients with NASH (n = 30) or patients with TIPS (n = 41), the PSFI remained as independ- ent prognosticator, whereas the VFI was lacking signifi- cance (Table S2). (46.6% vs. 27%, p < 0.001), arterial hypertension (54.3 vs. 34%, p < 0.001), and chronic kidney disease (42% vs. 28.3%, p = 0.001). They also had a higher BMI (27.8 kg/m² vs. 24.4 kg/m², p < 0.001), and they were also older (54.5 years vs. 51 years, p < 0.001) (Table 3). Due to the positive correlation between risk of com- plications and PSFI and VFI values, the upper tertile was chosen as the cutoff to define high and low fatty indices (PSFI male: 4.04 cm²/m²; PSFI female: 4.93 cm²/m²; VFI male: 58.61 cm²/m²; and VFI female: 49.97 cm²/m²). BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT Patients with high PSFI (HR = 1.743 [1.045, 2.905], p = 0.033) and high VFI (1.834 [1.105, 3.043], p = 0.019) were more likely to die after liver transplan- tation (Figure 5). Patients with a high PSFI suffered more often from diabetes mellitus (43.3% vs. 28.1%, p < 0.001), arterial hypertension (51.9% vs. 34.9%, p < 0.001), and chronic kidney disease (41.5% vs. 28.4%, p = 0.002). In addition, they had a higher BMI (27.7 kg/ m² vs. 24.4 kg/m², p < 0.001), and they were older (57 years vs. 50 years, p < 0.001). Likewise, patients with a high VFI suffered more often from diabetes mellitus A difference in severity of liver disease at transplan- tation between patients with high and low fat at evalua- tion might explain the effect on posttransplant outcome. Therefore, patient characteristics were calculated at the time point of liver transplantation and compared be- tween groups. Patients with high PSFI had increased creatinine levels, whereas all other organ function pa- rameters were not different between cohorts with high- fat and low-fat indices (Table S4). Fat indices and the risk of death after liver transplantation L E 3 Patients were divided into a cohort of low-fat and high-fat mass according to the gender-dependent, individual high-risk tertile (PSFI male: 4.04 cm²/m²; PSFI female: 4.93 cm²/ CFI male: 38.44 cm²/m²; SCFI female: 39.58 cm²/m²; VFI male: 58.61 cm²/m²; VFI female: 49.97 cm²/m²) line SCFI PSFI VFI roup Low High p Low High p Low High p D 15 (6–40) 15 (6–40) 0.232 15 (6–40) 17 (5–39) 0.101 15 (6–40) 16.5 (7–40) 0.128 es 119 (80.4%) 184 (62%) <0.001 253 (68.9%) 133 (66.5%) 0.552 254 (68.3%) 129 (67.2%) 0.792 33 (24.1%) 84 (29.9%) 0.215 100 (29.3%) 64 (35%) 0.184 96 (28.3%) 66 (36.3%) 0.062 es 108 (75.5%) 242 (82.6%) 0.082 293 (80.5%) 154 (81.1%) 0.875 288 (78.9%) 156 (93.9%) 0.163 bin (µmol/l) 44.2 (4.6–649.5) 45.3 (5.5–887.8) 0.146 48 (4.6–887.8) 52.8 (4.6–688.8) 0.759 50.7 (4.6–887.8) 46.8 (5.5–688.8) 0.665 inine (µmol/l) 84.5 (34–509) 79 (32–868) 0.563 76 (32–868) 96 (45–577) <0.001 76 (32–868) 93.5 (41–739) <0.001 1.4 (0.9–3.86) 1.4 (0.93–5.09) 0.227 1.4 (0.93–5.09) 1.38 (0.9–3.5) 0.615 1.4 (0.9–5.09) 1.36 (0.93–3.5) 0.554 (×109/l) 6.1 (1.2–20.3) 5.8 (1.3–36) 0.330 5.8 (1.2–36) 6.4 (1.3–25.5) 0.023 5.8 (1.2–20.8) 6.5 (1.3–36) 0.007 (mg/l) 14 (0.72–218.5) 7.2 (0.15–144-6) <0.001 9.2 (0.15–218.5) 9.6 (0.47–127.8) 0.096 9.24 (0.15–218.5) 8.4 (0.47–127.8) 0.122 etes mellitus 31 (22%) 103 (35.3%) 0.005 101 (28.1%) 84 (43.3%) <0.001 97 (27%) 89 (46.6%) <0.001 47 (34.1%) 112 (39.3%) 0.297 122 (34.9%) 96 (51.9%) <0.001 118 (34%) 100 (54.3%) <0.001 41 (28.5%) 100 (33.4%) 0.293 105 (28.4%) 81 (41.5%) 0.002 104 (28.3%) 81 (42%) 0.001 kg/m²) 22.4 (15.4–33.8) 26.7 (17.4–49) <0.001 24.4 (15.4–37) 27.7 (20.9–49) <0.001 24.4 (15.4–41.7) 27.8 (21.2–49) <0.001 years) 50 (21–71) 54 (27–71) 0.001 50 (21–70) 57 (27–71) <0.001 51 (21–71) 54.5 (27–71) <0.001 Groups were compared by Mann-Whitney U test or one-way analysis of variance and post hoc Dunnett’s test (unequal variances), depending on the number of groups. p-Values are displayed if significant in rison to the control group DISCUSSION Liver cirrhosis accelerates wasting, as exaggerated energy consumption requires amino acids and lipids \| 9 HEPATOLOGY COMMUNICATIONS 9 established prognostic markers for cirrhosis, to in- crease their prognostic accuracy. Ebadi et al. intro- duced the MELD-SATI, a combination of MELD and the SCFI, and it showed an increased c-index compared with MELD only (0.93 [0.87–0.99] vs. 0.85 [0.75–0.96]). However, data used to calculate the MELD-SATI were collected only in female patients,[11] questioning the gen- eralizability of these data especially as the gender did not play a role in our cohort. Previous attempts to add nutritional parameter to prognostic score, especially for sarcopenia, were of limited success.[24] Therefore, it might be necessary to validate such scores in a large- scale, multicenter setting. as additional energy sources,[18] and wasting is associ- ated with poor outcome.[8] The CT scan is a widespread and reliable technique to determine body composition. CT-derived fat mass parameters serve to identify sub- groups of patients with cirrhosis with high risks of com- plications before and after liver transplantation. This is a large retrospective study in patients with end-stage liver disease using three different CT-derived fat param- eters to explore their impact on developing cirrhosis- associated complications and on mortality before and after liver transplantation. Although patients with cirrhosis had lower fatty indi- ces compared with liver-healthy control patients, there was a great heterogeneity among individual fat com- partments to predict patients’ outcome, and only the SCFI but not the other parameters was associated with the development of complications such as bacterial in- fections, ascites, SBP, or death before transplantation. The lower the subcutaneous fat mass, the higher the risk of complications. Data emphasize that wasting in end-stage liver disease goes beyond sarcopenia as an indicator of disease severity,[8] and suggest that adipose tissue composition has the potential to serve as an al- ternative or additional marker for outcome in cirrhosis. To date there are several relevant studies evaluating the prognostic role of adipose tissue in liver disease. Ebadi et al.[11] showed in a large retrospective cohort of 677 patients that a decreased SCFI was associated with a higher mortality in female patients with cirrhosis awaiting liver transplantation. However, the impact of body fat composition after transplantation was not ex- plored. The second study presented by Tapper et al.[19] investigated the influence of fat and muscle density on mortality in a prospective cohort (n = 274). DISCUSSION This study showed that a decreased density of fatty tissue was associated with increased mortality and risk for decom- pensation. However, this analysis was performed in a univariate manner only, which in combination with the low sample size certainly questions the robustness of these results. The third study enrolled 104 patients with cirrhosis due to alcohol-associated liver disease and showed a shorter survival in patients with increased subcutaneous fat mass.[20] Finally, a fourth study eval- uated sarcopenia in 109 patients with CT who under- went hepatic vein pressure gradient measurement, and revealed sarcopenia as an independent risk factor for increased mortality.[21] Recently two other studies eval- uated fat mass and myosteatosis at L3 in especially the perioperative setting, implying that at the timepoint of transplantation, body composition acts as a prognos- tic factor.[22,23] Herein, the authors describe a poorer outcome and increased hospital and intensive-care- unit stay with a higher myosteatosis at the time point of transplantation. However, there was no differentiation of compartments. In light of the results presented here, it might be It would be interesting to understand whether a re- duced fat mass actively contributes to a progression of liver cirrhosis, thereby explaining its prognostic rele- vance. To answer this, it is important to note that CRP values as a marker of inflammatory response were increased in patients with low SCFI. Lipids are strong signals for intercellular communication, and a loss of fat mass in cirrhosis is associated with increased lev- els and impaired composition of circulating lipids.[18,25] In cirrhosis, lipids are inflammatory cues enhancing inflammation through alterations of toll-like receptor signaling.[26] Therefore, a loss of adipose tissue might enhance inflammation and organ injury in decompen- sated liver cirrhosis. Interestingly, the interplay between body fat compo- sition and patients’ outcome appears to be complex. A low SCFI increased the risk of complications, whereas patients suffering from HRS and varices were char- acterized by higher PSFI and VFI values. Although it remains speculative, previous observations in obese patients might provide one explanatory approach. ACKNOWLEDGMENT Indeed, several observations highlighted especially visceral adipose tissue being associated with the de- velopment of NASH, arteriosclerosis, and increasing insulin resistance.[37–39] However, there was no differ- ence between patients with low and high PSFI and SCFI concerning their cause of death (Table S5). The rather large number of unknown causes of death might influence the lack of significance. We thank Dr. Koji Moriishi and Dr. Atsuya Yamashita for their scientific advice and Ms. Takako Ohmori and Ms. Tomoko Nakajima for their technical assistance. Open Access funding enabled and organized by Projekt DEAL. DISCUSSION The presence of the metabolic syndrome was associated with an elevated sympathetic nerve activity in both muscles as in kidneys,[27] leading to a “sympathetic overdrive.”[28] This overdrive initiated an activation of the so-called hepatorenal reflex, leading to decreased renal blood flow, sodium retention, and increased por- tal vein pressure.[29] Therefore, it might be possible that an increase in visceral and paraspinal adipose tissue could enhance renal sympathetic activity. In addition, there were gender differences of fat composition, which prompted us to include gender as a co-founder in the multivariate regression model. Results did not provide evidence of a gender-related effect of fat composition on patient’s outcome. It is intriguing that while cachexia generally predicts outcome before transplantation, there is an opposite relationship between fat parameters and death after liver transplantation. Patients with high visceral and paraspinal muscle fat mass were more likely to die after transplantation. Therefore, a HR of 1.188 for PSFI translates into a risk increase to die after transplanta- tion of about 19% with every point of PSFI elevation. In addition to patient’s age, this was associated with In light of the results presented here, it might be tempting to add CT-derived fat parameters to already 10 BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT 10 the presence of diabetes mellitus, arterial hyperten- sion and chronic renal insufficiency, implying that the presence of other diseases in general and the meta- bolic syndrome in particular significantly contribute to this observation. This is well in line with studies eval- uating the outcome of obese patients who underwent liver transplantation. These patients were more likely to develop wound complications, postoperative in- fections, metabolic syndrome–related complications, and death.[16,30,31] Further studies also describe an in- creased risk of death after transplantation for patients with an increased visceral-subcutaneous fat tissue ratio,[32] an increased visceral fat area,[33] and with myosteatosis.[22,23,34] However, there are also contrary data showing that obesity does not affect survival after liver transplantation,[35,36] suggesting that the distinc- tion between different fat compartments might be the clue to reliably predict patients’ outcome in different disease scenarios. between muscle and fat composition in the context of liver cirrhosis, and further studies are needed to evalu- ate the interplay between fatty and muscle tissue. CONFLICT OF INTEREST R.J. owns stock in, is an employee of, consults, ad- vises, received grants, and holds intellectual property rights with Yaqrit Ltd. He consults, advises, and received grants from Grifols. C.E. own stock in and holds intellec- tual property rights with Hepyx. D.S. advises Sirtex and is on the speaker’s bureau of Astelas, Bayer, Eisai, and MSD/Merck. He advises and is on the speaker’s bureau of BTG, Johnson & Johnson, Novartis, and Olympus. There are several limitations to that study. The ret- rospective design might question the quality of data retrieval, especially if there are a considerable number of censored data included in the analysis. It might be arguable that there is a significant difference in age be- tween patients with cirrhosis and controls included in this study. Assuming that older muscle mass decreases and fatty tissue increases with age, this might interfere with the difference in fatty indices in this study. However, it appears unlikely, as multivariate analysis did not re- veal age or gender as a significant factor. Furthermore, it might be argued that patients with NASH-related cir- rhosis or patients with TIPS might have a significant influence on the prognostic impact of fat parameters in patients with cirrhosis. However, these subgroups represented only a minority in our cohort (NASH 4.9%, TIPS 6.7%), and the multivariate analysis in a cohort without patients with NASH confirmed the results from the overall cohort regarding mortality before and after liver transplantation, although the VFI was lacking sig- nificance for predicting death after transplantation. Finally, the categorized SCFI provided a HR for the risk of death before transplantation with borderline signifi- cance, while the metric SCFI variable proved to be a significant independent risk factor. Generally, metric variables provide statistically more robust results for this type of regression analysis. Adding the paraspinal muscle index as a confounder for 1-year mortality be- fore liver transplantation, the SCFI loses significance, which emphasizes the potential complex interplay CONCLUSIONS Distinct fat compartments impacted differently on patients’ outcome in cirrhosis. Although a low sub- cutaneous fat mass increased the risk of death and complications in patients with cirrhosis on the wait list, the risk of complications after liver transplantation was associated with high muscle and visceral fat. Therefore, it appears worthwhile to consider the nutritional state of patients awaiting liver transplantation as an important determinant of potential complications in relation to this intervention, and future risk indices may include fat in- dices as an essential confounder. ETHICS, CONSENT, AND PERMISSION Due to this retrospective, non-interventional, obser- vational study design, patients’ informed consent was not required. The study protocol conformed to the ethi- cal guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committee of the University of Leipzig (No. 356-10-13122010). Data shown in this manuscript can be requested from the corresponding author. The manuscript was compiled according to the STROBE guidelines. R E FE R E NCES 1. Rehm J, Mathers C, Popova S, Thavorncharoensap M, Teerawattananon Y, Patra J. Global burden of disease and in- jury and economic cost attributable to alcohol use and alcohol- use disorders. Lancet. 2009;373:2223–33. 19. Tapper EB, Zhang P, Garg R, Nault T, Leary K, Krishnamurthy V, et al. Body composition predicts mortality and decompen- sation in compensated cirrhosis patients: a prospective cohort study. JHEP Rep. 2020;2:100061. 2. Rehm J, Shield KD, Gmel G, Rehm MX, Frick U. Modeling the impact of alcohol dependence on mortality burden and the ef- fect of available treatment interventions in the European Union. Eur Neuropsychopharmacol. 2013;23:89–97. 20. Sakamaki A, Yokoyama K, Koyama K, Morita S, Abe H, Kamimura K, et al. Obesity and accumulation of subcutaneous adipose tissue are poor prognostic factors in patients with alco- holic liver cirrhosis. PLoS One. 2020;15:e0242582. 3. Sheron N. Alcohol and liver disease in Europe—simple mea- sures have the potential to prevent tens of thousands of prema- ture deaths. J Hepatol. 2016;64:957–67. 21. Cho YS, Lee HY, Jeong JY, Lee JG, Kim TY, Nam SW, et al. Computed tomography-determined body composition abnor- malities usefully predict long-term mortality in patients with liver cirrhosis. J Comput Assist Tomogr. 2021;45:684–90. 4. Artru F, Louvet A, Ruiz I, Levesque E, Labreuche J, Ursic- Bedoya J, et al. Liver transplantation in the most severely ill cirrhotic patients: a multicenter study in acute-on-chronic liver failure grade 3. J Hepatol. 2017;67:708–15. 22. Meister FA, Bednarsch J, Amygdalos I, Boecker J, Strnad P, Bruners P, et al. Various myosteatosis selection criteria and their value in the assessment of short- and long-term outcomes following liver transplantation. Sci Rep. 2021;11:13368. 5. D'Amico G, Garcia-Tsao G, Pagliaro L. Natural history and prognostic indicators of survival in cirrhosis: a systematic re- view of 118 studies. J Hepatol. 2006;44:217–31. 23. Czigany Z, Kramp W, Bednarsch J, Kroft G, Boecker J, Strnad P, et al. Myosteatosis to predict inferior perioperative outcome in patients undergoing orthotopic liver transplantation. Am J Transplant. 2020;20:493–503. 6. D'Amico G, Pasta L, Morabito A, D'Amico M, Caltagirone M, Malizia G, et al. Competing risks and prognostic stages of cirrhosis: a 25-year inception cohort study of 494 patients. Aliment Pharmacol Ther. 2014;39:1180–93. 24. van Vugt JLA, Alferink LJM, Buettner S, Gaspersz MP, Bot D, Darwish Murad S, et al. R E FE R E NCES A model including sarcopenia sur- passes the MELD score in predicting waiting list mortality in cirrhotic liver transplant candidates: a competing risk analysis in a national cohort. J Hepatol. 2018;68:707–14. 7. Engelmann C, Thomsen KL, Zakeri N, Sheikh M, Agarwal B, Jalan R, et al. Validation of CLIF-C ACLF score to define a threshold for futility of intensive care support for patients with acute-on-chronic liver failure. Crit Care. 2018;22:254. p 25. Phillips R, Ursell T, Wiggins P, Sens P. Emerging roles for lipids in shaping membrane-protein function. Nature. 2009;459:379–85. 8. Engelmann C, Schob S, Nonnenmacher I, Werlich L, Aehling N, Ullrich S, et al. Loss of paraspinal muscle mass is a gender- specific consequence of cirrhosis that predicts complications and death. Aliment Pharmacol Ther. 2018;48:1271–81. 26. Köberlin M, Snijder B, Heinz L, Baumann C, Fauster A, Vladimer G, et al. A conserved circular network of coregulated lipids modulates innate immune responses. Cell. 2015;162:170–83. 9. Figueiredo FA, De Mello PR, Kondo M. Effect of liver cirrhosis on body composition: evidence of significant depletion even in mild disease. J Gastroenterol Hepatol. 2005;20:209–16. 27. Mancia G, Bousquet P, Elghozi JL, Esler M, Grassi G, Julius S, et al. The sympathetic nervous system and the metabolic syndrome. J Hypertens. 2007;25:909–20. 10. Crawford DH, Shepherd RW, Halliday JW, Cooksley GW, Golding SD, Cheng WS, et al. Body composition in nonalco- holic cirrhosis: the effect of disease etiology and severity on nutritional compartments. Gastroenterology. 1994;106:1611–7. 28. Lambert GW, Straznicky NE, Lambert EA, Dixon JB, Schlaich MP. Sympathetic nervous activation in obesity and the meta- bolic syndrome—causes, consequences and therapeutic im- plications. Pharmacol Ther. 2010;126:159–72. 11. Ebadi M, Tandon P, Moctezuma-Velazquez C, Ghosh S, Baracos VE, Mazurak VC, et al. Low subcutaneous adiposity associates with higher mortality in female patients with cirrho- sis. J Hepatol. 2018;69:608–16. 29. Jalan R, Forrest EH, Redhead DN, Dillon JF, Hayes PC. Reduction in renal blood flow following acute increase in the portal pressure: evidence for the existence of a hepatorenal reflex in man? Gut. 1997;40:664–70. 12. Praktiknjo M, Book M, Luetkens J, Pohlmann A, Meyer C, Thomas D, et al. Fat-free muscle mass in magnetic resonance imaging predicts acute-on-chronic liver failure and survival in decompensated cirrhosis. Hepatology. 2018;67:1014–26. 30. van Son J, Stam SP, Gomes-Neto AW, Osté MCJ, Blokzijl H, van den Berg AP, et al. Post-transplant obesity impacts long-term survival after liver transplantation. Metabolism. 2020;106:154204. 13. Niklas F. Aehling https://orcid. org/0000-0002-2258-847X Niklas F. Aehling https://orcid. org/0000-0002-2258-847X Niklas F. Aehling https://orcid. org/0000-0002-2258-847X 18. Moreau R, Clària J, Aguilar F, Fenaille F, Lozano JJ, Junot C, et al. Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF. J Hepatol. 2020;72:688–701. AUTHOR CONTRIBUTIONS Cornelius Engelmann: Study concept and design, analysis, interpretation of data, draft of the manuscript; Niklas F. Aehling: Analysis, interpretation, draft of the manuscript; Stefan Schob, Ines Nonnenmacher, Luise Handmann: data collection, interpretation of data, criti- cal revision of the manuscript; Jane Macnaughtan, Adam Herber, Alexey Surov, Thorsten Kaiser, Timm Denecke, Rajiv Jalan, Daniel Seehofer, Michael Moche, Thomas Berg: interpretation of data, critical revision of the manuscript. 11 11 HEPATOLOGY COMMUNICATIONS 17. van der Poorten D, Milner K-L, Hui J, Hodge A, Trenell MI, Kench JG, et al. Visceral fat: a key mediator of steatohepatitis in metabolic liver disease. Hepatology. 2008;48:449–57. R E FE R E NCES Tachi Y, Kozuka A, Hirai T, Kojima Y, Ishizu Y, Honda T, et al. Skeletal muscle fat deposition is associated with hepatocellular carcinoma development in patients with chronic liver disease. Nutrition. 2018;54:83–8. 31. Barone M, Viggiani MT, Losurdo G, Principi M, Leandro G, Di Leo A. Systematic review with meta-analysis: post-operative complications and mortality risk in liver transplant candidates with obesity. Aliment Pharmacol Ther. 2017;46:236–45. 14. Montano-Loza AJ, Mazurak VC, Ebadi M, Meza-Junco J, Sawyer MB, Baracos VE, et al. Visceral adiposity increases risk for hepatocellular carcinoma in male patients with cir- rhosis and recurrence after liver transplant. Hepatology. 2018;67:914–23. 32. Hamaguchi Y, Kaido T, Okumura S, Kobayashi A, Shirai H, Yagi S, et al. Impact of skeletal muscle mass index, intramuscular adipose tissue content, and visceral to subcutaneous adipose tissue area ratio on early mortality of living donor liver trans- plantation. Transplantation. 2017;101:565–74. 15. García-Pajares F, Peñas-Herrero I, Sánchez-Ocaña R, Torrres-Yuste R, Cimavilla-Román M, Carbajo-López A, et al. Metabolic syndrome after liver transplantation: five-year prevalence and risk factors. Transplant Proc. 2016;48:3010–2. 33. Terjimanian MN, Harbaugh CM, Hussain A, Olugbade KO, Waits SA, Wang SC, et al. Abdominal adiposity, body compo- sition and survival after liver transplantation. Clin Transplant. 2016;30:289–94. 16. Spengler EK, O’Leary JG, Te HS, Rogal S, Pillai AA, Al-Osaimi A, et al. Liver transplantation in the obese cirrhotic patient. Transplantation. 2017;101:2288–96. 34. Czigany Z, Kramp W, Lurje I, Miller H, Bednarsch J, Lang SA, et al. The role of recipient myosteatosis in graft and patient 12 BODY FAT COMPOSITION AND OUTCOMES BEFORE AND AFTER LT 12 survival after deceased donor liver transplantation. J Cachexia Sarcopenia Muscle. 2021;12:358–67. 39. Husing A, Kabar I, Schmidt HH. Lipids in liver transplant recip- ients. World J Gastroenterol. 2016;22:3315–24. SUPPORTING INFORMATION Additional supporting information may be found in the online version of the article at the publisher’s website. p 35. Singhal A, Wilson GC, Wima K, Quillin RC, Cuffy M, Anwar N, et al. Impact of recipient morbid obesity on outcomes after liver transplantation. Transpl Int. 2015;28:148–55. 36. Ata N, Ayloo S, Tsung A, Molinari M. Recipient obesity does not affect survival after deceased donor liver transplantation for hepatocellular carcinoma. A national retrospective cohort study in the United States. HPB (Oxford). 2019;21:67–76. How to cite this article: Engelmann C, Aehling NF, Schob S, Nonnenmacher I, Handmann L, Macnaughtan J, et al. Body fat composition determines outcomes before and after liver transplantation in patients with cirrhosis. Hepatol Commun. 2022;00:1–12. https://doi.org/10.1002/ hep4.1946 37. Engin A. The definition and prevalence of obesity and meta- bolic syndrome. Adv Exp Med Biol. 2017;960:1–17. 38. Pelaez-Jaramillo MJ, Cardenas-Mojica AA, Gaete PV, Mendivil CO. Post-liver transplantation diabetes mellitus: a re- view of relevance and approach to treatment. Diabetes Ther. 2018;9:521–43. 39. Husing A, Kabar I, Schmidt HH. Lipids in liver transplant recip- ients. World J Gastroenterol. 2016;22:3315–24.
https://openalex.org/W2953409332	https://revista.cortesgenerales.es/rcg/article/download/48/117	es		La reforma del Reglamento del Congreso de los Diputados	Revista de las Cortes Generales	2,018	cc-by	18,176	Revista de las Cortes Generales. ISSN: 0213-0130 Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS Carlos Gutiérrez Vicén Letrado de las Cortes Generales RESUMEN La reforma del Reglamento del Congreso de los Diputados se ha convertido en una verdadera asignatura pendiente, tal y como puedan ser la reforma del Senado, la modificación del régimen constitucional en cuanto a la sucesión de la Corona o la aprobación del Reglamento de las Cortes Generales que para regir las sesiones conjuntas de ambas Cámaras prevé el artículo 72.2 de la Constitución. Este artículo pretende contribuir al homenaje que el Nº 100 de la Revista de las Cortes Generales quiere hacer a la Constitución de 1978 en el XL Aniversario de su promulgación. Se trata de exponer la líneas generales en las que se enmarca la reforma del Reglamento del Congreso, destacando el contraste entre el deseo manifestado prácticamente por todos los grupos políticos con representación parlamentaria de llevar a cabo una completa modificación del Reglamento y la falta de éxito en todos los intentos de conseguirlo, analizando las dificultades para lograrlo y recogiendo las distintas reformas parciales aprobadas hasta la fecha. Palabras clave: Reforma, Reglamento, Congreso de los Diputados, Grupos políticos, Representación, Reforma parcial, Procedimiento de reforma, Reserva reglamentaria, Control de constitucionalidad. ABSTRACT The reform of the Regulation of the Congress of Deputies has become a true pending subject, such as the reform of the Senate, the modification of the Constitutional Regime concerning the Succession to the Crown or the approval of the Regulation of the Spanish Constitution to regulate the joint sessions of both Chambers. 44 CARLOS GUTIÉRREZ VICÉN The present article aims to contribute to the tribute made to the Constitution of 1978 on the occasion of the 40th Anniversary of its enactment. It intends to draw the general guidelines that constitute the frame works for the reform of the Regulation of the Congress. The article highlights the contrast between the willingness to fully modify the Regulation, expressed by almost every political group with parliamentary representation, and the lack of success in every attempt to achieve. In this regard, it analyses the difficulties to make it possible, and gathers the different partial reforms which have been approved until the present day. Keywords: Reform, Regulation, Congress of Deputies, Political group, Representation, Partial reform, Reform procedure, Government’s power statutory regulations SUMARIO: I. Introducción. II. Procedimiento de reforma: antecedentes y derecho comparado. 1. Antecedentes. 2. Derecho Comparado. III. Procedimiento de reforma. IV. Posición en el ordenamiento, reserva reglamentaria y control de constitucionalidad. V. Reformas parciales e intentos de reforma global. 1. Reformas parciales. 2. Reforma global. VI. Conclusiones Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 45 I. Introducción Es un hecho notorio que el cuadragésimo aniversario de la Constitución coincide con una etapa de profundos cambios en nuestro país. En poco tiempo hemos vivido experiencias que hace unos años nos hubieran parecido hipótesis de laboratorio, más propias de la política-ficción que de la realidad cotidiana o probable. Así, por ejemplo, la fallida XI Legislatura y el año 2016 (en el que tuvieron lugar hasta tres sesiones de investidura) o la aprobación de una moción de censura por primera vez en nuestra experiencia constitucional el 1 de junio de 2018, no son sino el reflejo de la ruptura del sistema bipartidista de alternancia en el Gobierno con un Parlamento muy fragmentado como, por otra parte, está sucediendo en toda Europa. La división se manifiesta, asimismo, en lo que se refiere al modelo de distribución territorial del poder llegando a la puesta en marcha del mecanismo previsto en el artículo 155, a raíz de la declaración unilateral de independencia formulada en el Parlamento de Cataluña el 27 de octubre de 2017, con todas las peripecias posteriores a la celebración de las elecciones del 21 de diciembre de 2017. Acontecimientos en los que algunos han querido ver el agotamiento del modelo del Estado autonómico diseñado en el título VIII y la necesidad de su modificación. Y finalmente, también en cuanto a la propia reforma de la Constitución, conviven las propuestas de modificación parcial con las voces de quienes pretenden una completa sustitución de la misma. Desde esta última perspectiva no falta quien cuestione lo que se considera una interpretación ingenua, idílica, y a la postre falsa, de la etapa conocida como “la Transición” que se habría ido transmitiendo de forma acrítica desde entonces. Si, como decía Jefferson, una “generación viviente” dura diecinueve o treinta y cuatro años, habrían transcurrido algo más de una o dos generaciones, respectivamente, con un derecho consolidado a reformar la Constitución de 1978.1 Yo pertenezco a una de ellas pues entonces tenía 1 En la famosa carta que Thomas Jefferson escribió a James Madison desde París el 6 de septiembre de 1789, en plena Revolución Francesa, se contenía su célebre negativa a una Constitución perpetua que concluía con la afirmación: “Every constitution then, and every Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 46 CARLOS GUTIÉRREZ VICÉN catorce años y aunque, como es lógico a esa edad, ni yo ni la mayoría de mis amigos poníamos en el primer lugar de nuestras preocupaciones las cuestiones de política nacional, pronto comprendimos el significado que la Constitución tenía en la Historia de España. Más tarde, en la Facultad de Derecho o preparando las consabidas oposiciones, fuimos testigos de los importantes cambios que en poco tiempo permitió y así se asentó entre nosotros un fundado respeto, tanto por aquella época, como por el sistema constitucional a que dio lugar. Los años posteriores no han hecho sino confirmar esta opinión que es, por otra parte, poco original, pues las valoraciones positivas han sido muy numerosas desde las manifestaciones sobre su calidad técnica, hasta las que se han centrado en el hecho de que ha permitido uno de los más largos períodos de paz y estabilidad en nuestra convulsa historia constitucional. La mejor muestra de esa calidad es el tiempo de su vigencia sin apenas modificaciones, pues, como es sabido, la Constitución española se ha reformado tan sólo en dos ocasiones. Y, si se quiere, también es un buen indicio del sólido diseño constitucional de las instituciones su resistencia a las distintas pruebas a las que hacíamos referencia al comienzo, que han constituido un auténtico “test de estrés” del que han salido, como ha ocurrido con el Parlamento, notablemente reforzadas.2 Sin embargo, es evidente que este juicio no es unánime ni tiene por qué ser compartido. Especialmente por aquellas generaciones de españoles que no aprecian mayores méritos en el establecimiento de un régimen democrático ya que nacieron y se criaron en uno de ellos, rodeados además law, naturally expires at the end of 19 years”. Una generación abarcaría pues un período de 19 años. Sin embargo, en la carta que dirigió al Doctor Richard Gem el 9 de septiembre siguiente, entendía que este período debía ser de 34 años, considerando que este era el plazo máximo por el que se podían adquirir deudas. El texto completo de ambas cartas en The papers of Thomas Jefferson. Princeton University. Disponible en: https://jeffersonpapers.princeton.edu. 2 Algún autor ha hablado ya del “gran cambio”. Así, José Pablo Fernándiz. La década del gran cambio. El camino hacia el cuatripartidismo en España. Edit. Biblioteca Nueva, S.L. Madrid, 2018, que desde una perspectiva sociológica analiza la evolución de los resultados electorales desde el inicio de la crisis en la IX Legislatura hasta las elecciones generales del 20 de diciembre de 2015 en que se produce lo que llama la “materialización del cambio” y que podría resumirse en la ruptura del bipartidismo y la aparición de un nuevo panorama con cuatro partidos protagonistas principales de la escena política nacional. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 47 de Estados en los que la ausencia de libertad es, ciertamente, una excepción. Un buen resumen del variopinto panorama de opiniones al respecto se manifestó en la sesión del Pleno del Congreso de los Diputados del día 17 de octubre de 2017, con ocasión de la discusión de una Proposición no de Ley, presentada por el Grupo Parlamentario Popular, relativa a impulsar la celebración el 6 de diciembre de 2018 del Cuadragésimo Aniversario de la Constitución española de 1978. Y una iniciativa similar, la moción por la que se insta al Gobierno a impulsar las medidas necesarias para la celebración el 6 de diciembre de 2018 del 40 aniversario de la Constitución, fue aprobada en el Senado el 22 de noviembre de 2017. 3 En todo caso, nuestro propósito ahora es sumarnos al homenaje que este Número Extraordinario (nº 100) de la Revista de las Cortes Generales La defensa corrió a cargo del Sr. Posada Moreno (Grupo Parlamentario Popular), para quien se trata de la conmemoración de un hecho transcendental en nuestra historia, pues la Constitución “ha brindado a España el período de estabilidad democrática más prolongado de nuestra historia reciente, ha propiciado que España alcance cotas de progreso y bienestar homologables a las de los países más prósperos de Europa y ha ofrecido un fundamento sólido para progresar juntos y para dotarnos de un espacio de convivencia que todos los españoles pudiéramos compartir”. Frente a ello el representante del Grupo Mixto, Sr. Xuclà i Costa, afirmó que a pesar de que en 1978 su partido estuvo en el consenso constitucional hoy entiende que no hay nada que celebrar ya que para él existe una “incapacidad de la política española de afrontar las reformas de los marcos constitucionales en su momento”. Por su parte, el representante del Partido Nacionalista Vasco, Sr. Legarda Uriarte, tampoco ve motivos para la celebración “en coherencia con la postura de abstención que mantuvo en aquel referéndum del año 1978”. Para el Sr. Villegas Pérez, (Grupo Parlamentario de Ciudadanos), en cambio, “hay motivos para la celebración, pero también para la reflexión y para las reformas (…) Hace falta actualizar, modernizar y reformar nuestro modelo de convivencia para que esos mismos valores y principios vuelvan a brillar”. Mientras que para el Sr. Monereo Pérez, del Grupo Parlamentario Confederal de Unidos Podemos-En Comú Podem-En Marea, “mitificar, idealizar la Constitución es un inmenso error, en primer lugar, porque no fue verdad, y en segundo lugar, porque fue muy duro y hasta terrible” y aseguraba que “cada etapa política debe ganarse a los jóvenes de su generación”. Finalmente, para el Sr. Cámara Villar, (Grupo Parlamentario Socialista) pese a los logros de la Constitución, “debido al inexorable paso del tiempo y a la crisis económica, social, política, institucional y territorial que estamos viviendo, necesita ser reformada”. La Proposición no de Ley fue finalmente aprobada con 237 votos a favor, 74 en contra y 5 abstenciones. Diario de Sesiones del Congreso de los Diputados de 17 de octubre de 2017, nº 78, págs. 24 y ss. Los debates sobre la moción del Senado que reflejaron posturas similares pueden consultarse en el Diario de Sesiones de esta Cámara. Núm. 50, de 22 de noviembre de 2017, págs. 131 y ss. 3 Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 48 CARLOS GUTIÉRREZ VICÉN quiere hacer al cuadragésimo aniversario de la Constitución examinando la evolución durante este período de la reforma del Reglamento del Congreso de los Diputados. Cuestión aparentemente menor que, sin embargo, se ha convertido en una verdadera asignatura pendiente, tal y como puedan ser la reforma del Senado, la modificación del régimen constitucional en cuanto a la sucesión en la Corona o, más cercana a la nuestra, la aprobación del Reglamento de las Cortes Generales que para regir las sesiones conjuntas de ambas Cámaras prevé el artículo 72.2 de la Constitución. Como en todos estos casos, resulta curioso el contraste entre el deseo manifestado prácticamente por todos los grupos políticos con representación parlamentaria de llevar a cabo una reforma completa del Reglamento del Congreso de los Diputados y la falta de éxito en todos los intentos al respecto. Es llamativo, en cambio, que se hayan aprobado numerosas reformas parciales (un total de trece) y ninguna de ellas haya permitido introducir más cuestiones que las concretamente pactadas para asuntos muy limitados y particulares. Para entender este resultado, en cierto modo frustrante, debe partirse del procedimiento agravado que está previsto para las reformas reglamentarias, lógica consecuencia de su procedimiento de aprobación. II. Procedimiento de reforma: antecedentes y derecho comparado El establecimiento de un procedimiento específico dotado de especial rigidez para la reforma de los Reglamentos parlamentarios es una consecuencia lógica de la exigencia de algunos requisitos agravados para su aprobación. En efecto, el artículo 72 de nuestra Constitución determina en la proposición final del apartado primero que: “Los Reglamentos y su reforma serán sometidos a una votación final sobre su totalidad, que requerirá mayoría absoluta”. En desarrollo de esta exigencia la disposición final segunda del Reglamento del Congreso configura dicho procedimiento por remisión al de las proposiciones de ley de iniciativa del Congreso, reiterando la exigencia de una votación final de totalidad que reciba la aprobación de la mayoría absoluta; es decir, de al menos la mitad más uno de los diputados. Por su parte, el artículo 196 del Reglamento del Senado sigue una pauta similar al declarar de aplicación a las propuestas de reforma del ReRevista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 49 glamento lo dispuesto en él para las proposiciones de ley. Aunque, con un desarrollo algo más extenso, incluye un “trámite de admisión” por el Pleno de la Cámara y la remisión, una vez superado éste, a la Comisión de Reglamento para su estudio e informe. Finalmente, el apartado 3 contempla el debate en Pleno que se pronunciará sobre el dictamen de la Comisión y sobre los votos particulares “en la misma forma que en el procedimiento legislativo ordinario. Habrá una votación final sobre su totalidad, requiriéndose para su aprobación el voto favorable de la mayoría absoluta de Senadores”. Como es evidente, la cuestión procedimental se relaciona con el principio de autonomía parlamentaria. No en vano el artículo 72 de la Constitución destaca por consagrar aquélla del modo más completo, recogiendo no sólo la potestad autonormativa sino también otras manifestaciones como la autonomía financiera y la administrativa cuyo reconocimiento en el Derecho Comparado es frecuente dejar a normas de menor rango. También se vincula con la necesidad de proteger las normas rectoras de las Cámaras del juego de mayorías políticas ocasionales. Es decir, para evitar los llamados “golpes de mayoría” en una materia tan sensible para el funcionamiento de las Cámaras. Todo ello se relaciona al mismo tiempo con algunas cuestiones clave que han sido objeto de un extenso y profundo análisis doctrinal como son la naturaleza y la posición en el ordenamiento jurídico de los Reglamentos parlamentarios y, especialmente, el problema de la reserva reglamentaria y el papel de las otras fuentes del Derecho Parlamentario. 1. Antecedentes El examen de los antecedentes más relevantes debe abarcar tanto las previsiones constitucionales sobre la autonomía reglamentaria y la elaboración de los Reglamentos en general, como las referidas estrictamente al procedimiento de reforma que hacían los Reglamentos históricos. Debemos entonces remontarnos al artículo 127 de la Constitución de Cádiz. Con adelanto a muchos otros europeos, este texto recogía ambas cuestiones al disponer que: “En las discusiones de las Cortes, y en todo lo demás que pertenezca a su gobierno y orden interior, se observará el reglamento que se forme por estas Cortes generales y extraordinarias, sin perjuicio de las reformas que las sucesivas tuvieren por conveniente hacer en él.” Bien es cierto que las circunstancias históricas hacían preciso aclarar Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 CARLOS GUTIÉRREZ VICÉN 50 y dejar bien sentados tales extremos. Baste recordar al respecto cómo la más célebre de las construcciones doctrinales que sirvió de apoyo al carácter constituyente de las Cortes de Cádiz, la Teoría de las Cortes de Martínez Marina, fue también utilizada como armazón ideológico en defensa de los postulados realistas o absolutistas contrarios al régimen doceañista.4 Posteriormente empieza a reflejarse en el reconocimiento constitucional de la autonomía reglamentaria el carácter pendular de nuestro siglo XIX. Así, dicho reconocimiento está ausente en el Estatuto Real de Martínez de la Rosa en 1834 y vuelve a aparecer, junto a la atribución a las Cámaras de las funciones de tribunal de garantías electorales, en la Constitución de 18 de junio de 1837 cuyo artículo 29 disponía que: “Cada uno de los Cuerpos Colegisladores forma el respectivo reglamento para su gobierno interior y examina la legalidad de las elecciones y las calidades de los individuos que la componen.” Y la misma fórmula emplea el artículo 28 de la Constitución de 1845, aunque dado el carácter no electivo del Senado reservaba al Congreso la decisión sobre la legalidad de las elecciones de los diputados. Al amparo de esta norma se incluyó en el Reglamento de 4 de mayo de 1847 un Título XXI dedicado a las reformas del Reglamento donde, por primera vez, se establecían los trámites de las proposiciones de ley para las de reforma del Reglamento y se contemplaba la formación por la Secretaría de un “Apéndice” que debía recoger “las resoluciones del Congreso en casos Martínez Marina, F.: Teoría de las Cortes o Grandes juntas nacionales de los Reinos de León y Castilla: monumentos de su constitución política y de la soberanía del pueblo: con algunas observaciones sobre la lei fundamental de la monarquía española sancionada por las Cortes Generales y Extraordinarias, y promulgada en Cádiz a 19 de marzo de 1812. Imp. de Fermín Villalpando, Madrid, 1813. José Antonio Escudero ha puesto de relieve esta paradoja de modo particular por lo que se refiere a D. Bernardo Mozo de Rosales y los firmantes del llamado Manifiesto de los Persas: “Se trata, en resumen, de un ejemplo de cómo la misma tradición histórica puede ser manipulada por dos distintas interpretaciones políticas.”. Escudero, J.A.: Estudio Introductorio a la Teoría de las Cortes. Edición de la Junta General del Principado de Asturias. Oviedo, D.L. 1996, pp. CLXX-CLXXIV. Para un análisis en profundidad de ésta y otras influencias como el papel de D. Pedro Quevedo y Quintano, a la sazón Presidente de la Regencia, o la Instrucción Pastoral de los obispos refugiados en Mallorca, en el debate suscitado sobre la legitimidad de la convocatoria de Cortes, su composición y el alcance de su mandato debe verse el espléndido trabajo de Andrés Gambra Gutiérrez. Gambra Gutiérrez, A.: Los opositores a la Constitución de Cádiz. En Cortes y Constitución de Cádiz: 200 años. Espasa, Madrid, 2011, Vol. 2, pp. 496-518. 4 Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 51 omisos o dudosos” y que debía repartirse a los diputados al principio de cada Legislatura. Estos precedentes se aplicarían como adiciones provisionales al Reglamento en una especie de integración constante del mismo para su aplicación en casos análogos. En 1852, el proyecto constitucional de Bravo Murillo, proponía incluir entre las leyes fundamentales o “leyes del Estado” las de organización del Senado, elección de diputados a Cortes, de régimen de los Cuerpos colegisladores y de relaciones entre los dos Cuerpos colegisladores con un claro sentido restrictivo de la autonomía parlamentaria que llevaba a la aprobación de los Reglamentos por el Rey y al nombramiento por éste de los Presidentes de las Cámaras. El Reglamento Provisional de 9 de noviembre de 1854, que había de regir hasta la constitución definitiva de las Cortes convocadas después de la Vicalvarada, establecía en su artículo adicional 1º que una vez constituidas las Cortes se nombraría una Comisión permanente de Reglamento que debía ocuparse de examinar las adiciones y enmiendas presentadas por los diputados y de preparar el proyecto de Reglamento definitivo. Era la primera vez que se contemplaba una Comisión específica para preparar la labor del Pleno en esta materia. Sin embargo, tal previsión no llegó a cumplirse, como tampoco vio la luz la conocida como Constitución nonata de 1856. Más tarde el Acta adicional a la Constitución de 1845, impulsada por Narváez y aprobada por ley de 17 de julio de 1857, disponía en su artículo 28 que “Los Reglamentos del Senado y del Congreso serán objeto de una Ley”. Como es sabido, el Acta fue derogada por la Ley de 20 de abril de 1864 que restablecía la integridad de la Constitución de 1845 añadiendo solamente una disposición transitoria sobre nombramiento de Senadores. De esta forma, la Constitución de 6 de junio de 1869, de corte progresista como correspondía a un texto surgido tras la revolución de 1868, recuperaba la autonomía de las Cámaras en su artículo 45 donde reconocía a cada una por separado las facultades de formar su respectivo Reglamento, examinar la legalidad de las elecciones y la aptitud legal de los diputados y senadores, y nombrar a sus respectivos Presidente, Vicepresidentes y Secretarios. Dicha autonomía se mantendrá ya en la Constitución de 30 de junio de 1876, que regirá todo el período de la Restauración, en los artículos 34 y 35. Si bien presenta un rasgo conservador que se muestra en la mayor Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 CARLOS GUTIÉRREZ VICÉN 52 intervención real en el caso del Senado, desde su propia composición hasta la designación de su Presidente y Vicepresidentes, aunque la de los Secretarios sigue reservándose a la propia Cámara Alta. Es curioso destacar que durante este largo período y bajo la vigencia de tan diferentes textos constitucionales, la fórmula recogida por los distintos Reglamentos fue siempre la misma; es decir, la del Reglamento de 1847. El procedimiento ordinario de las proposiciones de ley y la formación del Apéndice se repitieron así en el Reglamento de 25 de junio de 1867 (Título XX, artículos 196 y 197); en el Reglamento del Congreso de los Diputados de 1847 restaurado con las modificaciones introducidas en 18 de junio de 1864, 16 de diciembre de 1878, 7 de abril de 1880, 10 de abril de 1894, 23 de febrero de 1895 y 24 de julio de 1896 (Título XXI, artículos 223 y 224, en el que se incorporó, efectivamente, el citado Apéndice recogiendo los acuerdos del Congreso sobre las materias más variadas),5 así como en el de 24 de mayo de 1918. El principio de autonomía reglamentaria desaparecía en el proyecto de Constitución de Primo de Rivera que en 1929 ensayaba unas Cortes pseudo corporativas, y se recuperaba de nuevo en el Parlamento unicameral de la II República. La Constitución de 9 de diciembre de 1931, disponía en su artículo 57 que: “El Congreso de los Diputados tendrá facultad para resolver sobre la validez de la elección y la capacidad de sus miembros electos y para adoptar su Reglamento de régimen interior.” El Reglamento que fue aprobado al amparo de este precepto el 29 de noviembre de 1934 dedicaba su Título XIII (artículo 136) a la “Reforma de este Reglamento”. Contenía una Desde el acuerdo adoptado en la sesión de 27 de diciembre de 1848 sobre el orden de intervención en los debates, hasta el del 9 de julio de 1904 en materia de tramitación de suplicatorios. Dicho apéndice puede consultarse en: Reglamentos (del Congreso de los Diputados y de las Cortes). Madrid: Cortes, Secretaría, 1977 (pp. 320 a 322). Esta Recopilación, presentada por D. Felipe de la Rica y Montejo, fue hecha con ocasión del mandato de la Ley para la Reforma Política de que las nuevas Cortes estableciesen su propio Reglamento. Resulta de gran utilidad para los estudios de carácter histórico pues, además de reunir el conjunto de todos los Reglamentos que han estado en vigor desde 1810, la mayoría de los cuales sólo puede consultarse en los archivos de su publicación oficial, incorpora el texto de otros documentos de interés como la Ley de Relaciones entre los Cuerpos Colegisladores, de 19 de julio de 1837, el Proyecto de Ley para el régimen de los Cuerpos Colegisladores, de 3 diciembre de 1852, o el Reglamento Provisional de la Asamblea Nacional, de 20 de septiembre de 1927. 5 Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 53 regulación algo detallada del procedimiento que era también más riguroso: comenzaba por una iniciativa reforzada de las proposiciones de reforma que debían “contar con diez firmas como mínimo y veinte como máximo.” Debían ser leídas en sesión pública, impresas y repartidas. El trámite de la toma en consideración exigía el voto favorable de 100 diputados, tras de lo cual la proposición pasaría a Comisión. Ésta no tenía carácter permanente, sino que habría de constituirse para la ocasión formada por 21 miembros designados en proporción a la fuerza numérica de las distintas fracciones políticas. La tramitación del dictamen, votos particulares, enmiendas y votación seguirían las normas correspondientes a los proyectos de ley. Como es de sobra conocido, el principio desaparecía con las Leyes Fundamentales y, en concreto, con la Ley de 17 de julio de 1942 que creó las Cortes Españolas. Éstas tenían una significación muy diferente a las que habían existido entre nosotros desde los comienzos de la era constitucional, estando plenamente sometidas al Poder Ejecutivo, como se observa, incluso, en el instrumento normativo mediante el que se aprobaban sus Reglamentos. La disposición adicional primera de la Ley de 1942 (elevada al rango de Ley Fundamental por la de 26 de julio de 1947) establecía que: “Las Cortes, de acuerdo con el Gobierno redactarán su Reglamento.” Pero antes de desarrollar este procedimiento mixto se publicó mediante una ley “dada” por el Jefe del Estado el Reglamento Provisional de 5 de enero de 1943 (al que se incorporaron las adiciones aprobadas en la sesión plenaria de 15 de julio de 1946) pues, según se diría más tarde, “como suele suceder en estos casos, se juzgó oportuno facilitar los primeros pasos del nuevo Cuerpo, suministrándole de oficio un primer Reglamento provisional. Sin embargo, la provisionalidad duró quince años, rigiendo el funcionamiento de las cinco primeras legislaturas hasta que se aprobó el Reglamento de las Cortes Españolas de 26 de diciembre de 1957 que inauguró el procedimiento mixto de elaboración que aseguraba el “acuerdo” mediante la presentación por el Gobierno de un proyecto de Reglamento que después era sometido a las Cortes y aprobado como Ley6. Así se aprobaron también el Reglamento de 22 de julio de 1967 y el de 15 de noviembre de 1971. Manuel Fraga Iribarne recordaba entonces que “también las Cortes Constituyentes de 1931 recibieron un Reglamento provisional, de un Gobierno igualmente provisional (18 de julio de 1931).” Y sostenía, con abundante cita doctrinal respecto a las características de tipo formal del Reglamento de 26 de diciembre de 1957, que no podía incluirse en la clásica 6 Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 54 CARLOS GUTIÉRREZ VICÉN En todos los casos se mantenía este esquema que permitía la influencia del Ejecutivo en las reformas del Reglamento: se preveía que el Reglamento podía ser modificado por las Cortes con el acuerdo del Gobierno y que “toda enmienda al mismo se tramitará con las formalidades que el propio Reglamento establece para las proposiciones de ley”, (disposiciones finales primera y segunda de los Reglamentos de 1957 y 1967). El Reglamento de 1971 establecía, además, entre las Comisiones Generales, la Comisión de Reglamento, presidida por el Presidente de las Cortes e integrada por cinco Presidentes de Comisiones y cinco Procuradores de los que uno tendría la condición de Ministro del Gobierno. A esta Comisión le correspondía tomar en consideración las proposiciones de ley de reforma del Reglamento que presentaran los Procuradores y la elaboración “de estudios e informes encaminados a mejorar el procedimiento parlamentario y, en su caso, de las proposiciones que inicien la reforma. Pero, como se encargaba de aclarar el artículo 28.3, estas atribuciones lo eran “sin perjuicio de lo dispuesto en la disposición adicional de la Ley de Cortes y en la final primera de este Reglamento.” Es decir, que en todo caso se requería el acuerdo del Gobierno para la tramitación de las reformas. Y, según disponía la disposición final tercera, una vez que las proposiciones de reforma fuesen tomadas en consideración por la Comisión de Reglamento, se tramitarían en una Comisión legislativa especial. división tripartita de Hatschek puesto que era un “acuerdo” entre las propias Cortes y el Gobierno. Lo que, a su juicio, aclaraba el problema de la naturaleza jurídica del Reglamento de las Cortes Españolas: era una norma auténtica, obligatoria para la Cámara en cuanto tal y para cada uno de sus miembros, y su rango de ley formal le daba un alcance general sólo limitado por su esfera de aplicación efectiva. Fraga Iribarne, M.: “Los Privilegios de los Procuradores y el nuevo Reglamento de las Cortes Españolas”. Revista de Estudios Políticos, nº 99, 1958, pp.57-83. Sin embargo, como decía Joan Corominas en polémica con María Rosa Lida de Malkiel sobre la prisión padecida por el Arcipreste de Hita y recoge Juan Luis Alborg, “es en vano siempre echar mano de la erudición contra la evidencia…” (Alborg, J. L.: Historia de la literatura española. Vol. I. Ed. Gredos, 2ª ed. Ampliada. Madrid, 1970, P.228) No puede desconocerse que esta construcción doctrinal era un ingenioso fruto de su época y en ese contexto debe analizarse. Pretendía encajar en las categorías jurídico-políticas al uso en los sistemas democráticos una realidad muy difícil o imposible de equiparar: la sumisión al Poder Ejecutivo (en todos los aspectos de su existencia) de unas Cortes no elegidas libremente impedía la formación de una voluntad autónoma que pudiese acordar nada u oponerse a cualquier decisión gubernamental. Y esto siguió siendo así hasta el Reglamento de 1971, por más que en él se apreciase algún avance o “apertura” admitiendo, al menos, la iniciativa parlamentaria de reforma. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 55 La tutela terminará con la Ley para la Reforma Política de 4 de enero de 1977 que recupera con decisión la autonomía parlamentaria al disponer en su artículo 2.5 que: “El Congreso y el Senado establecerán sus propios Reglamentos y elegirán sus respectivos Presidentes.” Así se aprobaron en las sesiones plenarias del 13 de octubre de 1977 el Reglamento Provisional del Congreso de los Diputados, y en la del 14 del mismo mes y año el Reglamento Provisional del Senado que rigieron las Cámaras durante la transición en las legislaturas Constituyente y Primera, tras la celebración de las elecciones generales de marzo de 1979.7 En concordancia con su carácter democrático, la disposición final segunda del Reglamento Provisional del Congreso eliminaba toda mención al Gobierno, señalando que: “La reforma de este Reglamento se tramitará conforme a lo establecido para las proposiciones de ley.” Mientras el artículo 155 del Reglamento Provisional del Senado disponía que: “En la tramitación de las propuestas de reforma del Reglamento se observará lo dispuesto en éste para el procedimiento legislativo ordinario, con las especialidades que pueden derivarse del carácter autonómico de esta Norma.” En fin, con la promulgación de la Constitución de 1978 cobró pleno vigor el mandato contenido en su artículo 72 que se desarrolló en primer término con la aprobación del Reglamento del Congreso de los Diputados el 10 de febrero de 1982 y el del Senado el 26 de mayo del mismo año. Ambos han sido objeto de numerosas reformas parciales, pero tan sólo el segundo ha conocido la aprobación de un Texto Refundido, el aprobado el 3 de mayo de 1994. Dicho texto deroga formalmente el Reglamento de 1982. Sin embargo, no se trataba de una reforma global del mismo ya que se mantenía prácticamente en su totalidad, incluso respetando la numeración de sus preceptos. Simplemente se buscaba integrar las reformas aprobadas hasta entonces, las de 11 de noviembre de 1992, 6 de octubre de 1993 y, sobre todo, la de 11 de enero de 1994 que creaba la Comisión General de las Comunidades Autónomas como órgano en el que se esperaba que recayese Reglamento Provisional del Congreso de los Diputados de 17 de octubre de 1977, (BOE nº 256, de 26 de octubre), fue modificado el 3 de mayo de 1979, sin embargo esta modificación sólo fue recogida por el Diario de Sesiones de aquella fecha (Diario nº 5 de 1979) y en la edición del Servicio de Estudios de Cortes Generales (Madrid, 1981) y Reglamento provisional del Senado de 18 de octubre de 1977 (BOE nº 256 de 26 de octubre), modificado el 27 de diciembre de 1978 y el 20 de noviembre de 1980. (BOE nº 287, de 29 de noviembre de 1980). 7 Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 CARLOS GUTIÉRREZ VICÉN 56 el peso fundamental del reforzamiento del papel del Senado como Cámara de representación territorial. Más tarde ha sido objeto también de distintas reformas parciales, la última de las cuales tuvo lugar en la sesión plenaria del Senado celebrada el 19 de junio de 2018. 2. Derecho Comparado Desde la perspectiva del Derecho Comparado, la autonomía reglamentaria de las Cámaras es un principio reconocido de modo casi general, de forma que cuando no es así, sufre el principio democrático y se refuerza el carácter autoritario del sistema político. Cosa distinta es que este reconocimiento tenga lugar de manera expresa en la Constitución o se entienda implícito en ésta. Oponiéndose así, en la clásica división de Hastchek, el sistema de reglas codificadas propias del Derecho Parlamentario continental a la “lex parliamenti” inglesa como suma de precedentes consuetudinarios y al modelo nórdico típico de la Ley constitucional que aprueba el Reglamento sueco. Aunque, la clasificación va perdiendo nitidez con el transcurso del tiempo.8 Son muy numerosos los ejemplos de recepción constitucional de la autonomía reglamentaria. Por limitarnos a unos cuantos entre los más clásicos dentro de la Unión Europea pueden citarse los casos de: Alemania (arts. 40.1 y 52.3 para el Bundestag y el Bundesrat, respectivamente); Austria (arts. 30.2 y 37.2 con la misma advertencia para el Nationalrat y el Bundesrat); Bélgica (art. 60); Dinamarca (art. 48); Grecia (art. 65); Irlanda (art. 15.10); Italia (art. 64.1 para la Cámara de diputados y el Senado); Países Bajos (art. 72 para ambas Cámaras, tanto por separado como reunidas en sesión conjunta); Polonia (art. 112 para la Cámara de diputados y art. 124 para el Senado); Portugal (art. 175 a). Pero, incluso en esta pequeña muestra, la casuística de supuestos particulares es muy variada: En Francia, es sabido, el artículo 61 de la Constitución de 4 de octubre de 1958 (reformada por última vez el 23 de julio de 2008) establece un control previo de constitucionalidad al disponer que los reglamentos de la Asamblea Nacional y del Senado, “antes de su aplicación deben ser sometidos al Consejo Constitucional, el cual se 8 Hastschek, J.: Das Parlamentsrecht (Das Parlamentsrecht Des deutschen Reiches). Leipsig, 1915. (Edición inglesa de Walter de Gruyter, 1973). Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 57 pronunciará sobre su conformidad con la Constitución”. En Luxemburgo la regulación de la organización y funcionamiento de la Cámara de diputados está compartida entre la Ley (artículo 51.2 de la Constitución de 17 de octubre de 1868, Texto refundido con la última reforma de 12 de marzo de 2009) y el Reglamento (artículo 70). Por otro lado, el modelo nórdico de aprobación del Reglamento mediante una ley constitucional va experimentando una cierta aproximación al sistema continental europeo clásico. Así ocurre en el caso de Suecia, donde la Ley del Riksdag, aprobada en 1974 (SFS 1974: 153) al amparo de los artículos 10 del cap. IV y 16 del cap. VIII del Instrumento de Gobierno, no se considera como éste una de las cuatro leyes fundamentales que integran la Constitución. Aunque, por su importancia, está a medio camino entre éstas, junto a las que suele citarse, y las leyes ordinarias, estando previsto un procedimiento de reforma agravado para alguna de sus partes por lo que podríamos hablar de ley semi-constitucional. Fue modificada por última vez mediante ley de 1 de agosto de 2010. De forma más clara en Finlandia se ha optado, frente al antiguo sistema de la Ley Orgánica del Parlamento de 1928, aprobada al amparo del artículo 17 del Instrumento de Gobierno de 17 de julio de 1919, por integrar en el texto constitucional las principales disposiciones sobre el Parlamento remitiéndose para una regulación más detallada al Reglamento que se aprobará conforme al procedimiento establecido para los proyectos de ley (artículo 52 de la Constitución finlandesa de 11 de junio de 1999, modificada y puesta al día el 12 de octubre de 2010). Por último, como es bien sabido, el peculiar caso del Reino Unido también ha experimentado un proceso de racionalización y sistematización que ha acercado a las Standing Orders de la Cámara de los Comunes y de los Lores a los Reglamentos parlamentarios continentales. Si bien es cierto que aquellas no son exhaustivas, ya que muchos de los procedimientos seguidos en las Cámaras tienen su origen en la costumbre y las prácticas consuetudinarias. En el caso de los Comunes existen dos compilaciones: una para Public Business y otra para Private Business cuyas últimas versiones fueron aprobadas, respectivamente, en mayo y octubre de 2005, aunque siguen añadiéndose nuevas standing orders desde entonces. En la Cámara de los Lores existe además el denominado Companion to Standing Orders aprobado en 2010 por el Procedure Committee. Esta es la vigésimo segunda edición desde que en 1862 Sir John Shaw-Lefevre, entonces Clerk of the Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 CARLOS GUTIÉRREZ VICÉN 58 Parliaments, hiciese la primera compilación de las reglas de procedimiento. Y otro tanto sucede en distintos países del entorno del Common Law, así por ejemplo la última versión consolidada de las standing orders de la Cámara de los Comunes canadiense fue aprobada el 18 de septiembre de 2017 y contiene no sólo una detallada descripción de las reglas que rigen el procedimiento legislativo, las funciones del Speaker, el calendario de sesiones y el funcionamiento de las comisiones o estatuto de los parlamentarios entre otros contenidos clásicos de los reglamentos continentales, sino que también incluye como apéndices el Código de Conducta de los miembros del Parlamento en cuanto a posibles conflictos de interés, acoso sexual, etc.9 En cuanto a los procedimientos, si nos atenemos al concepto estricto de reforma concebida como una alteración del texto del Reglamento acordada mediante el procedimiento específicamente establecido para ello y, en principio, con vocación de generalidad y permanencia, no son pocos los casos en que el procedimiento de reforma no se distingue del procedimiento legislativo ordinario. Por ejemplo, no existe ningún procedimiento específico, aunque se reconozcan ciertas peculiaridades que exigen las reformas reglamentarias en la Cámara de Representantes de Bélgica, el Folketing de Dinamarca, el Parlamento japonés, el Consejo Nacional y el Consejo de los Estados de Suiza. En otros casos se reconoce tan sólo la existencia de determinados requisitos, por ejemplo, en materia de iniciativa reservada a los parlamentarios como sucede en el Bundestag alemán, la Cámara de los Comunes canadiense o el Senado de Uruguay. Sin embargo, lo normal es que las Cámaras incluyan entre sus normas rectoras algunas relativas al procedimiento de reforma del Reglamento, configurándolo como un procedimiento especial aunque esté basado en sus líneas generales, como es lógico, en el procedimiento legislativo ordinario (así ocurre en España y también en Italia). De esta forma es frecuente la reserva de la iniciativa a instancias parlamentarias; bien a los miembros de la Cámara en general (Asamblea Nacional y Senado franceses); a un número determinado de miembros o a los Grupos Parlamentarios (la Asamblea de la República de Portugal exige una décima parte de los diputados); o a uno o varios órganos (en el Parlamento Europeo a la Mesa o una Comisión, en el Parlamento de 9 Puede consultarse en: www.ourcommons.ca/About/standingOrders/Index-e.htm. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 59 Finlandia al Speaker´s Council, en la Cámara de Diputados italiana a la Giunta per il Regolamento, y en el Sejm polaco al Presidium, al Committee on Procedure and Members Matters o un grupo de al menos 15 diputados. De las especialidades propias del procedimiento de reforma la más común es la de la existencia de una fase de Comisión en la que la Comisión de Reglamento o un órgano similar que cumpla sus funciones examina las propuestas y emite un informe que se conocerá por el Pleno. Así ocurre, por citar tan sólo algunos casos, en las dos Cámaras del Parlamento irlandés (por el Committee of Procedure and Privileges respectivo), el Knesset israelí (House Committee) y el Storting noruego (en donde además del informe de la Comisión se prevé también el del Presidium). En la práctica totalidad de los casos está previsto que los proyectos de reforma sean aprobados por mayoría de los miembros de la Cámara en sesión plenaria, exigiéndose en algún caso una mayoría cualificada como en el Senado italiano donde se requiere que la reforma sea aprobada por la mayoría absoluta de senadores. Finalmente, en algún caso están previstas varias lecturas para la aprobación de las reformas del Reglamento como sucede en las dos Cámaras de la Asamblea Federal suiza y el Sejm de Polonia, en que se exigen dos lecturas.10 III. Procedimiento de reforma Como hemos visto el procedimiento de reforma en el Reglamento del Congreso de los Diputados se basa en el procedimiento legislativo ordinario y, en particular, el de las proposiciones de ley de iniciativa de la Cámara. Haciendo un breve recorrido por sus fases y destacando sólo los Los datos para esta perspectiva comparada han sido extraídos, en el caso de las referencias constitucionales de los textos incluidos en: Ripollés Serrano, M. R. [Coord.]: Constituciones de los 27 Estados miembros de la Unión Europea. Congreso de los Diputados. Madrid, 2010. En el caso de las previsiones reglamentarias fueron recogidos del cuestionario aprobado en la reunión de la ASGP de abril de 1993 en materia de reforma de los Reglamentos parlamentarios y remitido a los Parlamentos miembros de la Unión Interparlamentaria ese mismo año. Sirvieron para presentar un informe al respecto que puede consultarse en la página web: http://www.asgp.info/en/publications/constitutional y están publicados por Alba Navarro, M.: “Procedimiento de reforma de los Reglamentos parlamentarios”. Revista de las Cortes Generales, nº. 30, 1993, pp. 7-51. 10 Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 CARLOS GUTIÉRREZ VICÉN 60 aspectos más sobresalientes habría que decir en primer lugar, que para la presentación de las propuestas de reforma son de aplicación los requisitos del artículo 126; es decir, que se excluye tanto la iniciativa gubernamental como la del Senado, la de las Asambleas Legislativas de las Comunidades Autónomas y la iniciativa legislativa popular. Las propuestas de reforma deben presentarse por un diputado con la firma de otros catorce miembros de la Cámara o por un Grupo Parlamentario con la sola firma de su portavoz.11 Por otra parte, las propuestas de reforma deben reunir los requisitos formales del artículo 124 del Reglamento, es decir, serán acompañadas de una Exposición de motivos y de los antecedentes necesarios para poder pronunciarse sobre ellas. Sin embargo, no resulta de aplicación a las propuestas de reforma reglamentaria el resto del artículo 126 relativo a la remisión de las proposiciones de ley al Gobierno al objeto de que manifieste su criterio respecto a la toma en consideración así como su conformidad o no con su tramitación. Este trámite se relaciona con el artículo 134.6 de la Constitución y la competencia del Gobierno en materia presupuestaria. Por eso limita su aplicación a las proposiciones de ley de iniciativa parlamentaria y a las enmiendas, pero no a las propuestas de reforma del Reglamento que, por otra parte, muy difícilmente podrían suponer un aumento significativo de los créditos o una disminución apreciable de los ingresos presupuestarios. Por ello, en el acuerdo de calificación de la Mesa junto a la admisión a trámite viene incluyéndose desde antiguo el traslado al Gobierno “a los efectos de su conocimiento”. Presentada la iniciativa tiene lugar el trámite de toma en consideración por el Pleno de la Cámara que, conforme a lo dispuesto en el artículo En la práctica cuando se trata de pequeñas reformas parciales sobre las que existe un acuerdo general, se busca el reforzamiento de éste y la agilización de todos los trámites mediante la presentación de una proposición suscrita por todos los Grupos Parlamentarios. Así, en la XII Legislatura se han presentado un total de 10 proposiciones de reforma del Reglamento. De ellas, tan sólo se han aprobado dos firmadas por todos o la mayor parte de los Grupos parlamentarios y en todos los casos mediante tramitación urgente por el procedimiento de lectura única, mientras otra, la presentada por el Grupo Parlamentario Confederal de Unidos Podemos-En Comú Podem-En Marea, para incluir en el artículo 46.1 del Reglamento a la Comisión para las Políticas Integrales de la Discapacidad, fue retirada. El resto, como se estudia más adelante, se encuentra en una u otra de las fases iniciales del procedimiento. 11 Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 61 126.4, se ajustará a lo establecido para los de totalidad. Concluido el debate y si la Cámara toma en consideración la propuesta de reforma, la Mesa debe acordar su envío a la Comisión de Reglamento y la apertura del plazo de enmiendas previsto en el artículo 110 sin que sean admisibles enmiendas de totalidad de devolución. La propuesta de reforma será primero examinada por una Ponencia designada en el seno de la Comisión de Reglamento conforme a las reglas del artículo 113. También en este caso y salvo declaración de urgencia o que se acuerde la tramitación por lectura única, será de aplicación el plazo de quince días para la emisión de su Informe. Este plazo podrá prorrogarse en los términos del apartado 2 del mismo precepto. Concluido el Informe de la Ponencia tiene lugar la fase de Comisión de acuerdo con lo previsto en el artículo 114 del Reglamento. En cuanto a la ordenación de los debates en la Comisión, debe tenerse en cuenta que, según el artículo 47, la Mesa de la Comisión de Reglamento está integrada por los miembros de la Mesa de la Cámara, siendo presidida por el Presidente del Congreso de los Diputados. El resto de los miembros de la Comisión se elige conforme a las normas del artículo 40 de manera que su composición, salvo en lo que se refiere a su órgano directivo, es idéntica a las restantes comisiones.12 El debate en Pleno sigue las reglas generales del procedimiento legislativo ordinario (artículos 117 a 119) con la especialidad de que concluida la votación de los votos particulares y el dictamen debe procederse a una votación final sobre la totalidad del texto que requiere su aprobación por mayoría absoluta. En este caso la práctica aconseja que, como ocurre con las votaciones previstas en el artículo 131 para las leyes orgánicas, dicha votación sea anunciada con antelación por la Presidencia, aunque tal 12 Debe reconocerse que la forma de trabajar en los distintos intentos de reforma global que se han llevado a cabo no siempre han respetado la ortodoxia de este esquema. Por ejemplo, en los trabajos de reforma llevados a cabo durante la VIII Legislatura se sucedió la labor de un Grupo de Trabajo y de la Ponencia formalmente constituida en el seno de la Comisión de Reglamento sobre un texto completo acompañado de unas llamadas ¨Normas de Funcionamiento¨ que no eran fruto de una proposición presentada por los Grupos parlamentarios, sino de un borrador encargado a los letrados de la Comisión. Por ello tan sólo pudo seguirse, aunque inspirado en los preceptos del Reglamento, un procedimiento informal que, como es sabido, no finalizó con éxito. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 62 CARLOS GUTIÉRREZ VICÉN anuncio no es preceptivo pues dicho artículo sólo lo prevé para el caso de los proyectos y proposiciones de Ley Orgánica. Finalizada la votación, y si el texto consigue la citada mayoría, se entiende aprobada la reforma y procede su publicación en el Boletín Oficial de las Cortes Generales y en el Boletín Oficial del Estado para su entrada en vigor. En este sentido la fórmula habitualmente empleada en las reformas aprobadas ha sido la de establecer, como ha hecho la última aprobada el 19 de junio de 2018, que la modificación entre en vigor el día de su publicación o el día siguiente a la misma en el Boletín Oficial de las Cortes Generales, aunque “también se publicará en el “Boletín Oficial del Estado”. Si bien no han faltado casos como el de la reforma por la que se modificaba el artículo 205, aprobada el 27 de junio de 2001, en la que tan sólo se preveía la publicación en el Boletín Oficial de las Cortes Generales (disposición final) aunque, de hecho, también se publicó en el Boletín Oficial del Estado (BOE, núm. 156, de 30 de junio de 2001). Ni como el de la reforma por la que se modificaban los artículos 79 y 82, aprobada el 21 de julio de 2011 que, sin mencionar su publicación, ordenaba la entrada en vigor a fecha fija, el 1 de septiembre de 2011. Este régimen, de publicación y entrada en vigor es idéntico al general que recoge el propio Reglamento de 1982 para sí mismo en su disposición final primera que, amparándose en la autonomía reglamentaria consagrada en el artículo 72 de la Constitución, se separa, como también lo hace el Reglamento del Senado, de la regla general fijada “por defecto” en el artículo 2.1 del Código Civil según el cual las leyes entrarán en vigor a los veinte días de su completa publicación en el BOE, si en ellas no se dispone otra cosa. Ello se debe, como han coincidido en señalar los distintos autores que se han acercado a esta cuestión tan concreta, a que los Reglamentos Parlamentarios no necesitan sanción ni promulgación para su entrada en vigor al amparo de la ya citada autonomía reglamentaria reconocida por la Constitución.13 13 Entre otros, pueden consultarse los estudios de: Rodríguez-Zapata Pérez, J.: “Los Reglamentos parlamentarios y su posición en el sistema de fuentes del derecho español”. En: I Jornadas de Derecho Parlamentario. Congreso de los Diputados, Secretaría General, Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 63 Algo más discutida es la exigencia de publicación, en cuanto requisito esencial para la seguridad jurídica que en nada compromete la potestad autonormativa de las Cámaras, especialmente si hablamos del Boletín Oficial editado por las Cortes Generales. La publicación en el Boletín Oficial del Estado, prevista en el Reglamento del Congreso en 1982 y realizada de hecho en el del Senado, tiene efectos simplemente amplificadores de su publicidad, aunque también sea una medida de puro sentido común. Y en el caso de un hipotético conflicto o discrepancia entre el texto aparecido en uno y otro diario oficial, no cabe duda, porque así se deduce de ambas disposiciones finales, de que prevalecería el texto del Boletín Oficial de las Cortes Generales.14 IV. Posición en el ordenamiento, reserva reglamentaria y control de constitucionalidad Dada la especial consideración de los Reglamentos Parlamentarios y su particular procedimiento de aprobación se plantea la cuestión de su naturaleza. El TC (en la STC 179/1989, de 2 de noviembre) dice que son normas “cuyo alcance es de mayor relevancia que la de un Reglamento interno” y “con valor de ley, aunque desprovista de la fuerza propia de la ley (STC 119/1990, de 21 de junio). No existe entre ellos y las leyes una relación jerárquica, sino sólo una división por el distinto ámbito de materias Madrid, 1985; Ripollés Serrano, M. R.: Los reglamentos parlamentarios en el sistema de fuentes de la Constitución Española de 1978. Dirección General del Servicio Jurídico del Estado. Instituto de Estudios Fiscales, Madrid, 1987. Vol. III, pp. 2047-2072; y, más recientemente, el Reglamento Parlamentario en el sistema español de fuentes del derecho. Cf.: Marco Marco, J.J.: El Reglamento Parlamentario en el Sistema Español de Fuentes del derecho. Cortes Valencianas, Valencia, 2000. Por su parte, A. Torres Moral se mostraba partidario de la sanción real en el caso de los Reglamentos del Congreso de los Diputados y del Senado porque: «la sanción regia es un acto debido, no acompañado de derecho de veto, y nada impide ni desaconseja que el Rey sancione los Reglamentos parlamentarios». Torres Del Moral, A.: “Naturaleza jurídica de los Reglamentos parlamentarios”. Revista de la Facultad de Derecho de la Universidad Complutense, nº 10, 1986, p.14. 14 Si en cambio nos planteamos la cuestión respecto a los reglamentos de las asambleas legislativas de las Comunidades Autónomas, donde no existe una cobertura constitucional directa como la del artículo 72, la respuesta es diferente. Al respecto pueden verse las reflexiones que suscita la STC. 179/1989, de 2 de noviembre. Gutiérrez Vicén, C.: “Disposición Final Primera”. En: Comentarios al Reglamento del Congreso de los Diputados. . Congreso de los Diputados, Madrid, 2012, pp.1373-1382. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 64 CARLOS GUTIÉRREZ VICÉN que pueden regular, de modo similar a cómo se articula la relación entre leyes orgánicas y ordinarias. Aunque, dado que se trata de las normas que fijan las reglas procesales para aprobar las leyes, su importancia en el conjunto de fuentes es capital. El Tribunal Constitucional también los ha reconocido como parámetro de constitucionalidad formal en cuanto a los vicios de procedimiento que pueden anular la validez de una ley en la STC 99/1987, de 11 de junio, si bien sólo cuando tales vicios alteren “de modo sustancial el proceso de formación de voluntad en el seno de las Cámaras”. Sin embargo, y aunque puedan servir para esta finalidad, no pueden considerarse automáticamente como una parte integrante del llamado bloque de constitucionalidad relativo al artículo 23 de la Constitución (STC 36/1990, de 1 de marzo). En inmediata conexión con lo anterior se encuentra la cuestión relativa a la existencia de una reserva reglamentaria de materias que no pueden ser reguladas por la Ley. También en este caso después de muchas discusiones teóricas se extiende la opinión de que esta reserva deriva directamente de la Constitución y marca el límite de los ámbitos materiales objeto de cada tipo de norma. Lo cual no quiere decir que no existan campos en los que la frontera de las distintas competencias sea algo borrosa, como sucede, por ejemplo, con la creación de las Comisiones parlamentarias por una norma de rango legal; práctica que, pese a las críticas que ha recibido se ha hecho relativamente frecuente. Del mismo modo que es aceptado que dentro de los llamados “interna corporis” existan algunas materias reguladas por otras fuentes del Derecho Parlamentario distintas de los Reglamentos como las normas complementarias, interpretativas o supletorias de éstos. Más recientemente el Tribunal Constitucional ha extendido esta doctrina a los llamados usos parlamentarios en la STC 57/2011, de 3 mayo. En este pronunciamiento estima el recurso de amparo planteado contra la Resolución de la Mesa del Congreso que inadmitía una solicitud de información de la Administración Pública formulada por un diputado sin la firma del portavoz de su Grupo Parlamentario, requisito que no está previsto en el Reglamento de la Cámara para este tipo de iniciativas, ya que su artículo 7 tan sólo exige “el previo conocimiento” del Grupo al que pertenezcan los diputados. Siendo así que los usos parlamentarios, al igual que el resto de las normas complementarias o resoluciones interpretativas dictadas por Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 65 los órganos de dirección de las Cámaras “tienen su límite inmediato en el Reglamento mismo; de manera que la práctica parlamentaria efectivamente instaurada no puede resultar tan restrictiva que impida u obstaculice desproporcionadamente las facultades reconocidas a los parlamentarios en el ejercicio de sus funciones constitucionalmente garantizadas (SSTC 177/2002, de 14 de octubre y 190/2009, de 28 de septiembre). En otras palabras: los usos parlamentarios son eficaces para la regulación del modo de ejercicio de las prerrogativas parlamentarias, pero no pueden restringir su contenido reconocido en la norma reglamentaria”. Y en este mismo sentido se manifiesta la reciente STC 34/2018, de 12 de abril de 2018 que resuelve el conflicto entre órganos constitucionales promovido por el Gobierno frente a los acuerdos de la Mesa del Congreso que rechazaba la disconformidad expresada por el Gobierno a la tramitación de la Proposición de ley orgánica presentada por el Grupo Parlamentario Socialista sobre la suspensión del calendario de implantación de la Ley Orgánica 8/2013, de 9 de diciembre, para la mejora de la calidad educativa (LOMCE). En esta Sentencia se afirma que: “hemos reiterado que la autonomía parlamentaria garantizada constitucionalmente (art. 72 CE) implica otorgar a los Parlamentos y, significativamente, a sus órganos rectores, un margen de aplicación en la interpretación de la legalidad parlamentaria que este Tribunal no puede desconocer [STC 215/2016, de 15 de diciembre, FJ 5b)]. Por ello mismo, es en todo caso limitado el valor que puede otorgarse a los usos parlamentarios, pues «[(n) o existe tampoco prescripción normativa alguna que imponga al Congreso de los Diputados la sujeción a sus precedentes en relación con las decisiones sobre los procedimientos de tramitación de las iniciativas legislativas, de modo que cualquier iniciativa de reforma de un mismo texto legal haya de ser tramitada a través de idéntico procedimiento parlamentario, como parece pretender el recurrente. De nuevo aquí entra en juego la libertad de opción de la Cámara sobre el procedimiento a seguir dentro del abanico de posibilidades que le brinda su Reglamento» (STC 215/2016, de 18 de diciembre, FJ 5 c)). En este caso, como afirma el escrito de la Letrada del Congreso, cada proposición es única y debe examinarse en conexión con el plan presupuestario del Gobierno, sin que quepa por ello calificar dicha actuación de uso parlamentario.” Quizá no quede más remedio que aceptar cierta indefinición en el diseño de la reserva reglamentaria como hace el propio Tribunal ConstitucioRevista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 66 CARLOS GUTIÉRREZ VICÉN nal en la STC 136/2011, de 13 de septiembre que, con abundante cita de su propia jurisprudencia, dice que en el ejercicio de la autonomía reglamentaria corresponde “una función ordenadora de la vida interna de las Asambleas” (…) “para regular su propia organización y funcionamiento” (…) “para ordenar los derechos y atribuciones que los parlamentarios ostentan” (…) “así como para articular las fases o procedimientos que se desarrollan en su seno”. Sin duda habrá oportunidad de que esta jurisprudencia se amplíe y complete cuando el Tribunal Constitucional tenga ocasión de pronunciarse sobre los recursos presentados contra el Real Decreto-Ley 4/2018, de 22 de junio, por el que se concreta, con carácter urgente, el régimen jurídico aplicable a la designación del Consejo de Administración de la Corporación RTVE y de su Presidente, así como contra los actos de ejecución del mismo. Una de las cuestiones nucleares en la fundamentación de estos recursos, aparte del empleo del instrumento del Decreto-Ley es, precisamente, la invasión de la autonomía parlamentaria y de los procedimientos previstos reglamentariamente para la elección de los consejeros y el Presidente de RTVE. No en vano en la Exposición de Motivos del Real Decreto-Ley se reconoce que el mismo “tiene como objetivo resolver una situación provocada por la falta de cumplimiento por parte de las Cámaras del mandato contenido en la disposición transitoria segunda de la Ley 5/2017, de 29 de septiembre, por la que se modifica la Ley 17/2006, de 5 de junio, de la radio y la televisión de titularidad estatal, para recuperar la independencia de la Corporación RTVE y el pluralismo en la elección parlamentaria de sus órganos. Los contenidos del artículo único resuelven con urgencia la situación permitiendo, de manera provisional, a la Corporación ejercer eficazmente su función de servicio público hasta la elección del nuevo Consejo de Administración a través del procedimiento que las propias Cámaras aprobaron en 2017”. Finalmente, la cuestión del control de constitucionalidad hace tiempo que tampoco ofrece dudas, aunque para la doctrina italiana fuese una cuestión muy debatida mezclada con la discusión sobre la verdadera naturaleza de los Reglamentos Parlamentarios como normas jurídicas y para la francesa suscitase algunas dudas la solución ofrecida por la Constitución de 1958 que, como vimos, supone el examen sistemático de los Reglamentos por el Consejo Constitucional antes de su entrada en vigor. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 67 En nuestro caso la posible sumisión de los Reglamentos al control del Tribunal Constitucional mediante la interposición del correspondiente recurso de inconstitucionalidad se establece en el art. 27.2 de la LO 2/1979, de 3 octubre del Tribunal Constitucional, que en su letra d) incluye entre las normas susceptibles de declaración de inconstitucionalidad a los Reglamentos de las Cámaras y de las Cortes Generales, mientras la letra f) hace lo propio con los Reglamentos de las Asambleas Legislativas de las Comunidades Autónomas. En fin, el TC con una jurisprudencia oscilante, ha ido sentando una doctrina primero extensiva del control de constitucionalidad mediante el recurso de inconstitucionalidad a las Resoluciones interpretativas de la Presidencia, dado su carácter normativo, para matizar después que ello no debía implicar la exclusión de la vía del recurso de amparo cuando se tratase de actos de aplicación de la norma que implicasen la violación de alguno de los derechos fundamentales susceptibles de dicho amparo, así como en los casos en que dichas resoluciones no se utilicen propiamente para suplir omisiones del Reglamento o para interpretarlo, sino para desarrollarlo o especificar sus prescripciones. Y ello porque la equiparación entre normas reglamentarias y Resoluciones presidenciales en cuanto a su posible impugnación no puede implicar una equiparación entre ambas a todos los efectos (SSTC 118/1988, de 20 de junio; 119/1990, de 21 de junio; 44/1995, de 13 de febrero y 12/1997, de 1 de julio, así como otros pronunciamientos citados en éstas). V. Reformas parciales e intentos de reforma global 1. Reformas parciales Como ya se ha señalado, en la práctica se busca un reforzamiento del acuerdo existente sobre las reformas, de manera que se constate el apoyo de los principales grupos parlamentarios. Esto provoca, de hecho, que se dé una mayor dificultad a la hora de conseguir el consenso para una reforma completa del Reglamento cuya necesidad, sin embargo, se viene invocando por dichos grupos desde hace décadas. Por ello todas las reformas que se han conseguido aprobar han sido reformas parciales dirigidas a adaptar algunos preceptos concretos a las nuevas normas o circunstancias políticas (por ejemplo el artículo 46 que enumera las Comisiones Permanentes Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 CARLOS GUTIÉRREZ VICÉN 68 Legislativas para adaptarlo a las distintas modificaciones de la planta ministerial) o el artículo 82.2 para permitir la emisión del voto por procedimiento telemático en los casos de embarazo, maternidad, paternidad o enfermedad grave que impida el desempeño de la función parlamentaria, como ocurrió con la reforma de 21 de julio de 2011. De esta forma, el Reglamento del Congreso de 1982 ha sido objeto de trece reformas parciales entre los años 1993 y 2018. En cambio, los intentos de aprobación de un nuevo texto completo del Reglamento no han concluido con éxito a pesar de que se vienen repitiendo desde la III Legislatura. Quizá la razón se encuentra, como opina Ignacio Astarloa, en que “los partidos políticos españoles, todos ellos sin distinción, no piensan lo mismo sobre el Parlamento cuando están en el Gobierno que cuando están en la oposición. Cuando tienen mayoría en las Cámaras rechazan lo mismo que propusieron cuando eran minoría y perseguían apretar al Gobierno mayoritario con todas las armas a su alcance.”15 La primera de las reformas parciales fue la aprobada el 23 de septiembre de 1993, por la que se modificaron los artículos 18, 20.1 y 46.1 y se deroga el apartado 3 del artículo 88 que estaba referido principalmente a la declaración de bienes y actividades, obligación para la que se remite a la Ley Orgánica 5/1985, de 19 de junio, del Régimen Electoral General y especialmente a su artículo 160, para cuya ejecución en su redacción actual existe un Acuerdo de las Mesas del Congreso y del Senado de 21 de diciembre de 2009, modificado en algunos aspectos por Acuerdo de 19 de julio de 2011 en materia de Registro de Intereses. Asimismo, se derogaba algún precepto relativo a las votaciones y se modificaba por primera vez el artículo 46.1 del Reglamento que es el que recoge la enumeración de las Comisiones Permanentes Legislativas. Para este autor “ha faltado durante todo este tiempo generosidad para perseguir el interés general y mayor responsabilidad institucional. Peor aún, se ha hecho política partidista con los Reglamentos parlamentarios, proponiéndose en ocasiones reformas que no tenían recorrido, con la única finalidad de poner en aprietos al adversario, sin preocuparse por el mejor funcionamiento de la institución”. Astarloa Huarte-Mendicoa, I.: El Parlamento moderno. Importancia, descrédito y cambio. Ediciones de El Cronista. Iustel. Madrid, 2017, p. 300. 15 Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 69 Este es el precepto del Reglamento que más veces ha sido modificado (ocho en total) con el objeto de adaptar el listado de Comisiones Legislativas a la estructura departamental y a su denominación. Lo cual, aunque puede tener una sencilla explicación práctica en la medida en que en cada Comisión sectorial se desarrolla la actividad legislativa y de control de cada Departamento ministerial y sus entes, no deja de reflejar un exceso de dependencia del Ejecutivo. Así el artículo 46.1 ha sido modificado en las siguientes reformas: 26 de septiembre de 1996, 11 de mayo de 2000, 28 de abril de 2004, 29 de abril de 2008, 15 de octubre de 2009, 11 de enero de 2012, 15 de diciembre de 2016 y el 19 de junio de 2018. Es decir, hasta por dos veces en una misma Legislatura, respondiendo de modo reflejo incluso a las crisis gubernamentales que se resolvieron con reordenaciones ministeriales o al cambio de Gobierno debido a la aprobación de una moción de censura en el último caso. Además de estas reformas, el 16 de junio de 1994 se aprobó una reforma sobre la publicidad de las Comisiones de Investigación estableciendo el carácter secreto de las reuniones preparatorias de su plan de trabajo o de deliberación interna y las de las Ponencias, pero no las restantes que se ajustarán a lo dispuesto en el apartado 1 del artículo 64; es decir el paradójico sistema que afirma que “las sesiones de las Comisiones no serán públicas”, aunque permite la asistencia de “los representantes debidamente acreditados de los medios de comunicación social, excepto cuando aquellas tengan carácter secreto.” Todo ello salvo que la comparecencia verse sobre materias que hayan sido declaradas reservadas o secretas conforme a la legislación vigente o cuando a juicio de la Comisión los asuntos a tratar coincidan con actuaciones judiciales que hayan sido declaradas secretas. Asimismo se modificaba el art. 85.1 descartando la votación secreta en los procedimientos legislativos y cuando los acuerdos se adopten en función del criterio del voto ponderado y el artículo 99.1.2º sobre la posibilidad de privar a un diputado de los derechos de los artículos 6 a 9 cuando quebrantaren el deber de secreto del artículo 16. La reforma aprobada el 27 de junio de 2001 modificaba el artículo 205 del Reglamento para incluir un nuevo apartado 2 regulando la Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 70 CARLOS GUTIÉRREZ VICÉN tramitación de la propuesta para el nombramiento de los seis Vocales del Consejo General del Poder Judicial que la Cámara debe realizar entre Jueces y Magistrados de todas las categorías judiciales. De conformidad con el sistema establecido por la Ley Orgánica 2/2001, por la que se modifica la Ley Orgánica 6/1985, de 1 de julio, del Poder Judicial, corresponde a los jueces y magistrados presentar hasta un máximo de 36 candidatos. Estos son sometidos directamente a la votación del Pleno, una vez comprobado por la Mesa de la Cámara que cumplen los requisitos constitucional y legalmente establecidos sin que proceda la comparecencia previa de los mismos. Los grupos explicarán su posición por tiempo máximo de 5 minutos y cada diputado podrá escribir en la papeleta hasta seis nombres. La reforma por la que se modifica el artículo 63.2 en materia de publicidad de los dictámenes de la Comisión del Estatuto de los Diputados sobre incompatibilidades aprobada el 29 de octubre de 2009. Para el resto de los debates plenarios sobre propuestas, dictámenes, informes o conclusiones elaboradas en la Comisión del Estatuto (distintas de incompatibilidades) se mantiene el carácter no público o secreto. Y en la disposición final de la reforma se establecía que la Mesa de la Cámara adoptaría las disposiciones necesarias para garantizar la publicidad de los dictámenes de esta Comisión en materia de incompatibilidades de los diputados, mientras en la Exposición de Motivos se entendía que el procedimiento de mayor accesibilidad y menos coste es que estos dictámenes se publiquen en la página web de la Cámara. Así se hace en la actualidad en base al Acuerdo de las Mesas del Congreso de los Diputados y del Senado en reunión conjunta de 21 de diciembre de 2009 por el que se aprobaron las Normas en materia de Registro de Intereses. Finalmente, la Reforma por la que se modifican los artículos 79 y 82 del Reglamento para permitir el voto por el procedimiento telemático en los casos de embarazo, maternidad, paternidad o enfermedad grave fue aprobada el 21 de julio de 2011, al final de IX Legislatura. Esta reforma trae causa de la naturaleza personal e indelegable del voto de los parlamentarios que dispone el artículo 79.3 de la Constitución y que reitera para los diputados el artículo 79.2 del Reglamento. Supuso la introducción en este último precepto de un nuevo apartado 3 según el cual: Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 71 “Se computarán como presentes en la votación los miembros de la Cámara que, pese a estar ausentes, hayan sido expresamente autorizados por la Mesa para participar en la misma.” Y un apartado 2 en el artículo 82 según el cual: “En los casos de embarazo, maternidad, paternidad o enfermedad grave en que, por impedir el desempeño de la función parlamentaria y atendidas las especiales circunstancias se considere suficientemente justificado, la Mesa de la Cámara podrá autorizar en escrito motivado que los diputados emitan su voto por procedimiento telemático con comprobación personal en las sesiones plenarias en aquellas votaciones que, por no ser susceptibles de fragmentación o modificación, sea previsible el modo y el momento en que se llevarán a cabo. A tal efecto, el diputado cursará la oportuna solicitud mediante escrito dirigido a la Mesa de la Cámara, quien le comunicará su decisión, precisando, en su caso, las votaciones y el período de tiempo en el que podrá emitir el voto mediante dicho procedimiento. El voto emitido por este procedimiento deberá ser verificado personalmente mediante el sistema que, a tal efecto, establezca la Mesa y obrará en poder de la Presidencia de la Cámara con carácter previo al inicio de la votación correspondiente”. En aplicación de esta reforma se aprobó la Resolución de la Mesa del Congreso de los Diputados, de 21 de mayo de 2012, para el desarrollo del procedimiento de votación telemática en la que se exige un acuerdo motivado de la Mesa caso por caso y en el que se establece el período por el que se concede este derecho al ejercicio del voto por procedimiento telemático (una sesión o por un mes como mucho, aunque en ocasiones se concede hasta el fin del período de sesiones correspondiente en función de la causa que lo justifica) y los puntos del orden del día sobre el que se puede ejercer: debates de totalidad, de proyectos de ley, toma en consideración de proposiciones de ley, convalidación de Reales Decretos-Ley, etc., pero no cuestiones como proposiciones no de ley, mociones, o dictámenes de Comisión sobre los que, hasta el último momento cabe aceptar enmiendas. Y el procedimiento comenzó a llevarse a cabo con el desplazamiento de un miembro del personal informático al domicilio del parlamentario, pero una vez desarrollada la correspondiente aplicación con un certificado electrónico se permite el voto desde la propia tableta o un dispositivo similar. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 CARLOS GUTIÉRREZ VICÉN 72 La reforma alcanzó un alto grado de consenso porque venía a permitir superar una situación derivada de la imposibilidad de sustituir a los diputados, dado, además, que su relación no se considera una relación laboral y que, por tanto, no contempla las bajas temporales por estos motivos. En el pasado, cuando se producían estas situaciones, se corregían normalmente mediante un pacto entre caballeros que vinculaba a los dos grandes partidos del arco parlamentario, de forma que en el contrario se producían las ausencias voluntarias necesarias que equilibraran las forzosas. De todas maneras es visible el carácter muy restrictivo con que se configura esta posibilidad lo cual también debe analizarse reflexionando sobre las posibilidades de la ciberdemocracia: si es posible que todos los diputados voten desde su casa… ¿cuál sería el siguiente paso? Y todo ello sin contar con otros problemas adicionales como los que han surgido con ocasión de la aplicación del artículo 155 de la Constitución en Cataluña y el planteamiento del posible voto electrónico de los diputados que se encuentran fuera de España o están sometidos a prisión provisional. En fin, todas estas cuestiones nos hacen pensar en las posibles consecuencias de la extensión creciente de este sistema de votación. Sin embargo, no parece ser un motivo de preocupación para los grupos parlamentarios que el 19 de septiembre de 2017 presentaron una Proposición de reforma suscrita conjuntamente por todos ellos para modificar el artículo 82 del Reglamento en materia de voto telemático por la que se extiende la posibilidad de la emisión del voto por este sistema “a los miembros de las Delegaciones Permanentes de las Cortes Generales en Asambleas Parlamentarias Internacionales cuando la participación en sus actividades oficiales les impide la asistencia a la votación en una sesión plenaria, siempre que dicha participación haya sido autorizada por la Mesa”. (BOCG. Congreso de los Diputados. Núm. 161-1, de 29 de septiembre de 2017). Sin embargo, en todo este tiempo tal Proposición de reforma no ha sido sometida al trámite de toma en consideración por el Pleno. 2. Reforma global En la III Legislatura la Propuesta de un texto articulado completo llegó a ser informada por la Ponencia de la Comisión de Reglamento y en la IV Legislatura se presentó otra propuesta similar que fue dictaminada por dicha Comisión en marzo de 1993. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 73 Tras una interrupción durante la V Legislatura, en la VI se reanudaron los trabajos y se constituyó una Ponencia encargada de elaborar un proyecto de Reforma del Reglamento del Congreso sobre la base de los anteriores trabajos, especialmente de los concluidos en 1993 como consecuencia de la disolución anticipada de la Cámara. Dicha Ponencia llegó a elaborar dos textos provisionales, aunque no pudo culminar su Informe. No obstante, sus deliberaciones y acuerdos sirvieron de punto de partida para el Grupo de Trabajo constituido en el seno de la Comisión de Reglamento al comienzo de la VII Legislatura. Este Grupo tuvo numerosas reuniones a puerta cerrada durante los años 2000 y 2001 y culminó su tarea con la aprobación de un texto provisional. Sin embargo, los avances se interrumpieron sin que hubiese lugar al nombramiento de una Ponencia encargada de emitir Informe sobre la propuesta de los Grupos. Esto no obstante, la existencia de algunos acuerdos sobre determinados aspectos puntuales llevó a la incorporación de los mismos a la práctica vigente como sucedió con: – – – Las interpelaciones urgentes y su debate en Pleno, del que se excluye desde entonces y por acuerdo de la Junta de Portavoces ya asentado como criterio general, el turno de fijación de posiciones del resto de grupos parlamentarios distintos a aquel del que proceda la interpelación que establece el artículo 183.2 lo cual agiliza el debate y lo sitúa entre el Gobierno y el interpelante. Asimismo la agilización de la tramitación de las mociones consecuencia de interpelación y de las proposiciones de ley en Pleno para las que se fijan turnos más breves (7 minutos el autor y 5 los enmendantes y los grupos que fijan posición). Las comparecencias durante la tramitación del Proyecto de Ley de Presupuestos Generales del Estado que originalmente se desarrollaban todas ante la Comisión de Presupuestos (como se sigue haciendo en el Senado) y ahora se distribuyen entre todas las Comisiones sectoriales por Departamentos y entidades públicas, Asuntos Exteriores, Justicia, Defensa, etc. En ciertos casos, además, dieron lugar a la aprobación de Resoluciones de la Presidencia, como la de 18 de junio de 1996, sobre desarrollo del artículo 188 del Reglamento, relativo a preguntas para respuesta oral Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 74 CARLOS GUTIÉRREZ VICÉN en Pleno, que incorpora la posibilidad de formular preguntas sobre asuntos de especial actualidad y fija el cupo de preguntas que corresponden a cada grupo. Y la de 26 de junio del mismo año, sobre el procedimiento de creación y reglas de funcionamiento de las Subcomisiones en el seno de las Comisiones de la Cámara que pone algo de orden en esta materia habida cuenta de su proliferación y atribuye la misma composición que las Ponencias en defecto de fijación de otra composición distinta, así como la necesidad de que se aprueben por el Pleno y por la Mesa, en su caso, el debate en Pleno de sus informes. En otras ocasiones el acuerdo se tradujo en la aprobación de reformas parciales concretas que plasmaban el consenso existente. Es el caso de la reforma del artículo 205 del Reglamento aprobada el 27 de junio de 2001 antes mencionado, y de la Resolución de la Presidencia del Congreso de los Diputados, de 25 de mayo de 2000, relativa a la intervención de la Cámara en el nombramiento de autoridades del Estado. Ambas reflejan el acuerdo básico existente entre los Grupos Parlamentarios en la materia regulada por su Título XII, relativo a las propuestas de nombramiento y designación de personas. Así se creó un órgano parlamentario de naturaleza consultiva, la Comisión Consultiva de Nombramientos que, integrada por representantes de los distintos Grupos parlamentarios, está encargada de informar al Pleno acerca de la idoneidad de las personas propuestas para acceder a los diferentes cargos previa comparecencia ante la Comisión, si se estima conveniente, de los candidatos. Algo parecido sucedió en la VIII Legislatura en la que, sobre la base del trabajo efectuado en pasadas legislaturas y, en particular, el texto provisional aprobado en 2001, el Grupo de Trabajo designado en la Comisión de Reglamento celebró doce reuniones entre el 2 de junio y el 14 de octubre de 2004, estableciendo los principios y líneas generales del texto que serviría para iniciar de nuevo las discusiones (por ejemplo, sobre el procedimiento legislativo, la reducción de tiempos en las intervenciones, etc.) Tras la conclusión de esta fase se convocó a la Comisión de Reglamento el 15 de noviembre de 2004 para proceder a la distribución y conocimiento por los Grupos Parlamentarios del Borrador de Propuesta Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 75 de Reforma del Reglamento del Congreso de los Diputados, elaborado por los Letrados de la Comisión a partir de los criterios adoptados en el Grupo de Trabajo. El texto estaba acompañado de una Propuesta de Normas de Funcionamiento cuyo objetivo era incorporar soluciones concretas a algunos problemas planteados por la práctica parlamentaria y que no encuentran acomodo en una norma de carácter general y mayor grado de abstracción como es el Reglamento de la Cámara. De esta forma en las Normas de Funcionamiento se incorporaban numerosos usos, precedentes y acuerdos de la Mesa y de la Junta de Portavoces que han servido para regular distintos aspectos de funcionamiento del Congreso en materias prácticas tan importantes como: – – – La elaboración del orden del día del Pleno (incluyendo el régimen de cupo, las reglas para su modificación, etc.). La ordenación de los debates de cada tipo de iniciativas de que conoce el Pleno. La convocatoria de las sesiones, o la tramitación de las preguntas orales en la Comisión de Control de RTVE, hoy en día Comisión Mixta de Control Parlamentario de la Corporación RTVE y sus Sociedades. Dada su naturaleza y la mayor facilidad de aprobación y modificación, estas Normas podrían servir para regular con mayor flexibilidad cualesquiera otras cuestiones no resueltas en el Reglamento. La Comisión acordó designar una Ponencia y abrir un plazo para la presentación de enmiendas por los Grupos Parlamentarios. Tras la presentación de las enmiendas y propuestas por parte de los Grupos Parlamentarios, la Ponencia celebró un total de 13 reuniones entre los días 24 de febrero y 10 de noviembre de 2004. Durante las mismas se fue avanzando en la adopción de acuerdos sobre la incorporación de las modificaciones, sin perjuicio del derecho a mantener vivas aquéllas enmiendas o propuestas que no fuesen incorporadas por parte de los Grupos autores de las mismas y sosteniendo, asimismo, el principio de que tan sólo podría hablarse de un acuerdo final, cuando se adoptase un acuerdo global sobre el conjunto del texto. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 76 CARLOS GUTIÉRREZ VICÉN De las reuniones celebradas en el Parador Nacional de Sigüenza (Guadalajara) surgió una propuesta en la que se incorporaban las enmiendas que habían recibido el apoyo mayoritario de los miembros de la Ponencia, dejando de lado algunas otras cuestiones concretas sobre las que no había sido posible llegar a acuerdos como, señaladamente, la cuestión del uso de las lenguas cooficiales distintas del castellano en el Congreso de los Diputados. Dicha propuesta abarcaba tanto al Reglamento del Congreso como al texto completo de las Normas de Funcionamiento que lo completaban. Posteriormente, la Ponencia se reunió en cuatro ocasiones más entre los días 21 de noviembre de 2006 y el 12 de septiembre de 2007 para debatir sobre la posibilidad de seguir avanzando en la reforma global del Reglamento del Congreso de los Diputados o, por el contrario, trabajar en orden a la aprobación de un conjunto de reformas parciales para abordar una serie amplia de cuestiones que se plantearon con la aprobación de nuevas leyes y que no habían sido tenidas en cuenta en la reforma de conjunto. Sin embargo, la Ponencia hubo de concluir sus trabajos constatando que, por el momento, no se daba el grado de acuerdo suficiente para emprender un proceso de reformas de esta naturaleza. No obstante, también en este caso se produjeron algunas modificaciones puntuales como consecuencia del estudio de estos aspectos susceptibles de reforma parcial en los que había, más o menos, coincidencia entre todos los Grupos parlamentarios. En esta línea habría que considerar la Resolución de la Presidencia del Congreso de los Diputados sobre Secretos Oficiales de 11 de mayo de 2004 que permitía ampliar el acceso a las materias clasificadas a todos los Grupos Parlamentarios. De otra parte, algunas de las innovaciones que se pretendían introducir en el Reglamento se incorporaron, de hecho, a la práctica parlamentaria por la vía de acuerdos de la Junta de Portavoces, como sucedió según vimos con las interpelaciones urgentes y su debate en Pleno; con la tramitación de las mociones consecuencia de interpelación (en relación con el debate de ambas iniciativas, la Junta de Portavoces, en su reunión del día 9 de mayo de 2001, acordó reducir el número y la duración de las intervenciones previstas en ambos debates, conforme a lo acordado en el Grupo de trabajo para la reforma del Reglamento). De manera similar, la Junta de Portavoces, por acuerdo de 21 de junio de 2005 acordó reducir el tiempo de las intervenciones en la tramitación de las Proposiciones no de Ley, tomando como Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 77 ejemplo los anteriores y a propuesta, asimismo, del Grupo de trabajo para el estudio de la Reforma del Reglamento. El texto mencionado de 2005 se ha tomado como punto de partida en posteriores intentos de reforma global por considerar que suponía un alto grado de acuerdo entre los grupos en cuestiones básicas como simplificación del procedimiento legislativo. Ha sido conocido después como “Propuesta Marín” por el empeño que puso este Presidente en sacarla adelante. Sin embargo, y sin perjuicio de que la excusa formal de su fracaso fuese la insistencia de las fuerzas nacionalistas en la admisión del uso de las lenguas distintas del castellano que tuvieran el carácter de cooficiales en alguna Comunidad Autónoma, lo cierto es que los dos grandes partidos nunca parecieron confiar en serio en que la reforma llegase a buen puerto y la mejor prueba de ello es que no hubo iniciativas de grupos parlamentarios como tales (proposiciones de reforma del Reglamento registradas) ni Informe de Ponencia que se pudiese elevar a la Comisión, sino que todos los esfuerzos se hicieron en grupos de trabajos de naturaleza informal. A la vista de lo anterior, en la IX Legislatura pareció haberse renunciado temporalmente al objetivo de la aprobación de una reforma global, sin que dejaran de aprobarse también modificaciones parciales del Reglamento sobre aspectos en los que había unanimidad como las citadas reformas del artículo 63.2 en materia de publicidad de los dictámenes de la Comisión del Estatuto de los Diputados sobre incompatibilidades en 2009, y de los artículos 79 y 82 sobre voto mediante procedimiento telemático en los casos de embarazo, maternidad, paternidad o enfermedad grave, del año 2011. Y en esta línea también se aprobaron algunas normas supletorias como la Resolución de la Presidencia del Congreso de los Diputados, de 10 de junio de 2008, sobre desarrollo del artículo 188 del Reglamento, relativo a preguntas para respuesta oral en Pleno que derogaba la anteriormente mencionada y que ha sido modificada por última vez el 15 de noviembre de 2016, fijando el número máximo de preguntas en 24: 9 del Grupo Parlamentario Popular, 6 del Grupo Parlamentario Socialista, 4 del Grupo Parlamentario Confederal Unidos Podemos-En Comú Podem-En Marea, 2 del Grupo Parlamentario Ciudadanos y 1 de cada uno del resto de grupos parlamentarios; la Resolución de las Mesas del Congreso de los Diputados y del Senado, reunidas en sesión conjunta de 26 de mayo de 2008, sobre composición de las Comisiones Mixtas Congreso-Senado; o el Acuerdo de Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 78 CARLOS GUTIÉRREZ VICÉN las “Mesas del Congreso de los Diputados y del Senado en reunión conjunta” de 21 de diciembre de 2009, por el que se aprobaban nuevas normas en materia de Registro de intereses. Asimismo debe mencionarse la creación de la Oficina Presupuestaria de las Cortes Generales por la Ley 37/2010, de 15 de noviembre, cuya composición y funcionamiento se regula por la Resolución de las Mesas del Congreso de los Diputados y del Senado, reunidas conjuntamente, de 19 de julio de 2011. Durante la X Legislatura el Pleno del Congreso de los Diputados, en su sesión de 29 de noviembre de 2012, con motivo del debate de la Moción consecuencia de interpelación urgente del Grupo Parlamentarios de IU, ICV-EUiA, CHA: La Izquierda Plural, acordó lo siguiente: El Congreso de los Diputados: 1. Manifiesta su voluntad de encomendar a la Comisión de Reglamento, de conformidad con los cauces reglamentarios oportunos, la puesta en marcha de los trabajos para impulsar, desde el mayor consenso posible, los cambios reglamentarios que contribuyan a mejorar el funcionamiento de la Cámara y a lograr una mayor identificación en su capacidad de representación de los ciudadanos. 2. Adopta el compromiso, desde los principios de transparencia, responsabilidad y fortalecimiento institucional, de impulsar y debatir, en su seno, medidas que incidan en la profundización y regeneración democrática tomando como base de discusión el informe que, a estos efectos, ha encargado el Gobierno al Centro de Estudios Políticos y Constitucionales. Asimismo, en el debate de política general sobre el estado de la Nación celebrado en 2013, se aprobaron (el 26 de febrero de 2013) distintas resoluciones presentadas por los Grupos Parlamentarios. Entre ellas, la Resolución Número 1, referente a Regeneración democrática y transparencia que incluía la previsión de estudiar en las correspondientes Comisiones parlamentarias los siguientes aspectos: I. Medidas oportunas a adoptar para regular las organizaciones de intereses o “lobbies”, con medidas que clarifiquen cuáles pueden ser sus actividades y cuáles deben ser sus límites. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 79 II. Forma de aplicación a Diputados y Senadores de las normas de transparencia incluidas en la futura Ley Reguladora del Ejercicio de las Funciones Políticas, estableciendo un procedimiento concreto de publicidad de las declaraciones tributarias, de actividades y bienes. III. Reformas necesarias para modernizar las campañas electorales (sondeos, tratamiento por los medios, debates e incluso duración). IV. Nuevas medidas a adoptar contra el transfuguismo político. V. Posibles reformas de la Ley Orgánica reguladora de la Iniciativa Legislativa Popular para facilitar su impulso y tramitación. VI. Posibles reformas de la Ley Orgánica del Régimen Electoral General para mejorar el procedimiento de votación de los ciudadanos españoles residentes en el exterior. VII. La puesta en marcha inmediata de la Oficina Presupuestaria de las Cortes Generales. La Ponencia encargada de la reforma en la X Legislatura se constituyó el 24 de abril de 2013 y celebró un total de nueve reuniones que finalizaron con la del 17 de diciembre de 2014 con la constatación de que no parecía posible avanzar en las negociaciones en busca de consenso. En las distintas reuniones se acordó trabajar sobre la base del texto de 2005 agrupando los temas tratados e introduciendo algunos nuevos. Así por ejemplo: – – – En cuanto al Estatuto del Diputado además de las cuestiones relativas al protocolo y la situación al cese y la suspensión y pérdida de la condición del diputado, el suplicatorio, el juramento y sus efectos, se presentaron nuevas propuestas respecto a la relación del diputado individual con el grupo, su derecho a la información, los derechos económicos, los deberes de asistencia, incompatibilidades y ética del cargo, declaraciones y Registro de intereses o incompatibilidades. Por lo que se refiere a los grupos parlamentarios, se mantuvieron los temas del número de portavoces, el Reglamento interno, el funcionamiento del Grupo Mixto, y el acceso a la información de Registros Públicos. Y además se incluyeron cuestiones como los requisitos de constitución y los diputados no adscritos. Respecto a la Mesa, se incorporaron temas como los relativos a la función de calificación de los escritos y documentos de índole parlaRevista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 80 – – – – CARLOS GUTIÉRREZ VICÉN mentaria que corresponde a este órgano y la cobertura y posibles ceses en la Mesa de la Cámara. En relación con las Comisiones, a los temas anteriormente discutidos se añadió el del funcionamiento general y la programación de los trabajos con reuniones periódicas de las Comisiones. Así como una propuesta de reforzamiento de la Comisión de Peticiones. En el punto relativo a las normas de funcionamiento, se incorporaron materias relativas a publicidad y transparencia; ordenación de los períodos de sesiones, y horarios y días de sesión, fijación del orden del día, regulación de turnos y tiempos de debate, votaciones, plazos, publicidad de actas, seguimiento del cumplimiento de acuerdos, especialmente de las proposiciones no de ley y las mociones por el Gobierno, y los grupos nacionalistas incluyeron un punto relativo al respeto a las minorías. Dentro del procedimiento legislativo, se añadió un cambio completo del procedimiento ordinario, la reforma de las proposiciones de ley y de la iniciativa legislativa popular; el acceso de los ciudadanos al procedimiento, revisión de la delegación en Comisión y del procedimiento de lectura única; cuestiones de técnica legislativa y evaluación de la calidad de las leyes. En materia de control al Gobierno, se plantean nuevas regulaciones de interpelaciones y mociones, la moción de censura o reprobación a los Ministros y la regulación de la intervención de los Ministros en Pleno y Comisión y nuevo régimen completo de las proposiciones no de ley o resoluciones. Se planteó, asimismo, la regulación del debate sobre el estado de la Nación, y se añadió también a la lista de cuestiones a tratar la definición de las delegaciones parlamentarias, las relaciones con la Unión Europea a través de la Comisión Mixta para la Unión Europea; el fomento de una mayor participación ciudadana con la creación de una Oficina del Ciudadano, la regulación de los lobbies y la integración de los medios y nuevas tecnologías, así como se reiteró la cuestión del uso de las lenguas cooficiales y la de la regulación de la autorización de las operaciones de las Fuerzas Armadas. En definitiva, se trabajó sobre la base de que todos los grupos deseaban conseguir un Congreso abierto y transparente, más cercano al ciudadano y más participativo así como más ágil y que elaborase leyes más eficaces16. 16 Un Parlamento abierto en el sentido en el que lo describen R. Rubio Núñez y R. Vela Navarro-Rubio, que aprovechando las facilidades que permiten las nuevas tecnologías Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 81 No se pudo llegar a un texto completo tampoco esta vez. No obstante, se consiguió poner en funcionamiento la Oficina Presupuestaria (Resolución de las Mesas del Congreso de los Diputados y del Senado, de 19 de julio de 2011, por la que se regula la composición y funcionamiento de la Oficina Presupuestaria de las Cortes Generales, creada por la Ley 37/2010, de 15 de noviembre, por la que se crea la Oficina Presupuestaria de las Cortes Generales) y también aprobar las Normas de la Mesa del Congreso de los Diputados, de 20 de enero de 2015, para la aplicación de las disposiciones de la Ley 19/2013, de 9 de diciembre, de transparencia, acceso a la información pública y buen gobierno a la Cámara, en relación con su actividad sujeta a Derecho Administrativo; y abrir el Portal de Transparencia en la página web de la Cámara. En la breve XI Legislatura, además de la solicitud de constitución de una Ponencia encargada de estudiar la reforma del Reglamento presentada por el Grupo Socialista, hay que citar dos proposiciones de reforma parcial. Concretamente: la Proposición de reforma para la inclusión de la Comisión para las Políticas Integrales de la Discapacidad en el artículo 46.1 presentada por el Grupo Parlamentario Mixto (BOCG. Congreso. Núm. B-37, de 15 de abril de 2016) y la Proposición de reforma para la creación del Registro de los Grupos de Interés o Lobbies presentada por el Grupo Parlamentario Catalán (Democràcia i Libertad) (BOCG. Congreso. Núm. B-36-1, de 15 de abril de 2016), que llegó a ser tomada en consideración en la sesión plenaria del 27 de abril de aquel año. Ambas fueron recuperadas en la XII Legislatura, en la que se han presentado un total de diez proposiciones de reforma del Reglamento del Congreso. De ellas dos estaban dirigidas a la adaptación de la enumeración de Comisiones Permanentes Legislativas del artículo 46.1 a la planta ministerial, fueron presentadas por la totalidad de los grupos parlamentarios y incorpore no sólo el principio de transparencia sino también y, de forma especial, los principios de participación y de colaboración de los ciudadanos permitiendo el acercamiento entre representantes y representados y la recuperación por parte de éstos de la confianza en la Institución parlamentaria. Rubio Núñez, R. y Vela Navarro-Rubio, R.: El Parlamento abierto en el mundo, evolución y buenas prácticas. 125 instrumentos de apertura parlamentaria. Fundación Giménez Abad de Estudios Parlamentarios y del Estado Autonómico, Zaragoza, 2017. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 82 CARLOS GUTIÉRREZ VICÉN aprobadas por el procedimiento de lectura única en 2016 y en junio de 2018 como ya quedó reseñado. Otras cuatro tan sido tomadas en consideración y se encuentran en plazo de presentación de enmiendas. En concreto son: – – – La proposición de reforma del Reglamento del Congreso de los Diputados para el control parlamentario de acuerdos parlamentarios sin fuerza de ley, presentada por el Grupo Parlamentario Vasco (EAJ-PNV) con la intención de incluir un nuevo artículo 203 bis que obligase al Gobierno a remitir un informe al principio de cada período de sesiones sobre el grado de cumplimiento de las proposiciones no de ley, mociones y resoluciones aprobadas por el Pleno y las Comisiones, así como a comparecer ante la Comisión de Reglamento para explicar dicho Informe (BOCG. Congreso. Núm. 80-1, de 30 de enero de 2017). La proposición de reforma del Reglamento del Congreso de los Diputados para la regulación de los Grupos de Interés, presentada por el Grupo Parlamentario Popular en el Congreso, que busca añadir un artículo 107 bis en el que se contemple la creación de un Registro Público de carácter obligatorio para la inscripción de los representantes de Grupos de Interés que mantengan encuentros con los Diputados y el personal de los grupos parlamentarios, regulando el contenido de dicho Registro y los datos de la inscripción así como las consecuencias de la infracción de sus previsiones. (BOCG. Congreso. Núm. 96-1, de 3 de marzo de 2017). Es similar a la Proposición de reforma del Reglamento para la creación del Registro de los Grupos de Interés o Lobbies presentada al comienzo de la XII Legislatura por el Grupo Parlamentario Mixto recuperando la iniciativa presentada en la XI Legislatura por el Grupo Parlamentario Catalán de Convergència i d´Unió, aunque regulando de forma más amplia el Código de conducta de los representantes de los grupos de interés, los derechos de acceso y la supresión del Registro. Sin embargo, esta Proposición (BOCG. Congreso. Núm. 28-1, de 9 de septiembre de 2016) no se ha sometido aún al Pleno para su toma en consideración. La Proposición de reforma por la que se modifican los artículos 111 y 126 del Reglamento del Congreso, presentada por el Grupo Parlamentario Confederal Unidos Podemos-En Comú Podem-En Marea (BOCG. Núm. 17-1, de 5 de mayo de 2017) fue tomada en consideración en Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS – – 83 la sesión celebrada el 24 de octubre de 2017 y tenía por finalidad la modificación de dichos preceptos para fijar los límites formales a los que el Gobierno debe atenerse para ejercer la facultad que le atribuyen para oponerse a la tramitación de proposiciones de ley o enmiendas que supongan aumento de los créditos o disminución de los ingresos presupuestarios, todo ello de conformidad con lo previsto en el artículo 134.6 de la Constitución. En el fondo de esta proposición de reforma se encuentra el problema planteado con el uso ciertamente amplio de esta facultad por parte del Gobierno de la primera mitad de la XII Legislatura y el planteamiento de sendos conflictos entre órganos constitucionales ante el Tribunal Constitucional por la negativa de la Mesa de la Cámara a aceptar dos de los llamados “vetos” del Gobierno a las proposiciones de ley orgánica de suspensión del calendario de la implantación de la LOMCE y de modificación del artículo 42.1 del Estatuto de los Trabajadores para garantizar la igualdad en las condiciones laborales de los trabajadores subcontratados presentadas por el Grupo Parlamentario Socialista. Como es sabido, el Tribunal falló a favor del Congreso de los Diputados desestimando el conflicto en las STCs. 34/2018, de 12 de abril de 2018 y 44/2018, de 26 de abril de 2018. Y la Proposición de reforma del Reglamento del Congreso de los Diputados sobre limitación de las prórrogas de plazos de enmiendas a las iniciativas legislativas (BOCG. Congreso. Núm. B-147-1, de 8 de septiembre de 2017) presentada por el Grupo Parlamentario Socialista que fue tomada en consideración en la Sesión Plenaria de día 30 de octubre de 2018. Con esta reforma se pretende incorporar al Reglamento el uso vigente en pasadas legislaturas según el cual las dos primeras ampliaciones del plazo de enmiendas se concedían siempre que lo pidiese algún grupo parlamentario, pero en cambio para las sucesivas ampliaciones se exigía que lo pidan, al menos, dos grupos que representen a la mayoría absoluta de la Cámara. Además, se han presentado dos proposiciones de reforma del Reglamento dirigidas a incluir entre las Comisiones Permanentes Legislativas la Comisión para las Políticas Integrales de la Discapacidad, retomando la iniciativa de la XI Legislatura antes reseñada, y siendo retirada la última de ellas presentada por el Grupo Parlamentario Confederal de Unidos Podemos-En Comú Podem-En Marea, para no duplicar la iniciativa Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 CARLOS GUTIÉRREZ VICÉN 84 presentada con anterioridad por el Grupo Parlamentario Mixto (BOCG. Congreso. Núm. B-50-1, de 21 de octubre de 2016). VI. Conclusiones Del examen de los distintos textos provisionales elaborados durante los trabajos desarrollados en el seno de la Comisión de Reglamento desde la VI a la X Legislatura, así como de los puntos esenciales de acuerdo entre los Grupos Parlamentarios que trascendieron y de las proposiciones de reforma que se han presentado durante la XI y la XII Legislaturas, pueden destacarse las siguientes conclusiones: 1. Existe un primer grupo de modificaciones respecto de las que parece existir un acuerdo básico en el que hay que incluir cuestiones muy diversas que van desde las que afectan a los Grupos Parlamentarios (como la regulación del funcionamiento interno del Grupo Mixto, o la de la figura de los Diputados no inscritos aunque existen divergencias respecto a su plasmación concreta); hasta las referidas al procedimiento legislativo, como la celebración obligatoria del debate de totalidad (inversión de la previsión del artículo 48), la eliminación del debate de enmiendas singulares en Pleno y la nueva regulación del debate presupuestario. Asimismo habría que mencionar, en relación con otros procedimientos, la mejora técnica general de la regulación de las comparecencias, del acceso a secretos oficiales y el control de gastos reservados, el reforzamiento de las facultades investigadoras de la Comisión del Estatuto de los Diputados, o el incremento de las funciones de la Comisión de Peticiones. 2. En cuanto a las reglas generales de funcionamiento, existía un alto grado de consenso sobre cuestiones como la de las reuniones de las Mesas de las Comisiones junto a los Portavoces de los Grupos Parlamentarios, la extensión del principio de publicidad de las sesiones de Comisión, la incorporación de la figura de las resoluciones internas o la regulación del Debate sobre el Estado de la Nación. 3. Mención aparte requerirían aquellas cuestiones relativas al Estatuto de los Diputados, como su retribución, protección social, garantías parlamentarias, regulación de su tratamiento protocolario y de sus deberes y régimen disciplinario respecto de los que se plantea la conveniencia de abordarlos conjuntamente con las que afectarían a los Senadores e, Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 85 incluso, como materia propia de un futuro Reglamento de las Cortes Generales. 4. En esta línea parece existir también consenso en la oportunidad de incluir algunas medidas que incidan en la consecución de un Parlamento más abierto y cercano a los ciudadanos como la introducción de mecanismos de participación y colaboración como la regulación del trámite obligatorio de comparecencias de expertos al principio de la tramitación de las iniciativas legislativas, y la regulación de las relaciones de los diputados y los grupos parlamentarios con los grupos de interés o lobbies. Asimismo existe un amplio grado de acuerdo en la necesidad de incorporar medidas de transparencia y de lucha contra la corrupción en la línea de las recomendaciones del Grupo de Estados contra la Corrupción creado en el seno del Consejo de Europa (GRECO). En este sentido cabe mencionar el inicio de algunos trabajos en el ámbito de la Comisión del Estatuto del Diputado en orden a preparar un borrador de Código ético de conducta de los diputados que pudiera vincularse de alguna manera al Reglamento de la Cámara. 5. Además de estos aspectos existe otra serie de cuestiones en las que tradicionalmente se han centrado las divergencias de más difícil resolución. Entre ellas están los requisitos para la constitución de Grupos Parlamentarios, así como diversos aspectos de la figura de los Diputados no inscritos y de la llamada “firma de complacencia”; la regulación definitiva de las Comisiones de Investigación; la de las preguntas al Presidente del Gobierno en el Pleno; o la posibilidad de incorporar al Reglamento las interpelaciones dirigidas directamente a aquél. Sin embargo, no es necesario decir que las condiciones no parecen las mejores para llegar a un acuerdo que finalice con éxito. Hoy en día la posibilidad de una reforma global del Reglamento del Congreso de los Diputados como otras tantas cosas, una reforma electoral, la reforma constitucional… parece más lejana que nunca. Bibliografía Alba Navarro, M.: “Procedimiento de reforma de los Reglamentos parlamentarios”. Revista de las Cortes Generales, nº. 30, 1993, pp. 7-51. Alborg, J. L.: Historia de la literatura española. Vol. I. Ed. Gredos, 2ª ed. Ampliada. Madrid, 1970. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 86 CARLOS GUTIÉRREZ VICÉN Astarloa Huarte-Mendicoa, I.: El Parlamento moderno. Importancia, descrédito y cambio. Ediciones de El Cronista. Iustel. Madrid, 2017. Escudero, J.A.: Estudio Introductorio a la Teoría de las Cortes. Edición de la Junta General del Principado de Asturias. Oviedo, D.L. 1996. Fernández, J.P.: La década del gran cambio. El camino hacia el cuatripartidismo en España. Biblioteca Nueva, S.L.,Madrid, 2018. Fraga Iribarne, M.: “Los Privilegios de los Procuradores y el nuevo Reglamento de las Cortes Españolas”. Revista de Estudios Políticos, nº 99, 1958, pp. 57-83. Gambra Gutiérrez, A.: Los opositores a la Constitución de Cádiz. En Cortes y Constitución de Cádiz: 200 años. Espasa, Madrid, 2011. Gutiérrez Vicén, C.: “Disposición Final Primera”. En: Comentarios al Reglamento del Congreso de los Diputados, Congreso de los Diputados. Madrid, 2012. Hastschek, J.: Das Parlamentsrecht (Das Parlamentsrecht Des deutschen Reiches). Leipsig, 1915. (Edición inglesa de Walter de Gruyter, 1973). Jefferson, T.: The papers of Thomas Jefferson. Princeton University. Disponible en: https://Jeffersonpapers.princeton.edu. Marco Marco, J.J.: El Reglamento Parlamentario en el Sistema Español de Fuentes del derecho. Cortes Valencianas, Valencia, 2000. Martínez Marina, F.: Teoría de las Cortes o Grandes juntas nacionales de los Reinos de León y Castilla: monumentos de su constitución política y de la soberanía del pueblo: con algunas observaciones sobre la lei fundamental de la monarquía española sancionada por las Cortes Generales y Extraordinarias, y promulgada en Cádiz a 19 de marzo de 1812. Imp. de Fermín Villalpando, Madrid, 1813. Reglamentos (del Congreso de los Diputados y de las Cortes). Cortes Generales, Secretaría. Madrid, 1977. Ripollés Serrano, M. R. [Coord.]: Constituciones de los 27 Estados miembros de la Unión Europea. Congreso de los Diputados. Madrid, 2010. Ripollés Serrano, M. R.: Los reglamentos parlamentarios en el sistema de fuentes de la Constitución Española de 1978. Dirección General del Servicio Jurídico del Estado. Instituto de Estudios Fiscales, Madrid, 1987. Rodríguez-Zapata Pérez, J.: “Los Reglamentos parlamentarios y su posición en el sistema de fuentes del derecho español”. En: I Jornadas de Derecho Parlamentario. Congreso de los Diputados, Secretaría General, Madrid, 1985. Rubio Núñez, R. y Vela Navarro-Rubio, R.: El Parlamento abierto en el mundo, evolución y buenas prácticas. 125 instrumentos de apertura Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87 LA REFORMA DEL REGLAMENTO DEL CONGRESO DE LOS DIPUTADOS 87 parlamentaria. Fundación Giménez Abad de Estudios Parlamentarios y del Estado Autonómico, Zaragoza, 2017. Torres Del Moral, A.: “Naturaleza jurídica de los Reglamentos parlamentarios”. Revista de la Facultad de Derecho de la Universidad Complutense, nº 10, 1986, pp. 7-22. Revista de las Cortes Generales Nº 104, Segundo Cuatrimestre (2018): pp. 43-87
https://openalex.org/W4303615142	https://vest.rea.ru/jour/article/download/1415/979	Russian	null	Risks Facing Russian Stock Exchange during Sanctions and Ways of their Overcomin	Vestnik Rossijskogo èkonomičeskogo universiteta imeni G. V. Plehanova	2,022	cc-by	3,738	Московский финансово-промышленный университет «Синергия», Москва, Россия Статья посвящена проблемам, стоящим перед российским фондовым рынком в условиях санкций, введен- ных западными странами в отношении организаций и физических лиц Российской Федерации. Серьезные санкционные меры, затронувшие ключевые финансовые институты России, не могли не оказать влияния на фондовый рынок, который находится в турбулентности с конца февраля 2022 г. Для снижения негативного влияния этих мер как правительству, так и регулятору (Центральному банку Российской Федерации) при- ходится реагировать принятием весьма жестких решений, направленных на стабилизацию положения в сфере финансов, среди которых следует отметить резкое увеличение ключевой ставки, закрытие торгов на Московской бирже, ограничения на операции с иностранной валютой, интервенции на фондовом рынке. В статье освещены проблемы, которые повлекли сильные колебания валютного курса на товарных и валют- ных рынках, вызванные также нестабильностью политической ситуации в мире. Авторами проведена оцен- ка перспектив дальнейшего движения рынка и возможных ответных контрсанкционных мер, которые долж- ны быть приняты как российским правительством, так и регулятором для сглаживания негативных послед- ствий в экономике страны. р Ключевые слова: фондовый рынок, санкции, биржа, Банк России, инфляция, курсы валют. ЭКОНО OI: http://dx.doi.org/10.21686/2413-2829-2022-5-5-11 РИСКИ РОССИЙСКОГО ФОНДОВОГО РЫНКА В УСЛОВИЯХ САНКЦИЙ И ПУТИ ИХ ПРЕОДОЛЕНИЯ ЭКОНОМИКА DOI: http://dx.doi.org/10.21686/2413-2829-2022-5-5-11 Keywords: stock exchange, sanctions, exchange, the Bank of Russia, inflation, currency rates. РИСКИ РОССИЙСКОГО ФОНДОВОГО РЫНКА В УСЛОВИЯХ САНКЦИЙ И ПУТИ ИХ ПРЕОДОЛЕНИЯ А. П. Гарнов, Е. В. Афанасьев Российский экономический университет имени Г. В. Плеханова, Москва, Россия RISKS FACING RUSSIAN STOCK EXCHANGE DURING SANCTIONS AND WAYS OF THEIR OVERCOMING Andrey P. Garnov, Evgeniy V. Afanasyev Plekhanov Russian University of Economics, Moscow, Russia Nadezhda P. Tishkina Moscow University for Industry and Finance «Synergy», Moscow, Russia Andrey P. Garnov, Evgeniy V. Afanasyev Plekhanov Russian University of Economics, Moscow, Russia Nadezhda P. Tishkina Moscow University for Industry and Finance «Synergy», Moscow, Russia The article deals with problems facing Russian stock exchange in conditions of sanctions imposed by western states on organizations and individuals of the Russian Federation. They are harmful and affect key financial institutions of Russia, thus they are bound to impact the stock market experiencing turbulence since late February 2022. In order to cut the adverse influence of such measures both the government and the regulator (the Central Bank of the Russian Federation) have to response by tough decisions aimed at stabilizing the situation in finance sphere, among which we should mention a sharp rise in the key interest rate, closing stock trading on the Moscow exchange, restrictions on foreign currency transactions and interventions on stock market. The article highlights problems that caused serious fluctuations of currency rate on commodity and foreign exchange markets brought about by unstable political situation in the world. The authors evaluated prospects of further movements of market and possible countermeasures, which should be taken both by the Russian government and the regulator to smooth negative after-effects in country economy. Keywords: stock exchange, sanctions, exchange, the Bank of Russia, inflation, currency rates. 5 Вестник РЭУ им. Г. В. Плеханова ● 2022 ● Том 19 ● № 5 (125) В ысокие темпы экономического роста, а также ускорение инфляции под действием временных и устойчивых факторов наряду с долгосрочными струк- турными тенденциями определяли вектор развития российских финансовых рынков в 2021 г. При этом российская экономика выросла на 4,7% после снижения в 2020 г. на 2,7%. ВВП достиг докоронавирусного уровня к середине года. Рост как россий- В ской, так и мировой экономики в 2021 г. оказался существенно выше прогнозов конца 2020 – начала 2021 г.: предприятия и население адаптировались к изменившим- ся условиям быстрее, чем ожидалось. Фондовый рынок является чутким ба- рометром состояния экономики. Поэтому в первых трех кварталах 2021 г. индекс МосБиржи рос довольно быстрыми темпа- ми (рис. 1). Рис. 1. Индекс МосБиржи в 2021–2022 гг. ММВБ – Индекс МосБиржи Рис. 1. Индекс МосБиржи в 2021–2022 гг. Источник: URL: https://ru.investing.com/indices/mcx Также следует отметить увеличение по- пулярности фондового рынка в целом в 2021 г. Число зарегистрированных физиче- ских лиц, несмотря на разворот ключевой ставки и рост доходностей депозитов, уве- личилось [4]. Однако отечественный фон- довый рынок к концу 2021 г. вступил в фа- зу серьезной коррекции, за счет которой потерял около половины всего роста, слу- чившегося за первые три квартала. Andrey P. Garnov, Evgeniy V. Afanasyev Plekhanov Russian University of Economics, Moscow, Russia Nadezhda P. Tishkina Moscow University for Industry and Finance «Synergy», Moscow, Russia Во мно- гом это было обусловлено периодически появлявшимися в информационных кана- лах сведениями о подготовке в рядах Рос- сийской армии специальной военной опе- рации. Тем не менее панических настрое- ний на рынке практически не отмечалось, хотя инвесторы-нерезиденты стабильно продавали акции российских компаний на сумму около 100 млрд рублей в месяц, что в итоге привело к смене ценового тренда на нисходящий. После осложнения политической ситу- ации в конце февраля 2022 г. произошло резкое падение цен российских активов. Индекс МосБиржи на эту дату вернулся к значениям 2015 г., снизившись почти в 2 раза. В то же время в отношении ряда лиц и финансовых институтов России были не- медленно введены санкции, ограничива- ющие или полностью исключающие про- 6 Гарнов А. П. и др. Риски российского фондового рынка в условиях санкций и пути их преодоления Гарнов А. П. и др. Риски российского фондового рынка в условиях санкций и пути их преодоления ведение ключевых операций с валютой и ценными бумагами. по сути, блокировку, находятся наиболее крупные системообразующие банки, такие как Сбербанк, ВТБ, Альфа-Банк и др. Учи- тывая, что именно банки являются глав- ными участниками фондового рынка, можно понять, что санкционные меры имеют серьезные последствия для даль- нейшего обращения капитала в России. В настоящее время практически ни од- но государство в мире не существует изо- лированно от других, между ними посто- янно происходят экономические, полити- ческие, культурные взаимодействия, ведет- ся обмен. Внешняя политика Российской Федерации предполагает экспорт товаров за рубеж, международную торговлю, при- влечение иностранных инвестиций и ис- пользование различных зарубежных фи- нансовых и других учреждений. Эта по- требность используется правительствами США и европейских стран как рычаг дав- ления. Практически сразу было приостановле- но сотрудничество санкционных банков с международными платежными системами Visa и Mastercard. Планируется отключе- ние Сбербанка, Московского кредитного банка и Россельхозбанка от платежной си- стемы SWIFT (в составе шестого пакета санкций). В марте Евросоюз уже отключил от SWIFT семь российских банков: ВТБ, «Россия», «Открытие», Новикомбанк, Промсвязьбанк, Совкомбанк и ВЭБ. В конце февраля – начале марта 2022 г. CША, Великобритания, страны Европей- ского союза разработали новые санкцион- ные меры, направленные на то, чтобы нанести еще более масштабный ущерб российской экономике, в первую очередь оборонной индустрии. По сути, эти новые меры во многом продолжаются и ужесто- чают секторальный санкционный режим, введенный администрацией Б. Обамы [5]. Дочерние компании банков также по- падают под санкции. Особенно пострада- ли клиенты брокеров банков из списка SDN: они не могут покупать и продавать иностранные акции. Andrey P. Garnov, Evgeniy V. Afanasyev Plekhanov Russian University of Economics, Moscow, Russia Nadezhda P. Tishkina Moscow University for Industry and Finance «Synergy», Moscow, Russia Брокеры под санкци- ями бесплатно переводят такие бумаги к свободным от ограничений брокерам, но выбор клиентам не предоставили. При этом в феврале – марте 2022 г. мно- гие участники рынка ожидали, что уже к лету – осени этого года санкционное дав- ление будет уменьшено, вследствие чего рынок постепенно, хоть и не сразу, придет в нормальное положение. Теперь таких иллюзий у большинства участников рынка больше нет: нам предстоит непростой путь перенастройки отечественной экономики в условиях новой реальности. Ситуация, сложившаяся на фондовом рынке 24–25 февраля, требовала быстрого реагирования от регулятора для предот- вращения еще более тяжких последствий для экономики. Поэтому Центральным банком Российской Федерации было при- нято решение о приостановке торгов на Московской бирже и существенных огра- ничениях для Санкт-Петербургской бир- жи. Разумеется, такое решение было срод- ни тому, как тяжелобольного вводят в ис- кусственную кому, чтобы дать организму силы и время справиться с недугом. Мож- но сказать, что Банк России временно пе- ревел российский фондовый рынок на полностью ручное управление. Быстрыми темпами происходит дегло- бализация финансовых рынков, начавшая- ся еще во время кризиса 2008 г. Самым важным индикатором финансовой глоба- лизации, как верно отмечают Е. В. Семен- кова и Е. Н. Власова, была возможность для компаний привлекать финансовый капи- тал по всему миру путем листинга соб- ственных акций на нескольких мировых биржах [3]. При этом также с 28 февраля 2022 г. был установлен запрет на продажу акций рос- сийских компаний нерезидентами Россий- ской Федерации. Это достаточно жесткий запрет, однако он был продиктован острой необходимостью: нерезидентам на данный Так, в 2022 г. под санкции попали 20 российских банков. При этом под самы- ми жесткими санкциями, означающими, 7 7 Вестник РЭУ им. Г. В. Плеханова ● 2022 ● Том 19 ● № 5 (125) не получить планируемую доходность, а то и вовсе остаться в убытке. момент по различным оценкам принадле- жит от 60 до 70% всех российских акций ведущих компаний, находящихся в сво- бодном обращении. Повышение ставок и проблемы в бан- ковском секторе уже отражаются на стои- мости жилой и коммерческой недвижимо- сти. Вложить капиталы в объекты станет значительно сложнее, существенно подо- рожают и ипотечные кредиты. Московская биржа открылась лишь спу- стя месяц с начала упомянутых событий, 24 марта. Торги открывались постепенно с постоянным мониторингом ситуации во- круг различных видов активов. Возможен рост стоимости активов то- варного рынка. Andrey P. Garnov, Evgeniy V. Afanasyev Plekhanov Russian University of Economics, Moscow, Russia Nadezhda P. Tishkina Moscow University for Industry and Finance «Synergy», Moscow, Russia Так, например, уже после объявления санкций существенно подоро- жал газ на европейских спотовых площад- ках, нефть надежно закрепилась над уров- нем 100 долларов за баррель (рис. 2), отме- чается повышение цен на пшеницу, титан, алюминий, палладий и т. д. Снижение цен облигаций и акций про- исходит не только из-за панических настроений на рынке и распродажи акций нерезидентами. Серьезные изменения, происходящие в российской экономике, требуют переоценки справедливой стои- мости акций российских компаний: какие- то из них лишатся зарубежных рынков сбыта, у других возрастут расходы и, сле- довательно, в санкционных условиях ста- нет меньше уровень прибыли. Пока труд- но строить прогноз относительно восста- новления стоимости компаний и перспек- тив их работы. Рис. 2. Уровень цен на нефть в феврале – мае 2022 г. Источник: URL: https://yandex.ru/news/quotes/1006.html В связи с финансовыми ограничениями нарушаются и товарные потоки – импорт зарубежных товаров и экспорт отечествен- ных. Это неизбежно вызовет рост цен и, как следствие, разгон инфляции. В резуль- тате неизбежны просадки фондового рын- ка. Дополнительными факторами здесь станут продажи иностранных инвесторов, открытие коротких позиций на опасениях ужесточения санкций. И хотя просадки могут быть весьма значительными, кризис, вероятно, будет преодолен за счет мер поддержки от Банка России и государ- ственного бюджета. Рис. 2. Уровень цен на нефть в феврале – мае 2022 г. Источник: URL: https://yandex.ru/news/quotes/1006.html Источник: URL: https://yandex.ru/news/quotes/1006.html Это, естественно, скажется на котиров- ках срочных контрактов, увеличит их во- латильность и, соответственно, уровень рисков для инвесторов (особенно с учетом доступной технологии маржинальных сделок с гарантийным обеспечением). Перспективы роста инфляции вынуди- ли Центробанк поднять ключевую ставку до 20% годовых. В результате существенно подорожают заемные средства для компа- ний, что затормозит развитие бизнеса и станет еще одним фактором для развития негативных сценариев на фондовом рын- ке. Рост ставки отразился и на стоимости облигаций: для обеспечения доходности цены долговых бумаг существенно снизи- лись. В результате инвесторы, желающие продать облигации до погашения, могут Высокая волатильность на всех рынках вынуждает Московскую и Санкт- Петербургскую биржи изменять расписа- ние торгов, ограничивать проведение ряда операций и задействовать другие рыноч- ные предохранители (перевод торгов в ре- жим дискретного аукциона, приостановку их и пр.). Это может обернуться для инве- 8 Гарнов А. П. и др. Риски российского фондового рынка в условиях санкций и пути их преодоления сторов как недополученной прибылью, так и реальными потерями. Банка России 28 февраля 2022 г. с 9,5 до 20%. В настоящее время ставка последова- тельно снижена сначала до 17%, а затем до 14% (рис. 3). Для поддержания курсов отечественных акций из Фонда национальной безопасно- сти был выделен 1 трлн рублей для осу- ществления интервенции на рынке в виде покупки государством акций наиболее уязвимых компаний. Таким способом уда- лось в краткосрочном периоде поддержать фондовый рынок, при этом не следует ис- ключать возможность для государства за- работать на разнице курсов, когда они вос- становятся. Можно сказать, что эти меры имели определенный эффект. Курс доллара США в настоящее время держится на от- метках ниже 70 рублей, что благоприятно для покупательной способности в отноше- нии товаров иностранного производства. Однако с точки зрения макроэкономики крепкий рубль снижает конкурентоспо- собность отечественной промышленности. Причина роста курса рубля по-прежнему в существенном превышении предложения валюты над спросом. В качестве меры, способной нивелиро- вать вред от отказа в сотрудничестве со стороны Visa и Mastercard, рассматривался переход к использованию китайской пла- тежной системы UnionPay. О данной си- стеме известны следующие факты: Рис. 3. Динамика ключевой ставки Центрального банка Российской Федерации в 2022 г. Источник: URL: https://cbr.ru/hd_base/KeyRate/ − она была учреждена по решению Народного банка Китая и Госсовета в 2000 г. и в 2005 г. стала международным сервисом; − она была учреждена по решению Народного банка Китая и Госсовета в 2000 г. и в 2005 г. стала международным сервисом; − система занимает 1-е место по коли- честву выпущенных пластиковых карт в мире; − пользоваться картами системы UnionPay можно более чем в 180 странах мира; Рис. 3. Динамика ключевой ставки Центрального банка Российской Федерации в 2022 г. − пластиковые карты этой системы вы- пускают более чем в 70 странах мира; − в России система работает офици- ально с осени 2013 г. через дочернюю ком- панию, зарегистрированную в стране, – ООО «Юнион Пэй». Большинство аналитиков объясняют сложившуюся ситуацию тем, что россий- ский экспорт превышает импорт, поэтому валютная выручка экспортеров давит на валютный рынок. Экспортеры продают валюты больше, чем покупают импортеры, в результате доллар падает. Вместе с тем по последней информации «Сбер», ВТБ, Альфа-Банк, «Открытие» и другие подсанкционные банки не смогут выпускать карты UnionPay: китайская пла- тежная система опасается сотрудничать с ними из-за риска вторичных санкций. По мнению ряда экспертов, к осени сле- дует ожидать падения курса рубля по от- ношению к основным мировым валютам. Этому будут способствовать введенные США и Европейским союзом эмбарго на некоторые экспортные отрасли, смягчение ограничений Центрального банка Россий- ской Федерации и частичное восстановле- ние импорта. Для поддержания курса национальной валюты по отношению к доллару США и евро были введены такие меры, как обяза- тельная продажа части валютной выручки компаниями-экспортерами; комиссия по приобретению валюты для физических и юридических лиц, одномоментное резкое повышение ставки рефинансирования 9 Вестник РЭУ им. Г. В. Плеханова ● 2022 ● Том 19 ● № 5 (125) Вестник РЭУ им. Г. В. Плеханова ● 2022 ● Том 19 ● № 5 (125) Таким образом, существует еще доволь- но большая потребность в принятии контрсанкционных мер как правитель- ством, так и Банком России в качестве ре- гулятора. В частности, звучат предложения разрешить негосударственным пенсион- ным фондам держать больше активов в акциях российских компаний, а также ин- вестировать в них до 10% средств Пенси- онного фонда Российской Федерации. Предлагаются меры стимулирования частных инвесторов, например, отмена налогообложения купонного и дисконтно- го дохода по облигациям, сохранение ин- вестиционного налогового вычета по ин- дивидуальному инвестиционному счету. Следует подвести итог и обозначить ос- новные перспективы, на которых хотелось бы остановить внимание. 1. Негативное влияние политической ситуации на экономику России сохраняет- ся (по некоторым данным – усугубляется). Пока нет ясной перспективы урегулирова- ния текущей ситуации на политической арене. 2. Планируется ввод шестого пакета санкций в отношении банковской сферы Российской Федерации. В него включат по- этапный запрет импорта нефти и отключе- ние от SWIFT Сбербанка (и еще двух круп- ных российских банков). Санкционное дав- ление нарастает, что не может не оказывать влияния и на фондовый рынок. При этом эмбарго на нефть – довольно чувствитель- ная мера для экономики России. Несомненно, следует согласиться с мне- нием А. Е. Абрамова, А. Г. Косырева, А. Д. Радыгина, М. И. Черновой, что в но- вых условиях поддержание стабильности на рынке акций российских компаний ста- новится столь же значимой задачей, как и обеспечение стабильности банковской си- стемы, макроэкономических параметров, в том числе целевого уровня инфляции и прогнозируемого валютного курса [1]. р 3. Курс доллара США колеблется на бирже на уровне 55–60 рублей, что, оче- видно, давит на наш рынок. Экспортеры будут чувствовать себя не очень хорошо с таким курсом. 4. Есть сомнения по возможной выплате дивидендов некоторыми российскими компаниями, которые также сильно по- страдали от введенных санкций. В частно- сти это касается таких крупных компаний, как «Газпром», «Лукойл», Сбербанк и ряда других, стабильно выплачивавших до 2022 г. сравнительно высокие доходы сво- им акционерам. Такое положение не мо- жет не повлиять негативно на цену акций. 4. Есть сомнения по возможной выплате дивидендов некоторыми российскими компаниями, которые также сильно по- страдали от введенных санкций. В частно- сти это касается таких крупных компаний, как «Газпром», «Лукойл», Сбербанк и ряда других, стабильно выплачивавших до 2022 г. сравнительно высокие доходы сво- им акционерам. Такое положение не мо- жет не повлиять негативно на цену акций. Вместе с тем следует учесть, что по- настоящему положительное влияние как на фондовый рынок, так и в целом на рос- сийскую экономику может оказать лишь глобальное изменение политической ситу- ации в положительную сторону, за кото- рым последуют постепенный отказ от санкционного давления и восстановление нарушенных экономических связей. References 1. Abramov A. E., Kosyrev A. G., Radygin A. D., Chernova M. I. Rossiyskiy rynok aktsiy v 2021 g. i v nachale 2022 g. [The Russian Stock Market in 2021 and at the Beginning of 2022]. Ekonomicheskoe razvitie Rossii [Economic Development of Russia], 2022, No. 1, pp. 36–42. (In Russ.). 1. Abramov A. E., Kosyrev A. G., Radygin A. D., Chernova M. I. Rossiyskiy rynok aktsiy v 2021 g. i v nachale 2022 g. [The Russian Stock Market in 2021 and at the Beginning of 2022]. Ekonomicheskoe razvitie Rossii [Economic Development of Russia], 2022, No. 1, pp. 36–42. (In Russ.). ( ) 2. Rossiyskiy rynok aktsiy k kontsu goda rasteryal polovinu rosta [The Russian Stock Market Lost Half of its Growth by the End of the Year]. (In Russ.). Available at: https://www.interfax.ru/business/813531 2. Rossiyskiy rynok aktsiy k kontsu goda rasteryal polovinu rosta [The Russian Stock Market Lost Half of its Growth by the End of the Year]. (In Russ.). Available at: https://www.interfax.ru/business/813531 p 3. Semenkova E. V., Vlasova E. N. Prichiny, faktory i posledstviya deglobalizatsii dlya rossiyskogo fondovogo rynka [Causes, Factors and Consequences of Globalization for the Russian Stock Market]. Izvestiya vysshikh uchebnykh zavedeniy. Seriya: Ekonomika, finansy i upravlenie proizvodstvom [News of Higher Educational Institutions. Series: Economics, Finance and Production Management], 2022, No. 1 (51), pp. 26–31. (In Russ.). p 3. Semenkova E. V., Vlasova E. N. Prichiny, faktory i posledstviya deglobalizatsii dlya rossiyskogo fondovogo rynka [Causes, Factors and Consequences of Globalization for the Russian Stock Market]. Izvestiya vysshikh uchebnykh zavedeniy. Seriya: Ekonomika, finansy i upravlenie proizvodstvom [News of Higher Educational Institutions. Series: Economics, Finance and Production Management], 2022, No. 1 (51), pp. 26–31. (In Russ.). 4. Chalenko D. D., Mballa Nomo Zh. F. Vozdeystvie makroekonomicheskikh faktorov na strukturu i dinamiku fondovogo rynka Rossii: institutsionalnye aspekty [The Impact of Macroeconomic Factors on the Structure and Dynamics of the Russian Stock Market: Institutional Aspects]. Finansovye rynki i banki [Financial Markets and Banks], 2022, No. 3, pp. 82–85. (In Russ.). 5. Ellen M.-I. Ukrainskiy konflikt i sanktsii Zapada. Instrument globalnoy ekspansii [The Ukrainian Conflict and Western Sanctions. A Tool of Global Expansion]. Svobodnaya mysl [Free Thought], 2022, No. 2 (1692), pp. 35–48. (In Russ.). 5. Ellen M.-I. Ukrainskiy konflikt i sanktsii Zapada. Instrument globalnoy ekspansii [The Ukrainian Conflict and Western Sanctions. A Tool of Global Expansion]. Svobodnaya mysl [Free Thought], 2022, No. 2 (1692), pp. 35–48. (In Russ.). Гарнов А. П. и др. Риски российского фондового рынка в условиях санкций и пути их преодоления Гарнов А. П. и др. Риски российского фондового рынка в условиях санкций и пути их преодоления References Андрей Петрович Гарнов Андрей Петрович Гарнов доктор экономических наук, профессор, профессор кафедры мировых финансовых рынков и финтеха РЭУ им. Г. В. Плеханова. Адрес: ФГБОУ ВО «Российский экономический университет имени Г. В. Плеханова», 117997, Москва, Стремянный пер., д. 36. E-mail: profgarnov@yandex.ru Андрей Петрович Гарнов доктор экономических наук, профессор, профессор кафедры мировых финансовых рынков и финтеха РЭУ им. Г. В. Плеханова. Адрес: ФГБОУ ВО «Российский экономический университет имени Г. В. Плеханова», 117997, Москва, Стремянный пер., д. 36. E-mail: profgarnov@yandex.ru Evgeniy V. Afanasyev Doctor of Economics, Professor of the Department for World Financial Markets and Fintech of the PRUE. Address: Plekhanov Russian University of Economics, 36 Stremyanny Lane, Moscow, 117997, Russian Federation. E-mail: e.v.afanasiev@mail.ru Евгений Васильевич Афанасьев доктор экономических наук, профессор кафедры мировых финансовых рынков и финтеха РЭУ им. Г. В. Плеханова. Адрес: ФГБОУ ВО «Российский экономический университет имени Г. В. Плеханова», 117997, Москва, Стремянный пер., д. 36. E-mail: e.v.afanasiev@mail.ru Список литературы 1. Абрамов А. Е., Косырев А. Г., Радыгин А. Д., Чернова М. И. Российский рынок акций в 2021 г. и в начале 2022 г. // Экономическое развитие России. – 2022. – № 1. – С. 36–42. 2. Российский рынок акций к концу года растерял половину роста. – URL: https://www interfax ru/business/813531 1. Абрамов А. Е., Косырев А. Г., Радыгин А. Д., Чернова М. И. Российский рынок акций в 2021 г. и в начале 2022 г. // Экономическое развитие России. – 2022. – № 1. – С. 36–42. 2. Российский рынок акций к концу года растерял половину роста. – URL: https://www.interfax.ru/business/813531 1. Абрамов А. Е., Косырев А. Г., Радыгин А. Д., Чернова М. И. Российский рынок акций в 2021 г. и в начале 2022 г. // Экономическое развитие России. – 2022. – № 1. – С. 36–42. 3. Семенкова Е. В., Власова Е. Н. Причины, факторы и последствия деглобализации для российского фондового рынка // Известия высших учебных заведений. Серия: Эконо- мика, финансы и управление производством. – 2022. – № 1 (51). – С. 26–31. 3. Семенкова Е. В., Власова Е. Н. Причины, факторы и последствия деглобализации для российского фондового рынка // Известия высших учебных заведений. Серия: Эконо- мика, финансы и управление производством. – 2022. – № 1 (51). – С. 26–31. у 4. Чаленко Д. Д., Мбалла Номо Ж. Ф. Воздействие макроэкономических факторов на структуру и динамику фондового рынка России: институциональные аспекты // Фи- нансовые рынки и банки. – 2022. – № 3. – С. 82–85. р 5. Эллен М.-И. Украинский конфликт и санкции Запада. Инструмент глобальной экс- пансии // Свободная мысль. – 2022. – № 2 (1692). – С. 35–48. р 5. Эллен М.-И. Украинский конфликт и санкции Запада. Инструмент глобальной экс- пансии // Свободная мысль. – 2022. – № 2 (1692). – С. 35–48. 10 Сведения об авторах Andrei P. Garnov Doctor of Economics, Professor, Professor of the Department for World Financial Markets and Fintech of the PRUE. Address: Plekhanov Russian University of Economics, 36 Stremyanny Lane, Moscow, 117997, Russian Federation. E-mail: profgarnov@yandex.ru Nadezhda P. Tishkina д д кандидат экономических наук, доцент кафедры управления человеческими ресурсами Университета «Синергия». Адрес: Негосударственное образовательное частное учреждение высшего образования «Московский финансово-промышленный университет "Синергия"», 105318, Москва, ул. Измайловский вал, д. 2. E-mail: tnadezd@yandex.ru 11
https://openalex.org/W4243401838	https://zenodo.org/record/1527888/files/article.pdf	English	null	The Delta Residence of the Ramessides	The journal of Egyptian archaeology/Journal of Egyptian Archaeology	1,918	public-domain	28,947	The Delta Residence of the Ramessides Author(s): Alan H. Gardiner Source: The Journal of Egyptian Archaeology, Vol. 5, No. 4 (Oct., 1918), pp. 242-271 Published by: Egypt Exploration Society Stable URL: http://www.jstor.org/stable/3853579 Accessed: 26-05-2016 10:11 UTC Egypt Exploration Society is collaborating with JSTOR to digitize, preserve and extend access to The Journal of Egyptian Archaeology 2 Recueil de Travaux, vol. xxxi, pp. 113 120, with full publication of the inscriptions of the obelisk. 3 So in the great Edfu geographical text, BRUGSCH, Dictionnaire Geographique, p. 1366; in the Edfu mythological text, ibid., 1388; in a Philae text, BRUGSCH, Geographische Inschriften, vol. in, P1. V; in two texts at Dendereh, DUMICHEN, Geographische Inschriften, vol. iii, P1. XXIII; op. cit., vol. I, P1. XCIX, 1. 21; and doubtless elsewhere. 4 Tuthmosis III, see SETHE, Urkunden, iv, 647; Sethos I, see LEPsIUS, Denkmdler, Part III, P1. 126 a, and above all P1. 128 b; Ramnesses II, see above p. 179, under no. 2. Egypt Exploration Society The Delta Residence of the Ramessides Author(s): Alan H. Gardiner Source: The Journal of Egyptian Archaeology, Vol. 5, No. 4 (Oct., 1918), pp. 242-271 Published by: Egypt Exploration Society Stable URL: http://www.jstor.org/stable/3853579 Accessed: 26-05-2016 10:11 UTC The Delta Residence of the Ramessides Author(s): Alan H. Gardiner Source: The Journal of Egyptian Archaeology, Vol. 5, No. 4 (Oct., 1918), pp. 242-271 Published by: Egypt Exploration Society Stable URL: http://www.jstor.org/stable/3853579 Accessed: 26-05-2016 10:11 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Egypt Exploration Society is collaborating with JSTOR to digitize, preserve and extend access to The Journal of Egyptian Archaeology Egypt Exploration Society is collaborating with JSTOR to digitize, preserve and extend access to The Journal of Egyptian Archaeology This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 242 1 PETRIE, Nebesheh and Defenneh, pp. 96 foll., and Plate LI. IV HERE at last we come to grips with the problem to which, in a sense, all the foregoing investigations have been prefatory-the localization of Pi-Rarmesse, the Delta Residence of the Pharaohs of the Nineteenth and early Twentieth Dynasties. of the Pharaohs of the Nineteenth and early Twentieth Dynasties. The discussion commenced under no. 39 of the last section provides us with a good point of departure. The researches of recent years have made it practically certain that ff^r ' \ Thel, the metropolis of the XIVth nome of ( 4 --.- Khant-yeb, was situated, not at Tanis, not at Tanis, but at Tell Abu Sefeh, an ancient site some 1 miles east of El- Kantareh. Mr Griffith, who in 1886 studied the neighbourhood of El-Kantareh with much care', found there part of an obelisk bearing the names of Ramesses I, Sethos I and Ramesses II, which Prisse d'Avennes had seen and published entire; a few years ago M. Cledat discovered the whereabouts of the missing portion and produced documentary evidence showing that the obelisk came from Tell Abu Sefeh (" du cote de l'Asie, vers l'est, a environ quatre kilometres ")2. At Tell Abu Sefeh itself Mr Griffith discovered, obviously in situ or almost so, a base for an image of some sort dedicated by Ramesses II, as well as a Latin inscription from the joint reign of Diocletian and Maximian recording the establish- ment there of a military station. Both the Ramesside monuments mentioned the god Ncc 3 ® "Horus of Mesen," often referred to as the god of the XIVth nome. This suggested the possibility that the place might be the capital of that nome3, namely Thel or, as it is often transcribed, Zaru, where was a fortress which Tuthmosis III, Sethos I and Ramesses II all had to pass when setting forth on their Asiatic campaigns4. Again, the Latin inscription made it appear likely that Tell Abu Sefeh was Selle, a town in the eastern Delta where, according to the Notitia Dignitatum (beginning of the Vth century) a Roman garrison was stationed. Now the Antonine Itinerary mentions a Sile at This suggested the possibility that the place might be the capital of that nome3, namely Thel or, as it is often transcribed, Zaru, where was a fortress which Tuthmosis III, Sethos I and Ramesses II all had to pass when setting forth on their Asiatic campaigns4. PETRIE, Nebesheh and Defenneh, pp. 96 foll., and Plate LI. Recueil de Travaux, vol. xxxi, pp. 113-120, with full publication of the inscriptions of the obeli This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 PETRIE, Nebesheh and Defenneh, pp. 96 foll., and Plate LI. 2 Recueil de Travaux, vol. xxxi, pp. 113-120, with full publication of the inscript IV Again, the Latin inscription made it appear likely that Tell Abu Sefeh was Selle, a town in the eastern Delta where, according to the Notitia Dignitatum (beginning of the Vth century) a Roman garrison was stationed. Now the Antonine Itinerary mentions a Sile at This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms THE DELTA RESIDENCE OF THE RAMESSIDES 243 a distance of 24 Roman miles, equivalent to about 221 English miles, from Pelusium on the road via Serapeum to Clysma (Suez), and this estimate is but a little in excess of the actual distance-some 19 English miles-between Farameh and Tell Abu Sefehl. The identification, thus obtained, of Tell Abu Sefeh with Sile or Selle suits the old Egyptian evidence admirably, for the name written with the signs T'-rw, contains actual distance-some 19 English miles-between Farameh and Tell Abu Sefehl. The identification, thus obtained, of Tell Abu Sefeh with Sile or Selle suits the old Egyptian evidence admirably, for the name written with the signs T'-rw, contains according to the so-called "syllabic" orthography current in the New Kingdom only two consonants that count, and of these t appears as a- in Greek (cf. Tb-ntr ;e/3evvv'ro), while the other consonant r may equally well stand for 1; Thel (or perhaps better Sel) is, accord- ingly, a perfectly legitimate equivalent for the old hieroglyphic name, and the identity of this with Sile, Sele, Selle is apparent. Mr Griffith was, therefore, fillly justified in identifying Tell Abu Sefeh with the Pharaonic fortress of Thel and the Romnan garrison-town of Sile; but he was less happy in the distinction he attempted to draw between the fortress of Thel and the town of the same name that was the metropolis of the XIVth nome. The reason assigned by him for this distinction was the fact that the XIVth nome of Lower Egypt seemed to correspond to Ptolemy's Sethroite nome, of which the capital, Herakleopolis Parva, ought probably, in his opinion, to be looked for at Tell Belim (Tell esh-Sherig)2; the town of Thel would, therefore, be situated at Tell Belim, on the edge of Lake Menzaleh, while the fortress of Thel was at Tell Abu Sefeh. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 4 Pap. Anastasi III, 1, 10. 5 See Zeitschriftfiir dgyptische Sprache, vol. xxvi (1888), p. 81. , p , p 4 Pap. Anastasi III, 1, 10. IV This theory overlooks the fact, of which we find further instances in the case of the Tanite and Arabian nomes, that an old metropolis was apt to be superseded by some other town that had come to surpass it in importance. In the case of the XIVth nome we may perhaps distinguish three successive capitals: Mesen, the home of the nome-god Horus; Thel, the frontier-town and military centre; and finally, in Graeco-Roman times, Herakleopolis Parva. For all we know, Herakleopolis Parva and Mesen may have been identical; they have no direct bearing on the problem before us, and we therefore cannot discuss them further; but there can be but little doubt that Thel, both town and fortress, is Tell Abu Sefeh3. About Thel we may gather the following additional information. It was on the very edge of the desert and almost out- side Egypt; this is indicated by the determinatives 1 accompanying its name in hieroglyphic writing. It was the boundary between Egypt and Syria (Khal), as appears from the epithet "Royal envoy [to the...] countries of Khal from Thel to Yupe4." Again, in Haremhab's time criminals were sent thither, docked of their noses, just as in later times they were sent to 'PtvoKopovpa, a place that owed its name to that barbarous form of punishment5. On the other hand, Thel lay on the border of the cultivation, for it pos- side Egypt; this is indicated by the determinatives 1 accompanying its name in hieroglyphic writing. It was the boundary between Egypt and Syria (Khal), as appears from the epithet "Royal envoy [to the...] countries of Khal from Thel to Yupe4." Again, in Haremhab's time criminals were sent thither, docked of their noses, just as in later times they were sent to 'PtvoKopovpa, a place that owed its name to that barbarous form of punishment5. On the other hand, Thel lay on the border of the cultivation, for it pos- 1 For the evidence as to Sile see KUTHMANN, Die Ostgrenze Aegyptens, pp. 5, 38-40. The distance from Clysma to Serapeum, according to the Antonine Itinerary, was 50 miles; the remainder of the road to Pelusium is given as follows: Iter a Serapiu Pelusio: Thaubasio . . . . . . . . Sile . . . . . . . . . . Magdolo . . . . . . . . . Pelusio m.p. 1 For the evidence as to Sile see KUTHMANN, Die Ostgrenze Aegyptens, pp. 5, 38-40. The distance from Clysma to Serapeum, according to the Antonine Itinerary, was 50 miles; the remainder of the road to Pelusium is given as follows: p 3 So KtTHMANN, op. cit., pp. 38-9. IV VIII ,, XXVIII XII XII Iter a Serapiu Pelusio: Thaubasio . . . . . . . . Sile . . . . . . . . . . Magdolo . . . . . . . . . Pelusio . . . . . . . . . 2 PETRIE N b h h d D f h 103 f t t 2 m.p. VIII ,, XXVIII XII XII 2 PETRIE, Nebesheh and Defenneh, p. 103, footnote 2. 3 So KtTHMANN, op. cit., pp. 38-9. 4 Pap. Anastasi III, 1, 10. 5 See Zeitschriftfiir dgyptische Sprache, vol. xxvi (1888), p. 81. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms ATLAN H. GARDINER 244 sessed vineyards whence wine was sent to the Theban Ramesseuml; and it was accessible by water2, probably by a canal which later, at least if local tradition, backed by some slight classical authority, can be trusted, extended to Ostracine, now El-Flusiyeh at the east end of Lake Sirbonis3. We must not forget to mention here the interesting sculptured scene on the north wall of Karnak, where the fortress of Thel is depicted as it was in the time of Sethos I4. In conclusion, reference must be made to some late sarcophagi recently found at Tell Abu Sefeh5; not only do their inscriptions mention Thel, but they also contain ample testimony as to the priestly and divine names connected with the XIVth nome. It is desirable for our purpose to form some idea as to the extent and boundaries of that nome. Its south-western end, Mr Griffith reasonably supposes, may have lain between Tell Defenneh (Daphnae) and El-Menagi, since at the latter place a block of Nektanebos was found on which some words are addressed to Buto of [ - 6~~ 'Imt; 'Imt is Neb8sheh, the capital of the XIXth nome of _ 'Imt-pehu "the lower 'Imt-nome," some six miles from El-Menagi'. The natural boundary between the XIVth and XIXth nomes would be the Pelusiac or Bubastite branch of the Nile, which, according to Mr Griffith, "running W. of Herakleopolis, seems to have turned to the N. IV a little before reaching Defenneh." Ptolemy makes the Nile the dividing-line between the nomes; between the Busiric and Bubastite river, he says, were (from north to south) the nome of Nesyt with its metropolis Panephysis, the Tanite nome with its metropolis Tanis, and It is desirable for our purpose to form some idea as to the extent and boundaries of that nome. Its south-western end, Mr Griffith reasonably supposes, may have lain between Tell Defenneh (Daphnae) and El-Menagi, since at the latter place a block of Nektanebos was found on which some words are addressed to Buto of [ - 6~~ 'Imt; 'Imt is Neb8sheh, the capital of the XIXth nome of _ 'Imt-pehu "the lower 'Imt-nome," more recent excavations (Recueil de Travaux, vol. xxxviii, pp. 21-32) produced no inscriptions. 6 The following bibliographical notes may be useful. Brugsch at first (Geographische Inschriften, vol. I, pp. 262-3) identified Thel with Heroonpolis and placed it at the mouth of the Wady Tfmilat; later, as we have seen, he changed his view and located it at Tanis (see especially Dictionnaire Geo- graphique, pp. 992-7, 1356), J. de Roug6 following suit (Geographie de la Basse-Žgypte, p. 95). Diimichen, however, laid great stress on a passage in the Edfu texts relating to the myth of Horus, and hence sought to show that Thel lay near Ismailtyeh (Geschichte Aegyptens, pp. 257-60); though he himself subsequently modified his view, placing the town somewhat further north (Zur Geographie des altens Aegyptens, p. 33); this opinion was retained by German scholars until quite recently (e.g. STEINDORFF, Die dgyptischen Gaue, p. 6, footnote 2, in Abhandl. d. phil.-hist. Klasse d. kon. Sacs. Ges. d. iss., vol. xxvii, vol. 1909), when it was disposed of by Kiithmann (op. cit., p. 49). MAX MULLER, Proceedings of the Society of Biblical Archaeo- logy, vol. x, pp. 467-77, ignores Griffith, but correctly remarks that Thel would be best located " where the great road from the East crosses the narrowest part of the water between the Birket BallAh and the lake of Menzaleh"; his further suggestion (p. 478) that Thel is identical with Shuiir, Gk. zovp, of the Bible, is less fortunate. To complete the documentation of Thel a few references that do not occur elsewhere in this article may here be added: "split in-fishes of Thel," Pap. 1 SPIEGELBERG, Hieratic Ostraca, nos. 163, 189, 203, 211. 2 See above, p. 132, under the letter H. 3 Annales du Service, vol. xvi, p. 8. 4 LEPSIUS, Denzkmdler, Part III, P1. 128, b. 5 Annales du Service, vol. xii, pp. 69-76; Bulletin de l'lnstitut Franfais, vol. xi, pp. 29-38. Still more recent excavations (Recueil de Travaux, vol. xxxviii, pp. 21-32) produced no inscriptions. 6 The following bibliographical notes may be useful. Brugsch at first (Geographische Inschriften, vol. I, pp. 262-3) identified Thel with Heroonpolis and placed it at the mouth of the Wady Tfmilat; later, as we have seen, he changed his view and located it at Tanis (see especially Dictionnaire Geo- graphique, pp. 992-7, 1356), J. de Roug6 following suit (Geographie de la Basse-Žgypte, p. 95). Diimichen, however, laid great stress on a passage in the Edfu texts relating to the myth of Horus, and hence sought to show that Thel lay near Ismailtyeh (Geschichte Aegyptens, pp. 257-60); though he himself subsequently modified his view, placing the town somewhat further north (Zur Geographie des altens Aegyptens, p. 33); this opinion was retained by German scholars until quite recently (e.g. STEINDORFF, Die dgyptischen Gaue, p. 6, footnote 2, in Abhandl. d. phil.-hist. Klasse d. kon. Sacs. Ges. d. iss., vol. xxvii, vol. 1909), when it was disposed of by Kiithmann (op. cit., p. 49). MAX MULLER, Proceedings of the Society of Biblical Archaeo- logy, vol. x, pp. 467-77, ignores Griffith, but correctly remarks that Thel would be best located " where the great road from the East crosses the narrowest part of the water between the Birket BallAh and the lake of Menzaleh"; his further suggestion (p. 478) that Thel is identical with Shuiir, Gk. zovp, of the Bible, is less fortunate. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 SPIEGELBERG, Hieratic Ostraca, nos. 163, 189, 203, 211. , , 2 See above, p. 132, under the letter H man of Thel, see SPIEGELBERG, Correspondances des Temps des Ro 7 PETRIE, Nebesheh and Defenneh, P1. XLIII, and pp. 46, 107. , p , 3 Annales du Service, vol. xvi IV Anastasi IV, 15, 7; the fortress of Thel is named in the well-known journal of a frontier-official, Pap. Anastasi III, verso 3,5; at Serabit el-KhAdim was found the stele of a mayor of Thel from the reign of Tuthmosis IV, see GARDINER-PEET, Inscriptions of Sinai, vol. I, P1. XIX, no. 59, and a stele apparently of the same man is now in Leyden (V43, see BOESER, Denknmaler des Neuen Reichs, Part iII, P1. XIII, no. 22). There also occurs the personal name Tlry, doubtless meaning " a man of Thel " see SPIEGELBERG Correspondances des Temps des Rois-Prgtres Index s v t'h Sketch Map of the EASTERN DELTA Reconstructed physical details are marked in red. The Roman numnbers in red are those of the Egyptian nomes, to twhich the Graeco-Roman names are attached. 2·9 4./.- 4 zS' TANIS · - , TANIS e-,a San el-Hagarj XIX. HE rTA Nebshehe ' - Me I# I Sledllll ' ° PHARBAETHUS Horbet ¢ ,0 ,. $2 # - 1 " Pi- Ba(r)st """ J BUBASTUS, Tell Basteh -" XVlIII. Pi-' S a "" ? ',^, , c "T'r c'. ARABIA?THt.OU9? -ASTITES Saftel-Henineli 0 6 . ,o0\\1 Tell Abu Sulmn;n"' ." 0 Belbes,' - kh e t a e I1 d - o f - Z o a nJ IRACLEOPOLIS PARVA? el// esh-Sher^g, Tell Beln is NIE.S T A7gi , " : , , -. [ " ^ - l ihiyeh 0t ^ gPHACUSA, Fakus ARABIA? ' , ' XX. AR^ A' "IA '5-? o ....? ,,,, ^ -..,,,,,,,,..,,,, ; o4 ... 4 e g' x,, A< , \i f, Ix,\9P, 8 ',,,,. % \! ,,,.~t,,I t,,, : . .... -PI ' English Miles o 5 o10 I . ,. , 15 I o IoI Kilometres 20 Roman Miles ... I5 I. I Egyptian names Classical , . Arabic ,, .- ----- - ' - MAGDOLON ? , ,~ ..7 .... ... . "i- Tell el-Her i-i: -. XIV, S E'¥H K. 7 Ml Abu Sefeh ^f - LAKE BALLAH y -? 3 o0 45' 1 32 o3 PLATE XXXV ,5 3' 4S ES L- road f, c-i- . 1- K , 1 s 'I-, - Af ' k I 4, UlB 30 3O ,. .».' Isnmaili'yeh ...'.o ..""". I VIII. ,. J ' ,.,,,N.- .,....,,,,%D,l ,1- {, ,\M«\ \l, \\s, . Y -.c^ ' P1 ." ",",,,,.,,.,,,,,... ....... , , p 4 Prof. Naville (Goshen, pp. 14 foll.) contends that Phacfisa must be the modern Saft el-Henneh, since Strabo (p. 805) states that the canal running from the Nile to the Red Sea branches off from the river at Phacusa; this, says Prof. Naville, would be a physical impossibility if Phacusa were the modern Fakfis. It seems to me easier to suppose that Strabo has confused the earlier and later capitals of the nome than that Fakfis should not be the modern equivalent of Phacuisa. The capital of the nome seems to have been called Arabia in late classical times; see KUTHMANN, op. cit., pp. 9-10. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 The name of the Pharbaethite nome survives in the modern place-name Horb6t, which possibly marks the site of the capital Svdnw, the cult-place of Har-merti; see DE RoUGA, Geographie de la Basse- Egypte, pp. 66 foll., and Annales du Service, Index to vols. I-x, p. 29, s.v. Horbelt. The nome was a late creation, which took in some lists the place of the old nome of 1sb (XI), though this was probably situated in a quite different part of the Delta. IV the Pharbaethite nome with its metropolis Pharbaethosl; to the east of the Bubastite river were the Sethroite nome with its metropolis Herakleopolis Parva, the nome of Arabia with its mnetropolis Phacusa, the Bubastite nome with its metropolis Bubastus2, and the Heliopolitan nome with its metropolis Oniou. Doubtless this account is too schematic even for Graeco-Roman times, and there is the additional difficulty that the old Egyptian nomes cannot be legitimately equated in all details with those recorded by the classical writers. Still, if it is roughly permissible to identify the XIVth nome with the Sethroites of Ptolemy, then surely we must go further and identify the XIXth nome with Ptolemy's Tanites. As the pivot on which the former identification revolved was the localization of Thel at Tell Abu Sefeh, so that on which the latter identification turns is the localization of 'Imt at Nebesheh. This was sufficiently proved by Professor Petrie's excavations in 1886, and the same scholar was amply justified in explaining3 the Greek namne of the nome as due to the fact that Tanis was a city of more recent origin which in course of time had come to overshadow the old religious centre of 'Imt. So too, probably, Phakuisa (Fafkuis) later took the place of Pi-Sapt (Saft el-Henneh) as the capital of the XXth nome of Arabia4, and we have seen that the Sethroite nome may have possessed three successive capitals. p It thus appears that Brugsch, in identifying the XIVth nome with the Tanites and the XIXth with the Sethroites, exactly reversed the truth. Some modern writers on the subject, like Professor Naville5, seem clear on the point that the XIVth is the Sethroite nome, but an Egyptologist as well acquainted with Delta geography as M. Daressy still maintains that though Tell Abu Sefeh was the earlier capital of the nome, later it was eclipsed in importance by Tanis, which then took its place as the metropolis6. How improbable a view this is may be seen from a mere inspection of the map; Tanis is some ten miles to the north-west of Nebesheh, the capital of the XIXth nome, while Tell Abu Sefeh is about thirty miles due east of the latter place. If there is no very definite q p 2 Bubastus is the form of the place-name preferred by almost all the classical geographers except Herodotus, in contradistinction to the goddess Bubastis. to have been called Arabia in late classical times; see KUTHMANN, op. cit., pp. 9 10. 5 Proceedings of the Society of Biblical Archaeology, vol. xxxiv (1912), p. 310. The proposal there made to identify Thel with the Zoar of Genesis xix, 20 is phonetically quite impossible. IV I"'l, SERAPEI Gebel Map LAjE TIMSA H live ,/4 15 20 3° 40 30, 30 _F,, SERAPEUM? ''(Descr. de 1' Eg.) BITTER . LA KES .TANIS ..Belbes I. t , "I I I 'oc, I -- - e I .._ "II. I I . I . . I I ............I . I ...I _/f PHAR BA ET , TE S: - I 20 I 'J ,, .1!;, :'." /Sff I PLATE XXXV I Sketch Map of the EASTERN DELTA Reconstructed physical details are marked in red. The Roman numnbers in red are those of the Egyptian nomes, to twhich the Graeco-Roman names are attached. LAjE TIMSA H This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms THE DELTA RESIDENCE OF THE RAMESSIDES 245 the Pharbaethite nome with its metropolis Pharbaethosl; to the east of the Bubastite river were the Sethroite nome with its metropolis Herakleopolis Parva, the nome of Arabia with its mnetropolis Phacusa, the Bubastite nome with its metropolis Bubastus2, and the Heliopolitan nome with its metropolis Oniou. Doubtless this account is too schematic even for Graeco-Roman times, and there is the additional difficulty that the old Egyptian nomes cannot be legitimately equated in all details with those recorded by the classical writers. Still, if it is roughly permissible to identify the XIVth nome with the Sethroites of Ptolemy, then surely we must go further and identify the XIXth nome with Ptolemy's Tanites. As the pivot on which the former identification revolved was the localization of Thel at Tell Abu Sefeh, so that on which the latter identification turns is the localization of 'Imt at Nebesheh. This was sufficiently proved by Professor Petrie's excavations in 1886, and the same scholar was amply justified in explaining3 the Greek namne of the nome as due to the fact that Tanis was a city of more recent origin which in course of time had come to overshadow the old religious centre of 'Imt. So too, probably, Phakuisa (Fafkuis) later took the place of Pi-Sapt (Saft el-Henneh) as the capital of the XXth nome of Arabia4, and we have seen that the Sethroite nome may have possessed three successive capitals. PETRIE, Nebesheh and Defennek, p. 6. made to identify Thel with the Zoar of Genesis xix 6 Bulletin de l'lnstitut Franfais, vol. xi, p. 35. q p 2 Bubastus is the form of the place-name preferred by almost all the classical geographers except Herodotus, in contradistinction to the goddess Bubastis. The prototypes of the Tanite and Bubastite nome together originally formed the large nome of 'Imt, part of which was to the right and part to the left of the Bubastite Nile-arm. At an early date the nome of 'Imt seems to have been divided into two, known respectively as the Upper and Lower 'Imt-nomes ('Imt-khant, 'Imt-pehu). In Graeco-Roman timnes the Pharbaethite norne may have been cut partly out of the early Bubastite nomne ('Imt-khant); see last note. 1 The name of the Pharbaethite nome survives in the modern place-name Horb6t, which possibly marks the site of the capital Svdnw, the cult-place of Har-merti; see DE RoUGA, Geographie de la Basse- Egypte, pp. 66 foll., and Annales du Service, Index to vols. I-x, p. 29, s.v. Horbelt. The nome was a late creation, which took in some lists the place of the old nome of 1sb (XI), though this was probably situated in a quite different part of the Delta. 2 Bubastus is the form of the place-name preferred by almost all the classical geographers except Herodotus, in contradistinction to the goddess Bubastis. The prototypes of the Tanite and Bubastite nome together originally formed the large nome of 'Imt, part of which was to the right and part to the left of the Bubastite Nile-arm. At an early date the nome of 'Imt seems to have been divided into two, known respectively as the Upper and Lower 'Imt-nomes ('Imt-khant, 'Imt-pehu). In Graeco-Roman timnes the Pharbaethite norne may have been cut partly out of the early Bubastite nomne ('Imt-khant); see last note. PETRIE, Nebesheh and Defennek, p. 6. 4 Prof. Naville (Goshen, pp. 14 foll.) contends that Phacfisa must be the modern Saft el-Henneh, since Strabo (p. 805) states that the canal running from the Nile to the Red Sea branches off from the river at Phacusa; this, says Prof. Naville, would be a physical impossibility if Phacusa were the modern Fakfis. It seems to me easier to suppose that Strabo has confused the earlier and later capitals of the nome than that Fakfis should not be the modern equivalent of Phacuisa. The capital of the nome seems to have been called Arabia in late classical times; see KUTHMANN, op. cit., pp. 9-10. 5 Proceedings of the Society of Biblical Archaeology, vol. xxxiv (1912), p. 310. The proposal there made to identify Thel with the Zoar of Genesis xix, 20 is phonetically quite impossible. 6 Bulletin de l'lnstitut Franfais, vol. xi, p. 35. made to identify Thel with the Zoar of Genesis xix, 20 is phonetically quite impossible. 6 Bulletin de l'lnstitut Franfais, vol. xi, p. 35. see too Annales du Service, vol. nII, p. 27 3 BRUGSCH, op. cit., pp. 1369, 1389. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms IV The prototypes of the Tanite and Bubastite nome together originally formed the large nome of 'Imt, part of which was to the right and part to the left of the Bubastite Nile-arm. At an early date the nome of 'Imt seems to have been divided into two, known respectively as the Upper and Lower 'Imt-nomes ('Imt-khant, 'Imt-pehu). In Graeco-Roman timnes the Pharbaethite norne may have been cut partly out of the early Bubastite nomne ('Imt-khant); see last note ALAN H. GARDINER 246 evidence beyond its proximity-this indeed would seem enough-to prove that Tanis was in the same nome as Nebesheh, there is no really valid argument to show that it belonged to the same nome as Thel. Since Brugsch's error still appears to exercise considerable influence, and since a decision on this point has an important bearing upon the problem of the position of Pi-Racmesse, his arguments for identifying the XIVth nome with the Tanites must receive very careful examination. y Let us remember that Brugsch, when compiling the Dictionnaire Geographique, had as yet no means of locating 'Imt, the capital of the XIXth nome; nor does he seem ever to have realized the import of Professor Petrie's researches at Nebesheh, since in his latest utterance (1891) on these geographical problems he still clings to his conjecture that 'Imt signified "the mud-city" (Coptic ozue, lutum) and hence was to be equated with Pelusiuml. We must take the Dictionnaire Geographique as the clearest expression of Brugsch's opinions. Handicapped by his ignorance of the true whereabouts of 'Imt, he would naturally attach the greater importance to his apparent evidence for the identity of Thel with Tanis. This evidence appears to consist of the following points: (1) > . \ (a Sht-DCnt " Field-of-Zacanet," which is obviously identical with the "Field-of-Zoan" (i.e. Field of Tanis) of the Bible, occurs on several occasions in place of the more usual ,000 ~ Sht-Dc "Field-of-ZaC," which is the pehu or " Hinterland " of the XIVth nome; (2) monuments have been found at Tanis which appear to connect that town with Thel (Sile), because they bear upon them the name either of the town itself or of its gods. ( ), y p g (1) The first point does indeed seem to be a formidable one, and requires the most careful consideration. 3 BRUGSCH, op. cit., pp. 1369, 1389. 4 For .Hbyt, the modern Bekbet, see particularly ROEDER, Zeitschrift fur dgyptische Sprache, vol. XLVI (1909), pp. 62 foil. 1 BRUGSCH, Dictionnaire Geographique, p. 1373. A few other references, DUMICHEN, Geographische Intschriften, vol. I, P1. XIV (Edfu); P1. XXVIII (Dendereh); vol. iv, P1. CXX (Dendereh); GRIFFITH- PETRIE, Two Hieroglyphic Papyri, PI. X, frag. 18. 2 See Bulletin de l'Institut Franvais, vol. xi, p. 36, for a full bibliography of this monument. 3 Recueil de Travaux, vol. xv, p. 150. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms ETRIE, Two Hieroglyphic Papyri, PI. X, frag. 18. 2 See Bulletin de l'Institut Franvais, vol. xi, p. 36, for a full bibliography of this monument. 3 Recueil de Travaux, vol. xv, p. 150. 1 BRUGSCH, Dictionnaire Geographique, p. 1373. A few other references, DUMICHEN, Geographische Intschriften, vol. I, P1. XIV (Edfu); P1. XXVIII (Dendereh); vol. iv, P1. CXX (Dendereh); GRIFFITH- PETRIE, Two Hieroglyphic Papyri, PI. X, frag. 18. Intschriften, vol. I, P1. XIV (Edfu); P1. XXVIII (Dendereh); vol. iv, P1. CXX (Dendereh); GRIFFITH PETRIE, Two Hieroglyphic Papyri, PI. X, frag. 18. 2 See Bulletin de l'Institut Franvais, vol. xi, p. 36, for a full bibliography of this monument. 3 Recueil de Travaux, vol. xv, p. 150. IV The resemblance of "Field-of-ZaCanet" to the W^-lnw "Field-of-Zoan," n-X8iov Tave'ws, TcoC)ige 1i1e&&", TROI RiX&InH of Psalm 78 was far too striking to be fortuitous, so that Brugsch could conclude with some show of reason that the nome to which " Field-of-Za'," "Field-of-ZaCanet" belonged was the nome of Tanis. That "Field-of-ZaCanet" is no mere mistake on the part of a scribe is shown by the facts that a Ptolemaic stele in Cairo2 couples the goddess C ~ ©} "Nebhotpe in the territory of RaCnufer, arising in Field-of-Zacanet" with the god of the fourteenth nome " Horus of Mesen, the noble flying scarabaeus, protecting the two lands, great god of Thel," and that a third instance of the expression " Field-of-Zacanet" occurs on a late statue pub- lished by M. Daressy3 in a more or less close association with Thel. The text inscribed on the statue is very obscure, but it is somehow narrated of its owner, the general Amenpiom, that his business was to superintend the irrigation of the north-eastern nomes: · @ ^ ° ^^c nnn^ 5,o '^ [l @ ^ <=, the king appointed him as the governor over the province (?) of Field-of-ZaCanet in order to pre- vent the oppression of ..... in their oppressions (?)." Then follow some extremely unintelli- gible sentences, the text of which, according to M. Daressy, runs as follows: <o=>N_ that his business was to superintend the irrigation of the north eastern nomes @ ^ ° ^^c nnn^ 5,o '^ [l @ ^ <=, the king appointed him as the governor over the province (?) of Field-of-ZaCanet in order to pre- vent the oppression of ..... in their oppressions (?)." Then follow some extremely unintelli- gible sentences, the text of which, according to M. Daressy, runs as follows: <o=>N g g y I I I ,a (l A r 'K 0 = 31 = 1 l 1 -" ^^I>: ww - ^AAAA---H--TT.T A ^ V>? / 00^ ^ <^> H H fl^^-^31^^___ 0 ___^A AAAAM w /ww^s W\ -^ Avesv^/ ]\^nO °UUU -3V>~)X ^It is <=>/^won^TJE , fv,/~ 1 © , ~ o 1s / , ,/ , difficult to believe that the published text is quite correct, though its obscurity may well be due in some part to the ancient sculptor. IV The name Q "Field-of-ZaC " occurs first of all amid a series of representations of inundated districts and the like depicted upon a rlined wall of the temple at Memphis which Ramesses II dedicated to Ptah2. The adjacent names are: (a) I O- Sha'usef, the flooded land of the IXth, Busirite, nome; (b) = e © Sha'mentet, a designation unknown from any other source, which might, however, mean the land flooded by the waters V mnwt of the XVth Hermopolite nome3; (c) l, roite nome. The general sequence seems to be from west to east along the northern ex- tremity of the Delta. The addresses to Ptah which accompany these personifications of geographical areas are, as a rule, without interest; alone in the case of " Field-of-Za¢ " does the accompanying legend contain references that may be of topographical significance. The address in question runs thus: "Ramesses II comes to thee, 0 Ptah, he brings to thee Field-[of-Z This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms THE DELTA RESIDENCE OF THE RAMESSIDES 247 Ramnesses II gives to thee .........." To these words I shall return later. In Graeco-Roman times "Field-of-ZaC" is constantly named as the pehu of the XIVth nomel; thus in the great Ramnesses II gives to thee .........." To these words I shall return later. In Graeco-Roman times "Field-of-ZaC" is constantly named as the pehu of the XIVth nomel; thus in the great Ramnesses II gives to thee .........." To these words I shall return later. In Graeco-Roman times "Field-of-ZaC" is constantly named as the pehu of the XIVth nomel; thus in the great geographical text of Edfu we read V~ : 7. _> f z D X "he (the nome-god Horus of Mesen) inundates the Boinu-land (Phoenix-land) at (its) season of the year, he pours its fresh water into the pehu of Field-of-ZaC." To repeat what was said under no. 39 of the last section (p. 200) the title f- LI Oss"he who is in the regions of Field-of-ZaCanet" occurs on one of the statues of Teos among titles that point unequivocally to the nome of Khant-yeb (XIV) and not to that of 'Imt-pehu (XIX). 1 See ERMAN, Agyptische Grammatic3, § 236. 2 See H. RANKE, Keilschriftliches Material zur altdgyptischen Vocalisation, p. 34, in Abh. d. ko'n. Preuss. Acad. d. Wiss., 1910, where, however, S a'anu is not properly distinguished from Si'inu, guessed by Prof. Spiegelberg to be Pelusium; see below p. 253. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms IV If then "Field-of-ZaCanet" is to be accepted, like "Field-of-Za,") as a name of the pehu of the XIVth nome, we must face the fact that the nome whose capital was at Tell Abu Sefeh possessed a pehu having its name compounded with the name of Tanis-Zoan. For the identity of "Field-of-ZaCanet" with the Biblical "Field-of-Zoan" is beyond dispute, and the LXX and Coptic equivalents y p , p q prove that the latter contained the name of Tanis; on the other hand \ {3 . 2n \" x-~ r Dent is really the Egyptian name of Tanis, though this fact was until comparatively recently merely a deduction based by Brugsch on the term ijJs D "Field-of-Zacanet." The Annals of Assurbanipal had indeed afforded the Assyrian transcription Sa'anu2, but the 9 A WV\ AM W name w © Dent was actually first found as the name of the city of Tanis in M. Golenischeff's papyrus containing the romantic story of the mission of Unamfin to Phoenicia; here it appears as the seat of government of Smendes, the founder of Manetho's XXIst Tanite government, and as a port where the prince of Dor alone had twenty trading- vessels (1, x + 23). The city-name DCnt-Zacanet has since been encountered in the Golenischeff glossary (above no. 38), on the Stele of the Adoption of Nitokris, and in the Demotic tale of Petubastis. Now since the name DCnt-ZaCanet for Tanis is conspicuously absent from texts of earlier date-at Tanis itself it does not occur at all except in "_Field-of-ZaCanet" on the statue of Teos-it seems likely that it owes its origin to the proximity of the " Field-of- Za¢," which may well be a rather wide expression signifying the region of Lake Menzaleh. How great was the extension of the "Field-of-Za " we do not know; if any importance is to be attached to the words on the Memphite wall addressed to Ptah in connection with it, then we should have to conclude that it covered the whole marshy region lying between the region north of Tell Abu Sefeh and the Phatnitic mouth; the statue of Amenpiom, however, indicates a less extension. IV The following rendering will, however, give some notion of its contents: "in order to punish the deed of him who commits it and who transgresses (??) it, in order to free ([s]sw ?) (it) from ruins (?) which fail to receive mud (i.e. he had to remove brick ruins which prevented the inundation from spreading its mud over the area in question ??) and in order to divide (?) ...... near the ...... of Thel. He made pro- lific the good territories in front of them, Sha'sef (there is an intrusive m, which makes it 248 ATIAN H. GARDINER doubtful whether Sha'usef, the flooded land of the Busirite nome mentioned above, is really meant) being with (?) their waters, and from the territory of the Mendesian nome as far as Field-of-ZaCanet being inundated every [year]." After making all allowances for mistrans- lation, it is clear that the general Amenpiom was charged with the duty of looking after the irrigation of the extreme north, from the Phatnitic mnouth eastward to "Field-of- ZaCanet"; the latter is somehow connected with the name of Thel. These three instances of "Field-of-ZaCanet" appear to me to place its equivalence with "Field-of-ZaC " beyond a doubt. "Field-of-Za¢" appears, so far as our evidence goes, to be the older form ; the later variation can only be explained by taking D as a femi- nine adjective agreeing with sht "field" in place of the old genitive D _d, "Zac." Egyptian does display analogous adjectival formations, cf n. mhnt " the Northern House" and the like; compare the Semitic terminations, Arabic CO-, Hebrew lb-. If then "Field-of-ZaCanet" is to be accepted, like "Field-of-Za,") as a name of the pehu of the XIVth nome, we must face the fact that the nome whose capital was at Tell Abu Sefeh possessed a pehu having its name compounded with the name of Tanis-Zoan. For the identity of "Field-of-ZaCanet" with the Biblical "Field-of-Zoan" is beyond dispute, and the LXX and Coptic equivalents These three instances of "Field-of-ZaCanet" appear to me to place its equivalence with "Field-of-ZaC " beyond a doubt. "Field-of-Za¢" appears, so far as our evidence goes, to be the older form ; the later variation can only be explained by taking D as a femi- does display analogous adjectival formations, cf n. mhnt " the Northern House" and the like; compare the Semitic terminations, Arabic CO-, Hebrew lb-. 1 See ERMAN, Agyptische Grammatic3, § 236. IV In any case, it is necessary to emphasize the fact that because the pehu of a nome has a name that can be associated with a place which This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms THE DELTA RESIDENCE OF THE RAMESSIDES 249 would otherwise be considered to lie outside that nome, this is no reason whatsoever for in- cluding the said place in the norne to which the pehu in question belongs'. We know hardly anything as to the real nature of the pehu; the word seems to mean " Hinterland" or " back-lying district"; in the great Edfu geographical inscription it would appear to signify the region into which the river of the nome pours its waters (see above, p. 247, top). Even this is doubtful, however, for the pehu of the Arabian nome (XX) bears the samine name a i--r A , AAA 'Ity as the "river" of the Heliopolitan nome (XIII) farther upstream2, though \\ AWA this seeming contradiction may be reconciled by supposing that 'Ity was one name of the entire stretch of the Bubastite river from the Heliopolitan nome to below that of Arabia. What is more illuminating for our present argument, the pehu of the IVth nome, that of the Southern Neith, bears the name (iAc CAnz, which is obviously inseparable from the name and the deity of the IXth, the Busirite, nome 3 that lay wholly or partially to the north of the IVth nome. - _ (2) It cannot, therefore, be proved from the term " Field-of-ZaCanet " that ZaCanet- Tanis lay within the XIVth nome, and so far as the testimony of this expression is concerned we may still regard the Bubastite river as the dividing line between the Sethroites (XIV) and the Tanites (XIX). It remains, however, to examine the second argument that seems to have influenced Brugsch, namely the fact that Thel, or the god of Thel, is named on certain monuments discovered at Tanis, whence it might seem natural to identify Tanis and Thel. Little weight can be attached to the application of the title "Commander of the Fortress of Thel" to the Vizier Pracmesse who accompanies Ramesses II on the Stele of Four Hundred Years, where Ramesses II is depicted offering to Setekh-of-Ramesses4. 1 In my discussion of the town of Avaris (Journal of Egyptian Archaeology, vol. iI, pp. 99-101) I failed to realize this point, and the remarks there made on the relationship between the XIVth and XIXth nomes consequently require revision. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 In my discussion of the town of Avaris (Journal of Egyptian Archaeology, vol. iI, pp. 99-101) I failed to realize this point, and the remarks there made on the relationship between the XIVth and XIXth nomes consequently require revision. 2 BRUGSCH, Dictionnaire Geographique, pp. 1369, 1373. 3 See GRIFFITH, Proceedings of the Society of Biblical Archaeology, vol. xxi, pp. 278--9, for the reading for these names. 4 The most accessible reproduction is BUDGE, History of Egypt, vol. in, p. 157. 5 PETRIE, Tanis II, P1. X, nos. 168, 170. Op. cit., P1. X, nos. 164, 165. She occurs also on a statue usurped by Meneptah, Tanis I, P1. I, no. 3, B. This stele has been already quoted in another connection, see above p. 247, with footnote 2. 8 Unamun, II, 35. Journ. of Egypt. Arch. v. 33 q y q 2 BRUGSCH, Dictionnaire Geographique, pp. 1369, 1373. 2 BRUGSCH, Dictionnaire Geographique, pp. 1369, 1373. 3 See GRIFFITH, Proceedings of the Society of Biblical Archaeology, vol. xxi, pp. 278--9, for the reading for these names. 4 The most accessible reproduction is BUDGE, History of Egypt, vol. in, p. 157. I failed to realize this point, and the remarks there made on the relationship between t XIXth nomes consequently require revision. p XIXth nomes consequently require revision. IV It must be admitted, on the other hand, that the statues of Teos come from Tanis, and that one or two Ptolemaic stelae found in the excavations there 'either mention Thel or are dedicated to the Horus of Mesen5. Other Ptolemaic stelae from Tanis, however, are dedicated to Buto of Nebesheh6, though her name scarcely occurs upon the earlier monuments from that site. M. Daressy tells us that the chief deity of Tanis was Amen-ReC, and he accordingly quotes a late stele from Memphis where the divinities of Thel are associated with Amen-ReC as proof that Tanis later replaced Thel as capital of the XIVth nome7. As a matter of fact Amen-R6C hardly occurs on the Tanite monuments, though the rulers of the XXIst Dynasty, spoken of as " the officers whom Amen-ReC has given to the north of his land8," may well have shown to the god of 33 33 33 Journ. o ALAN H. GARDINER 250 their Theban home more consideration than previous Pharaohs had done. To tell the truth, the documents at our disposal do not permit us to decide what special cults were most in favour at Tanis; Mr Griffith, who made a careful investigation of this questioln, summed up his opinion as follows': "The search for a local mythology and really local worship has not been successful. When we have sifted out the national gods who change with the dynasties (the Ptah, Osiris, and Sokar of the Middle Kingdom; Set of the Hyksos; Set, Harmachis, Turn, Tathnen and Amen of the Ramessides) there is no resi- duum left sufficient to certify a local worship. Set, Uati ap taui and Horus of the foreigners, wear a semblance of localization." g , The uncertainty of which Mr Griffith here complains would be much increased if a plausible theory recently put forward by M. Daressy were to prove true2. Combating the late Sir Gaston Maspero's hypothesis of a special Tanite school of Art, he argues that the Ramesside and later Pharaohs who adorned the great temple of Tanis transported thither, and particularly from Memphis, many of the older monuments found on the site; only thus, he considers, can we explain the prominence of the Memphite Ptah on a number of the early statues excavated at Tanis. M. 1 Tanis II, p. 34. 2 Annales du Service, vol. xvii, pp. 164-176. 3 Journal of Egyptian Archaeology, vol. iII, pp. 99-101. 1 Tanis II, p. IV Daressy does not extend his theory to the monuments naming Seth of Avaris, since though he does not admit the identity of Avaris and Tanis, he thinks that those towns were quite close to one another. Manetho, however, distinctly says that Avaris lay to the east of the Bubastite river and in the Sethroite nome; I have tried elsewhere3 to make it probable that Avaris was Pelusium or at all events near it, a view for which, as we shall see, there is evidence in tradition. Still, just as Thel was close enough to Tanis for its gods occasionally to be mentioned there, so may Avaris also have been. Whether or no, therefore, we suppose that the monuments discovered at Tanis and bearing the name of Seth of Avaris were moved thither by the kings of the Nineteenth or of the Tanite Dynasty, no serious evidence is forthcoming for the view that Seth was the principal god of Tanis. The same argument applies to the monuments naming the Seth of Meneptah, who, as will be shown subsequently, was merely the Seth of Pi-Racnesse under another nane. From the coins of the Tanite nome it appears that its predominant cult was that of the falcon-god Horus. pp p g To sum up the results of the last few pages, there is no evidence favouring the view that Tanis was in the nome of Khant-yeb which can outweigh the inherent impro- bability of such a supposition. The actual topographical conditions demand imperatively that Tanis should belong to the nome of which Nebesheh was, at one time at least, the capital; we are, therefore, justified in identifying the Tanites with the nomne of 'Imt-Pehu (XIX) and in assuming, until contradictory facts make their appearance, that in the Pharaonic times, as also later, the Bubastite branch formed the boundary between the XIVth and the XIXth nomes. This elaborate discussion of the relationship between the two north-eastern nomes of the Delta may have seemed irrelevant to the particular question here at issue; in reality it has cut away the ground beneath Brugsch's identification of Pi-Racmesse with Tanis. That identification rested to no small extent on the occurrence of the title "priest of Amfin of RaCmes of Pi-RaCmesse" on the statue of Teos found at Tanis (above p. 199, no. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 Tanis II, p. 34. 2 Annales du Service, vol. xvii, pp. 164-176. 3 Journal of Egyptian Archaeology, vol. iII, pp. 99-101. p 3 Journal of Egyptian Archaeology, vol. iII, pp. 99-101. IV Knowing what we now know about the relations of the XIXth and XIVth, the fTanite and the Sethroite, nomes, the close association of the title "priest of Amien of Racmes of Pi-RaCmesse" with titles belonging to the XIVth nome either indicates nothing at all or else indicates that Pi-RaCmesse is to be sought eastward of the Bubastite branch of the Nile. It is true that the statue of Teos was found at Tanis, and its owner may therefore have held some offices there. But that would not prove that the Tanitic and Sethroitic nomes were in any way fused or inseparable; the general Nektanebos who lived under Ptolemy Soter was governor not only in Thel (Sile), but also in 'Imt (Nebesheh) and Thebnute (Sebennytos)'. Similarly the owner of the Sarcophagus Louvre D 1 (quoted above p. 130, under C) appears to have held priesthoods in various cities in by no means close proximity to one another. Numberless other examples of the same kind could be quoted. p q If no convincing argument can be based on the testimony of the statue of Teos adduced by Brugsch, another piece of evidence that he employed can now be made to weigh heavily against him. In the long description contained in Pap. Anastasi III (above p. 184, no. 15) of the delights and riches of Pi-Racmesse, a number of localities are named which must necessarily lie in the neighbourhood, though possibly not in the immediate neighbourhood, of that city. Unhappily only one of the names is identifiable: in 2, 8-9 it is said that "The- Waters-of-Horus yield salt," and three lines further on we find the sentence, "The reed-swamps come to it (Pi-RaCmesse) with papyrus, and The-Waters-of- Borus with rushes." Now the name I-: l ; P, Sz-Hr, here rendered "The- Waters-of-Horus," is obviously the same as the ^ - S-i-Hr " Waters-of- Horus" frequently mentioned by the Graeco-Roman texts in connection with the XIVth nome2. , , , 2 E.g., Dendereh, DiUMICHEN, Geographische Inschriften, vol. iv, P1. CXX; MARIETTE, Denderah, vol. I, P1. 66 a, 7. At Edfu, cf. DtMICHEN, op. cit., vol. iII, P1. XXII ; DE ROUGE, Edfou, P1. XXVI; op. cit., P1. LXIII; PIEHL, Inscriptions HIieroglyphiques, vol. i, P1. CII, F. Only indisputable references to the SA-JHr of the XIVth nome have here been quoted; for other localities with the same or a similar name, cf. BRUGSCH, Dictionnaire Geographique, pp. 519, 1261. IV 39), where it was associated with various priesthoods connected with Thel and with This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms THE DELTA RESIDENCE OF THE RAMESSIDES 251 the XIVth nome. We now realize that Thel was Tell Abu Sefeh and not Tanis, as Brugsch supposed; but still if Tanis had been in the same nome as Thel the title in question might have seemed to point naturally to the equation of Pi-RaCmesse with Tanis, the more so because Tanis possesses an abundance of Ramnesside monuments and was also virtually a sea-port, as we have learnt from the narrative of Unamuan. Knowing what we now know about the relations of the XIXth and XIVth, the fTanite and the Sethroite, nomes, the close association of the title "priest of Amien of Racmes of Pi-RaCmesse" with titles belonging to the XIVth nome either indicates nothing at all or else indicates that Pi-RaCmesse is to be sought eastward of the Bubastite branch of the Nile. It is true that the statue of Teos was found at Tanis, and its owner may therefore have held some offices there. But that would not prove that the Tanitic and Sethroitic nomes were in any way fused or inseparable; the general Nektanebos who lived under Ptolemy Soter was governor not only in Thel (Sile), but also in 'Imt (Nebesheh) and Thebnute (Sebennytos)'. Similarly the owner of the Sarcophagus Louvre D 1 (quoted above p. 130, under C) appears to have held priesthoods in various cities in by no means close proximity to one another. Numberless other examples of the same kind could be quoted. the XIVth nome. We now realize that Thel was Tell Abu Sefeh and not Tanis, as Brugsch supposed; but still if Tanis had been in the same nome as Thel the title in question might have seemed to point naturally to the equation of Pi-RaCmesse with Tanis, the more so because Tanis possesses an abundance of Ramnesside monuments and was also virtually a sea-port, as we have learnt from the narrative of Unamuan. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 SETHE, Urkunden, II, 24-6. 2 E.g., Dendereh, DiUMICHEN, Geographische Inschriften, vol. iv, P1. CXX; MARIETTE, Denderah, vol. I, P1. 66 a, 7. At Edfu, cf. DtMICHEN, op. cit., vol. iII, P1. XXII ; DE ROUGE, Edfou, P1. XXVI; op. cit., P1. LXIII; PIEHL, Inscriptions HIieroglyphiques, vol. i, P1. CII, F. Only indisputable references to the SA-JHr of the XIVth nome have here been quoted; for other localities with the same or a similar name, cf. BRUGSCH, Dictionnaire Geographique, pp. 519, 1261. 3 BRUGSCH, op. cit., p. 1369. 4 At Edfu see PIEHL op cit P1 CVIII L; cf also MARIETTE op cit vol I P1 10 3 BRUGSCH, op. cit., p. 1369. 4 At Edfu, see PIEHL, op. cit., P1. CVIII, L; cf. also MARIETTE, op. cit., vol. I, P1. 10. 1 SETHE, Urkunden, II, 24-6. 1 SETHE, Urkunden, II, 24-6. IV In the great geographical inscription of Edfu "Waters-of-Horus" is the "river" ( lr--t) of the XIVth nome-the stream or strip of water upon which'floated the sacred bark "Beautiful-is-Mesen3." Most of the references to "Waters-of-Horus" in the late temples are devoid of geographical interest, but once4 in a legend that accompanies a figure of the king pouring libations we read, "These thy libations come forth from Ele- phantine, A I \ ~r I they arrive at Waters-of-Horus, that thou mayst drink of them," etc.; apparently the purpose of the writer was to say that the Nile is offered to the god in its full extent, from Elephantine and the First Cataract down to " Waters-of- 33-2 This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms ATLAN H. GARDINER 252 Horus," its northernmost reach. From this and various other passages it seems evident that "Waters-of-Horus" must be a stretch of running water; it cannot (e.g.) be equated with Lake Ballah, nor again is it likely to have anything to do with the canal, already mentioned, which connected Thel with the Nile. For the Egyptian the only drinking- water that seemed desirable was that of the Nile, and it is therefore necessary to regard " Waters-of-Horus" as name of a part of the Nile itself. From the circumstances of the case it can only be the name of that stretch of the Bubastite branch which we must pre- sume to have formed the western boundary of the XIVth nome, in other words the stretch which debouched at or near Pelusium. This conclusion is confirmed in a quite remarkable manner by the place-name Shihor familiar from the Old Testament. The Hebrew name 'min" Shihor, once (Joshua xiii, 3) preceded by the definite article inwn Hash-Shihor, is an impeccable equivalent of the Egyptian 2Si-Hr " Waters-of-Horus" or :cxa xx , Pi Si-Hr " The- Waters- of-Horus'." Two of the passages where Shihor occurs prove it to be a name of the Nile or a part of it, and the other two equally clearly show that it marked the potential or actual boundary between Egypt and the land of Israel. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 3 BROWN-DRIVER-BRIGGS, Hebrew Lexicon, s.v. Shihor, takes the correct view, as does also E. MEYER, Die Israeliten und ihre lVachbarstdimme, p. 457, footnote 1. CEEYNE, Encyclopaedia Biblica (s.v.), deals with the name in a characteristically fanciful way. gyp g ( ), p ( p , p , ) g y 2 R.V. gratuitously inserts after Shihor " the brook." This insertion rests on the supposed identity of Shihor with " the brook of Egypt" namned (e.g.) Numbers xxxiv, 5, and generally taken to be the WAdy el-eArish. 1 The equation is perhaps due to Brugsch, the first mention of it that is known to m Aegyptologie (1891), p. 451. Dr Ktithmann (op. cit., p. 41, footnote 2) has rightly revived it. THE DELTA RESIDENCE OF THE RAMESSIDES THE DELTA RESIDENCE OF THE RAMESSIDES 253 As we have seen, it was Chabas who first voiced the possibility that Pi-RaCmesse was Pelusium or in its neighbourhood; apart from Max Miiller, whose adhesion to this theory seems to have been quite transitory, it has found no supporters. Chabas attached much weight to the passage in the " Poem of Pentaur" (no. 2), whence we learn that Ramesses II passed the fortress of Thel before reaching the town of Ramesses; but in view of the defective text, and the consequent uncertainty whether the particular town of Ramesses there meant was the Delta capital, we have pledged ourselves not to use this passage otherwise than as corroborative evidence. What further arguments can be adduced in favour of our thesis ? Thus far we have ascertained that Pi-RaCmesse was intimately associated with Shihor, a name of the lower reaches of the Bubastite Nile-arm; further, that it was near the sea and was distinct from Thel. Certain epithets, moreover, indicate that it lay on the extreme eastern border (p. 252). But it did not lie on the route between Thel and Raphia; else it would have been named in the comprehensive list of names of places on that route contained in Pap. Anastasi I, 27, 3-8. Beyond these details and the fact that Pi-RaCmesse was accessible by water from Heliopolis (no. 8) we have no direct or certain information as to its whereabouts. But there remains a certain amount of indirect and not quite demonstrable testimony which, taken in bulk, renders it exceedingly probable that Pi-RaCmesse stood on the site known to the Greeks as Pelusium, "the city of mud." y Pelusium, as has been said already, was situated at the approximate distance of 24 Roman miles from Sile (Thel) and must have lain in the same nome. The ruins, now known as Tell Farameh, have been described by Mr Griffith1 and, more recently, by M. C14dat2. Thus far the only monuments of Pharaonic date discovered there have been a weight bearing the name of the Pharaoh Nektanebos, part of a sarcophagus of the same period, and a small fragment of a temple scene (or the like) that appears to belong to Ra- messide times3. There exists evidence, however, that the town was a very old one. 1 PETRIE, Nebesheh and Defenneh, pp. 99 101. 2 Annales du Service, vol. xII, pp. 79-85, with a plan of the ruins. The general position of the town is best shown by Clddat's map, op. cit., vol. x, unnumbered Plate referring to the article, pp. 209-237. 3 Recueil de Travaux, vol. xxxvii, pp. 33-4. IV In Isaiah's "Burden of Tyre" (xxiii, 3) we read, "And on great waters the seed of Shihor, the harvest of the Nile, was her revenue "; and the prophet Jeremiah asks (ii, 18), "And now what hast thou to do in the way to Egypt, to drink the waters of Shihor?" Again, when Joshua was grown old, there re- mained yet much land to be possessed by Israel, " all the regions of the Philistines, and all the Geshurites; fromn the Shihor which is before Egypt, even unto the border of Ekron north- ward " (Joshua xiii, 3); and in the history of David, "So David assembled all Israel together, from Shihor of Egypt2 even unto the entering in of Hamath" (I Chronicles, xiii, 5). It is strange that these very explicit passages could ever have been misinterpreted3; neither the Hebrew Shihor nor the Egyptian "Waters-of-Horus" can well be understood otherwise than as designations of the lower reaches of the Bubastite or Pelusiac Nile-arm. g Thus the association of " Waters-of-Horus" with Pi-RaCmesse indicates the identity of the latter, not as Brugsch supposed with Tanis, but rather with Pelusium or at all events with some town situated near the sea on the Bubastite branch of the Nile. With the Biblical passages representing Shihor as the boundary of Israel onie may compare the Egyptian texts describing Pi-Ra(messe as "betwixt Zahi and Egypt" (no. 17) and as "the forefront of every land, the end of Egypt" (no. 16). That Pi-RaCmesse was a sea-port, whether actually beside the sea or some little distance inland beside the river-mouth, is shown by the words "the harbourage of thy ships' troops" (no. 16); " its ships fare forth and return to port" (no. 15). p In parting company once and for all with Brugsch and his theory of the identity of Pi-Racmesse and Tanis, let us recall the decisive evidence of the Golenischeff Glossary (no. 38), where Pi-Racmesse is distinct both from Thel and from Tanis. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms is best shown by Clddat s map, op. cit., vol. x, unnumbered Plate referring to the articl 3 Recueil de Travaux, vol. xxxvii, pp. 33-4. 1 PETRIE, Nebesheh and Defenneh, pp. 99-101. 1 PETRIE, Nebesheh and Defenneh, pp. 99 101. 2 Annales du Service, vol. xII, pp. 79-85, with a plan of the ruins. The general position of the best shown by Clddat's map op cit vol x unnumbered Plate referring to the article pp 209- 2 Annales du Service, vol. xII, pp. 79-85, with a plan of the ruins. The general position of the town is best shown by Clddat's map, op. cit., vol. x, unnumbered Plate referring to the article, pp. 209-237. 3 Recueil de Travaux vol xxxvii pp 33-4 t shown by Clddat s map, op. cit., vol. x, unnumbered Plate referring to the article, pp. 209-237 Recueil de Travaux, vol. xxxvii, pp. 33-4. 1 PETRIE, Nebesheh and Defenneh, pp. 99-101. THE DELTA RESIDENCE OF THE RAMESSIDES Its original name may have been [1 S()nw, this occurring already in the Pyramid Texts as the provenance of a particularly good kind of wine; the name possibly means " stronghold " (1+ + i e . The native Egyptian inscriptions of the Pharaonic period do not allude to inw except in this connection, but the Annals of Assurbanipal name Sharludari, prince of Si'inu, as one of the Delta chieftains subjugated by the Assyrians; Si'inu here must be a city distinct from Sa'anu-Tanis, since the prince of that city bore the name of Petubishti (Petubastis). In Demnotic two references to a town of similar name are found: the Vienna novel of Petubastis sends that warrior "into the provinces of Egypt from Elephantine to Swn," where Swn must obviously be a place in the extreme north; in the geographical manual Pap. Cairo 31169, 3, 26 a place o) ot Swn is named in a position such that we must assume it to be just outside the borders of Egypt. A few years ago Professor Spiegelberg, combining these data with the prophecy Ezechiel xxx, 15, " I will pour my anger upon Sin, the strength of Egypt," where the Vulgate has Pelusium for the Hebrew IP (probably to be vocalized catv with the Septuagint), made out a plausible case for the This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms ALAN H. GARDINER 254 old Slnw of the Pyramid Texts being Pelusiumn. The Greek name l1Xovo-tov "mud- city" would on this view be a popular etymology from the Egyptian word [1 sin " clay," " mud." Modern scholars have always adhered to the Vulgate's identification of pD in Ezechiel with Pelusium, both because Pelusiumn suits the requirements of the passage well and because of the Syriac word sCydna " mud," which seemed to furnish the Semitic equivalent of the Greek name2. Professor Erman attempts to reconcile these conflicting etymologies by regarding the Egyptian word sin " mud " as the source both of the Syriac scydna, which has no good Senmitic affinities, and also of the Greek translation Hf\XoVi3tov. y g Leaving now these rather doubtful philological details, we may take it as possible that Sainu or some similarly vocalized name was the commonest Egyptian designation of the important and historic town of Pelusium3. 1 Zeitschrift fur agyptisclhe Sprache, vol. XLIX (1911), pp. 81-4. Spiegelberg makes out a fairly good case for transcribing the old Egyptian name as Sinw,'though in a number of instances Swn, with w, is written. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 Zeitschrift fur agyptisclhe Sprache, vol. XLIX (1911), pp. 81-4. Spiegelberg makes out a fairly good case for transcribing the old Egyptian name as Sinw,'though in a number of instances Swn, with w, is written. 2 MAX MtLLER, Encyclopaedia Biblica, art. Sin (written before the appearance of Spiegelberg's essay), rejects the identification of the Sin of Ezechiel with Pelusium. 3 For the history of Pelusium in classical and medieval times, see a good popular account by Dr W. F. Hume in the Cairo Scientific Journal, vol. ix (1917), pp. 54-63. 4 Journal of Egyptian Archaeology, vol. in, pp. 99-101. rejects the identification of the Sin of Ezechiel with Pelusium. 3 For the history of Pelusium in classical and medieval times, see a good popular account by Dr W. F. Hume in the Cairo Scientific Journal, vol. ix (1917), pp. 54-63. 4 J l f E ti A h l l i 99 101 2 MAX MtLLER, Encyclopaedia Biblica, art. Sin (written before the appearance of Spiegelberg rejects the identification of the Sin of Ezechiel with Pelusium. THE DELTA RESIDENCE OF THE RAMESSIDES II) called " House-of-Ramesses-Prince-of-Heliopolis (R III)-in-the-House-of-Setekh ;" this possessed 106 serfs, no 29 y p q p "Setekh-great-of-valour-of-Ramesses-Beloved-of-Amiun" (R. II), nos. 12, 30; "Setekh-great-of-valour- son-of-Nut" named immediately after Arfin and Ptah of R. II, no. 4. Of-Pi-RaCmesse it is said: " Its southern part is the house of Setekh," no. 17. In the reign of R. III, Pi-Ratmesse had a temple of " Setekh-of-Ramesses-Beloved-of-Amun " (R. II) called " House-of-Ramesses-Prince-of-Heliopolis (R. III)-in-the-House-of-Setekh ;" this possessed 106 serfs, no. 29. (R. III) in the House of Setekh ; this possessed 106 ser "PrSe-of-Ramesses-Beloved-of-Amin," only nos. 30 and 31. , y In addition to these deities, it is once said of Pi-Raemesse that "Astarte is in its Orient, and Buto in its northern part," no. 17. On the stele of the Hittite treaty (no. 4), R. II is described as doing the behests of certain gods in the Residence-city; Amen-Re¢, Harakhte and Atum are here named before Amuin of R. II, Ptah of R. II and Setekh, great of valour, son of Nut, but there is no reason for thinking that the first three gods were specially worshipped in Pi-RaCmesse. , y In addition to these deities, it is once said of Pi-Raemesse that "Astarte is in its Orient, and Buto in its northern part," no. 17. On the stele of the Hittite treaty (no. 4), R. II is described as doing the behests of certain gods in the Residence-city; Amen-Re¢, Harakhte and Atum are here named before Amuin of R. II, Ptah of R. II and Setekh, great of valour, son of Nut, but there is no reason for thinking that the first three gods were specially worshipped in Pi-RaCmesse. g g It is extremely interesting to find that on several monuments usurped by Meneptah he describes himself alternately as (a) " loved of Seth, lord of Avaris," (,3) " loved of Seth, great of valour," and (y) " beloved of Seth-of-Meneptah." The monuments in question are: base of statue, PETRIE, Tanis I, P1. II, no. 5 A (a, /3, 7 together); colossus originally of Sesostris I, now in Berlin, Aeg. Inschr. aus den kon. Mus., vol. II, pp. 19-22 (a, y together); statue originally of Amenemmes III, now in Berlin, op. cit., vol. Il, p. 18 (/3 only); statue originally of Sesostris I, now in Cairo, Annales du Service, vol. XVii, p. 170, no. THE DELTA RESIDENCE OF THE RAMESSIDES There are, however, certain indications which make it by no means improbable that Pi-RaCmesse stood on the site of the old Avaris, and consequently is none other than Pelusium itself; possibly even the story told by Josephus may contain a hint of this, for that story describes how numbers of Egyptians and foreigners were gathered together into Avaris-Pelusium during the Ramesside age, just as it is said in a poem about Pi-Racmesse THE DELTA RESIDENCE OF THE RAMESSIDES 255 translated above (no. 17): "Everyone hath left his town and settled within its territory." Apart from this, however, the promninence of Setekh in both Avaris and Pi-Racmesse is a point strongly in favour of the identity of the two places. The gods of Pi-RaCmesse, as we learn from the evidence set forth in the last section, were as follows: "Amiin-of-Ramesses-Beloved-odf-Amun" (R. II), nos. 1, 4, 17, 20, 30, 31. Of Pi-RaCmesse it is said: "Its western part is the house of Amun," no. 17. His temple, no. 1. "Anman-of-Ramesses-Prince- of-Heliopolis" (R. III); his temple described, no. 26; it possessed 7872 serfs, no. 27. In late times, "Amnin-of-RaCmes" (sic), no. 39. "Amiin-of-Ramesses-Beloved-odf-Amun" (R. II), nos. 1, 4, 17, 20, 30, 31. Of Pi-RaCmesse it is said: "Its western part is the house of Amun," no. 17. His temple, no. 1. "Anman-of-Ramesses-Prince- of-Heliopolis" (R. III); his temple described, no. 26; it possessed 7872 serfs, no. 27. In late times, "Amnin-of-RaCmes" (sic), no. 39. , ( ), "Ptah-of-Ramesses-Beloved-of-Amun" (R. II), nos. 1, 4, 19, 20, 30, 31. His temple, no. 1. "Ptah-of- Ramesses-Prince-of-Heliopolis" (R. III); his temple is described in no. 28, and was apparently attended by 23 (16+ 7) persons, but it is not quite certain that this temple was in Pi-Ra'messe. , ( ), "Ptah-of-Ramesses-Beloved-of-Amun" (R. II), nos. 1, 4, 19, 20, 30, 31. His temple, no. 1. "Ptah-of- Ramesses-Prince-of-Heliopolis" (R. III); his temple is described in no. 28, and was apparently attended by 23 (16+ 7) persons, but it is not quite certain that this temple was in Pi-Ra'messe. y ( ) p , q p "Setekh-great-of-valour-of-Ramesses-Beloved-of-Amiun" (R. II), nos. 12, 30; "Setekh-great-of-valour- son-of-Nut" named immediately after Arfin and Ptah of R. II, no. 4. Of-Pi-RaCmesse it is said: " Its southern part is the house of Setekh," no. 17. In the reign of R. III, Pi-Ratmesse had a temple of " Setekh-of-Ramesses-Beloved-of-Amun " (R. 1 See above, pp. 249-251. 2 The most handy reproduction of this curious monument is to be found in BUDGE, History of Egypt, vol. III, p. 157. On the Seth of Ramesses II, see Griffith's article in Proceedings of the Society of Biblical Archaeology, vol. xvi, pp. 87-89, where it is pointed out that he is associated with Buto of Nebesheh not only in the titles of the Stele of Four Hundred Years but also on a monument from Nebesheh itself. 3 A 21, see SHARPE, Egyptian Inscriptions, vol. ii, P1. 44; also PETRIE, Tanis, I, P1. II, nlo. 25. THE DELTA RESIDENCE OF THE RAMESSIDES But Egyptian towns have, as has been noted previously, a way of possessing several different names, and it is by no means unlikely that this may have been the case with Pelusium. In the confused and un- doubtedly composite story told by Josephus about the expulsion of the Hyksos and the Exodus of the Jews, Pelusium is several times mentioned in a mnanner that leaves no doubt in the mind that it was thought synonymous with Avaris. The Heliopolitan priest Osarsiph had been made ruler of the lepers set apart by king Amenophis in Avaris (contra Apione7n, ed. NABER, I, 238-9); Osarsiph now sends to Jerusalem to the descendants of the Hyksos who had been driven out of Avaris by Tethmosis, and begs them to return thither (op. cit., I, 242-3). This they do, but at a subsequent stage of the narrative it is at Pelusium, and not at Avaris, that the army of the son of Amenophis, according to Manetho, encounters the lepers (op. cit., I, 274). In the still more muddled version of the legend ascribed by Josephus to Chairemon, Moses-Tisithen and Joseph-Peteseph arrive at Pelusium and find 380,000 men left there by Amenophis, he not wishing to take them into Egypt; Moses and Joseph enter into a league of friendship with them and join them in an expedition to Egypt (op. cit., I, 290-1; cf. also 302); Pelusium is here what Avaris is in the other accounts, the place of assembly for foreigners and rebels engaged in warfare against Egypt. g g Now as I have shown elsewhere4, the scanty data we possess concerning the position of Avaris point to its having been situated in the neighbourhood, if not on the actual site, of Pelusium, so that the tradition as to the identity of the two places handed down by Josephus is entitled to a respect which would not have been due to it had it stood alone. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 See above, pp. 249-251. THE DELTA RESIDENCE OF THE RAMESSIDES 4 (,3 only); statue originally of Sesostris I, now in Cairo, loc. cit., nos. 5 and 8 (/3 and 7 together). Those statues here marked with an asterisk are known to have come from Tanis, and the same may be true of the others, as well as of several related monuments yet to be quoted; but we have seen that not much importance is to be attached to this fact in connection with our topographical probleml. Now Seth-of-Meneptah is obviously identical with Seth-of- Ramesses-Beloved-of-Amun, who is depicted on the Stele of Four Hundred Years found at Tanis2, and named on a sphinx from the same place now in the Louvre3. Thus one of the principal gods of Pi-RaCmesse-we shall return to the others further on-is also the principal god of Avaris, a fact which seems to point to the identity of Avaris and Pi-RaCmesse; but if Avaris is Pelusium, then Pi-RaCmesse will have to be looked for at Pelusium too. With this fact would agree, further, the prominence of Amufin at Pelusium. In Coptic times the town was called nepeao-n, which must surely correspond to an old Pr-'Imn This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms ALAN H. GARDINER 256 " House-of-A man,," whatever the reluctance of Egyptologists to recognize the fact1; and it is this name which gave rise, it is believed, to the namine Tell Farameh borne by the mounds now concealing the remains of Pelusium2. In Roman times the Pelusiac Amfin was called Zeus Kasios, and the remains of his temple have been recently excavated in the middle of the western mound of Tell Farameh3. It is probably the merest coincidence that Pap. Anastasi III, describing Pi-RaCmesse, tells us that " its western part is the house of Amun." It cannot be denied that the deities of Pi-RaCmesse were contemporaneously wor- shipped, or at least commemorated, in various other cities besides Pi-Rarmesse itself. Seth-of-Ramesses has been met with at Tanis, and it must now be added that he occurs in company with Ptah-of-Ramesses also at Bubastus4; Ptah-of-Ramesses is named on a palette fronm Thebes5 and at es-SebOca in Nubia6, and Ptah-of-Meneptah, evidently the same god, is recorded on a lintel from Memphis7. At Tell-el-Yahudiyeh Amfn-of-Meneptah and Ptah-of-Meneptah are associated with the erased Seth-of-Meneptah8. Lastly, Amun-of- Usimarc¢-setpenrec is depicted at Abu Simbel9. 1 AMALINEAU, La Geographie a l epoqtue Copte, pp. 319 20. 2 A different and impossible origin is proposed by BRUGSCH, Dictionnaire Geographique, p. 1089; rightly combated by Maspero in Zeitschrift fir dgyptische Sprache, vol. xxi (1883), pp. 63-4. 3 Annales du Service, vol. xviii, pp. 79-85. For references to the temple in classical writers see WIEDEMANN, Herodots Zweites Buch, p. 63. 4 NAVILLE, Bubastis, P1. XXXVI, F, H. 5 Berlin 6764; see Zeitschrift fur dgyptisc]he Sprache, vol. xxx (1892), pp. 43-6. 6 GAUTHIER, Le Temple de Ouadi es-Seboud, p. 151. 7 PETRIE, Palace of Apries, P1. XXI. 8 PETRIE, Hyksos and Israelite Cities, P1. XVI. 9 CHAMPOLLION, Notices Descriptives, vol. I, p. 73. 10 See BREASTED, Battle of Kadesh, p. 11. 1 GARDINER, Th/e Stele of Bilgai, in Zeitschriftfi'r dgyptische Sprache, vol. L (1912), pp. 49-57. 12 NAVILLE, Bubastis, P1. XLVI, B. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 5 Berlin 6764; see Zeitschrift fur dgyptisc]he Sprache, vol. xxx (1892), pp. 43-6. p GARDINER, Th/e Stele of Bilgai, in Zeitschriftfi'r dgyptische Sprache, vol. L (1912), pp. 49-57. 1 GARDINER, Th/e Stele of Bilgai, i 12 NAVILLE, Bubastis, P1. XLVI, B. 1 AMALINEAU, La Geographie 'a l'epoqtue Copte, pp. 319-20. , p , 8 PETRIE, Hyksos and Israelite Cities, P1. XVI. , / g , gyp p , ( ), pp 12 NAVILLE, Bubastis, P1. XLVI, B. p 7 PETRIE, Palace of Apries, P1. XXI. THE DELTA RESIDENCE OF THE RAMESSIDES The two gods in question occur among the delicately sculptured figures of This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms THE DELTA RESIDENCE OF THE RAMESSIDES 257 F THE RAMES ompanied by the l z-of-Ramesses-(" 8 f h 0 -..l f0 r." If the argument at Pi-RaCmesse was s " The- Waters-of-Rec us passages recorded Pi-Racmesse lay bes e- Waters-of-the- to which they appe know about t" The- W t of nomes, inundate os I at Abydos: the l h," [j Q "The- W of-the-Sun" and a s from the Ramesse Thebes), which is in in the Golenischeff THE DELTA RESIDENCE OF THE RAMESSIDES 257 numerous local deities, and their images are accompanied by the legends: Jj z mutually confirm one another and the conclusion to which they appeared to p acquires some degree of solidity. What then do we know about t" The- Waters-of acquires some degree of solidity. What then do we know about t The Waters of the Sun ? The earliest occurrence of this name is in a list of nomes, inundated districts and river- arms depicted on the walls of the temple of Sethos I at Abydos: the list concludes with ~Aw A O "The Waters of Ptah " [j Q "The Waters of Mac q g y The earliest occurrence of this name is in a list of nomes, inundated districts and river- arms depicted on the walls of the temple of Sethos I at Abydos: the list concludes with p p y ~Aw A O "The- Waters-of-Ptah," [j Q "The- Waters-of-M ~Aw A O "The- Waters-of-Ptah," [j Q "The- Waters-of-Mac- ~Aw A O "The- Waters-of-Ptah," [j Q "The- Waters-of-Mac- v ~\wA' "The-Waters-of-the-Sun" and another name that is illegible,. On a number of fragmentary wine-jars from the Ramesseum we read of wine from " the great orchard of the Ramesseum (i.e. of Thebes), which is in The- Waters-of-the- Sun2." The same branch of the river is mentioned in the Golenischeff Glossary and in the analogous catalogue of place-names, the Demotic Pap. Cairo 311693. In Pap. Harris 10, 7-11 is a list of herds of cattle belonging to the estate of Amrun; each herd has a name, and after three of them is an indication of the district where they were pastured ("in the Great-River," "in The- Waters-of the-Sun," "in the Great-River"). 1 MARIETTE, Abydos, vol. I, P1. 14, c; DUMJCHEN, Geographische Inschriften, vol. I, PI. XCII. " The- Waters-of-Ptah" are named on the fragments of two wine-jars, SPIEGELBERG, Hieratic Ostraka, nos. 197, 292. The destroyed name may have been " The- Waters-of-Amrln " named on the fragments, op. cit., nos. 153, 225, 228. Stress must be laid on the importance of these fragments of wine-jars for a knowledge of the names of Nile-arms and canals in the Delta; they have already been quoted several times in this connection. Note that Li "Ka"' in nos. 186, 209, 217, 218, 221, 243, 269, 289, 292 looks like the original of the " Agathodaemon " river in Ptolemy 1 MARIETTE, Abydos, vol. I, P1. 14, c; DUMJCHEN, Geographische Inschriften, vol. I, PI. XCII. " The- Waters-of-Ptah" are named on the fragments of two wine-jars, SPIEGELBERG, Hieratic Ostraka, nos. 197, 292. The destroyed name may have been " The- Waters-of-Amrln " named on the fragments, op. cit., nos. 153, 225, 228. Stress must be laid on the importance of these fragments of wine-jars for a knowledge of the names of Nile-arms and canals in the Delta; they have already been quoted several times in this connection. Note that Li "Ka"' in nos. 186, 209, 217, 218, 221, 243, 269, 289, 292 looks like the original of the " Agathodaemon " river in Ptolemy. 2 Op. cit., nos. 143, 231, 276; for no. 264, which may have named Pi-RaCmesse, see no. 37 of last section. 3 Sphinx, vol. xiv, p. 163. 4 Ancient Records, vol. iv, § 224. 5 See above, p. 134, under M. Journ. of Egypt. Arch. v. 34 This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms Ancient Records, vol. iv, § 224. 5 See above, p. 134, under M. THE DELTA RESIDENCE OF THE RAMESSIDES So devoted, indeed, was Ramesses II to his four gods Preg, Amufin, Ptah and Seth that he not only honoured them in his Residence, but also named the four divisions of his army after them10. A temple of Prc¢-of-Ramesses situated to the south of Memphis has been discussed above (p. 133, under I), but apart from this we have no evidence of an established cult of the gods of Pi-RaCmesse elsewhere than at that city itself. For it is reasonable to conjecture that the chapel of Amun-of-Usimare-setpenrec dedicated by a queen who may have been Tewosret ("Tausert") of the Nineteenth Dynasty'1 was at Pi-Racmesse itself or quite close to it; this is the more likely, since it was placed in charge of an official known as " The Commander of the Fortress of the Sea." But even if we suppose the gods of Pi-RaCmesse to have possessed temples and priests of their own in other towns beside Pi-Rarmesse, it is highly improbable that such cults survived Ramesside times. In Pi-RaCmesse itself they may have lingered on, the priesthoods sustaining themselves on the relics of the vast heritage with which Ramesses II endowed them; indeed we know for a fact from the statue of Teos (no. 39) that Amiln-of-Ramesses was attended in Pi-Racmesse by a priest as late as the fourth century B.C. The foregoing argument enables us to estimate at its true value the mention of two of the gods of Pi-RaCmesse on a shrine of red granite dedicated by the Pharaoh Nekht- harhebet (Dyn. XXX), the fragments of which were discovered by Professor Naville at Bubastus12. original of the Agathodaemon river in Ptolemy. 2 Op. cit., nos. 143, 231, 276; for no. 264, which may have named Pi-RaCmesse, see no. 37 of last section. 4 See above, p. 249. Since the first part of this article was in print, I have noticed that the inscrip- tion on the Carnarvon scarab, p. 131, under E, ought probably to be translated: " The-Castle-of-Ramesses- Beloved-of-Amrtln-Beloved-like-T7mr on the west of the 'Ity-waters." Where this temple was situated is un- known. There is as yet no reason to think that a town of Ramesses was built around it. gyp g 2 Skn is probably a derivative of Hebrew 1:r in spite of the determinatives of water; these may be borrowed fiom 'Ity by a kind of anticipatory attraction; so BURCHARDT, Die altkanaandischen Fremnd- worte, no. 885. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms g p g g p 7 The fictitious writer of the satirical letter contained in Pap. Anastasi I is of mythological name and parentage; he was called Hori, son of Onnophris of Abydos, " born of Tewosret (" the powerful," i.e. Isis) in the district of Brst, the chantress of Bast in Field-of-the-God." See GARDINER, Egyptian Hieratic Texts, vol I p 5 and p 7* notes 2 and 3 8 BRUGSCH Dictionnaire Geographique p 1328 5 BRUGSCH, Dictionnaire Geographique, p. 197. 6 NAVILLE, Goshen, p. 26; ID., Mound of the Jew, pp. 24-5; PETRIE, Hyksos and Israelite Cities, p. 65. It is true that the few fragments of inscription found by Professor Naville at Belbes did mention the goddess Bast and places belonging to the Bubastite nome; but this does not prove that Brst is Belb8s. vol. I, p. 5 and p. 7 , notes 2 and 3. 8 BRUGSCH, Dictionnaire Geographique, p. 132 9 BENEDITE, Le Tombeaut de Ne/erhotpou, P1. III, in Memoires de la Mission Francaise, vol. y 5 BRUGSCH Dictionnaire Geographique p 197 1 Latest and best edition, MAX MULLER, Egyptological Researches, vol. I, Pls. 17-32. 1 Latest and best edition, MAX MULLER, Egyptological Researches, vol. I, Pls. 17-32. 2 Skn is probably a derivative of Hebrew 1:r in spite of the determinatives of water; these may be THE DELTA RESIDENCE OF THE RAMESSIDES 7) it is said that foreign invaders had pitched p , 1 , I!IlI r-^-\ I _IA I I , ,II "tents in front of Pr-Brst and had made (their) habitation2 in the tract of 'Ity3." Now the name 'Ity is determined with the signs for water, and "the tract of 'Ity" must therefore be the region watered by that r k ^ / 'Ity which, as we have seen, is described by the great Edfu geographical text as the " river " of the XIIIth (Heliopolitan) and the pehu of the XXth (Arabian) nome4; hence it is plausible to suppose that 'Ity was the name given to the portion of the Bubastite Nile-arm adjacent to those two nomes. Brugsch bears the responsibility for the conjecture that Brst or Pr-Brst is the modern Belbes (Coptic teX2ec5), a conjecture that has received a quite unmerited degree of acceptance6, seeing that it rests on nothing more than a superficial consonantal resemblance. There is no evidence that the Bubastite branch ran so far east as Belbes, and it may be guessed that Brst and Pr-Brst are but variant names, due to some cause unknown, of Bst and - r( Pr-B?st respectively. At all events it is a fact that in two of the three7 instances where Brst, Pr-Brst occurs it is in connection with the goddess of Bubastus. Brugsch and others have quoted a goddess j% <> nj Brst8; I am unable to lay my hands on the evidence for her, but if such exists, she is probably only Bast with the name abnormally spelt. This view is confirmed by the occurrence of a writing \|\| \1 -. j that is obviously intermediate between Brst and B?st9. The conclusion to be drawn, then, 258 ALAN H. GARDINER 258 Professor Breasted is obviously in error, as also a passage now to be quoted tends to show. In the list of dedications made to the less important local deities by Ramesses III show. In the list of dedications made to the less important local deities by Ramesses III are named 169 serfs who were placed <=> 01 ~ . J f ® l~ OS I l X"iln the House of Bast, lady of Brst, in The-Waters-of-the-Sun" (Pap. Harris, 62a, 2). The locality Brst is referred to again in the great Karnak inscrip- tion of Meneptahl, where (1. 3 Probably so to be read, though the bird-sign actually employed is / and not tlw. THE DELTA RESIDENCE OF THE RAMESSIDES The conclusion to be drawn, then, I!IlI r-^-\ I _IA I I , ,II "tents in front of Pr-Brst and had made (their) habitation2 in the tract of 'Ity3." Now the name 'Ity is determined with the signs for water, and "the tract of 'Ity" must therefore be the region watered by that r k ^ / 'Ity which, as we have seen, is described by the great Edfu geographical text as the " river " of the XIIIth (Heliopolitan) and the pehu of the XXth (Arabian) nome4; hence it is plausible to suppose that 'Ity was the I!IlI r-^-\ I _IA I I , ,II "tents in front of Pr-Brst and had made (their) habitation2 in the tract of 'Ity3." Now the name 'Ity is determined with the signs for water, and "the tract of 'Ity" must Brst and Pr-Brst are but variant names, due to some cause unknown, of Bst and - r( Pr-B?st respectively. At all events it is a fact that in two of the three7 instances where Brst, Pr-Brst occurs it is in connection with the goddess of Bubastus. Brugsch and others have quoted a goddess j% <> nj Brst8; I am unable to lay my hands on the evidence for her, but if such exists, she is probably only Bast with the name abnormally spelt. This view is confirmed by the occurrence of a writing \|\| \1 -. j that is obviously intermediate between Brst and B?st9. The conclusion to be drawn, then, 5 BRUGSCH, Dictionnaire Geographique, p. 197. 6 NAVILLE, Goshen, p. 26; ID., Mound of the Jew, pp. 24-5; PETRIE, Hyksos and Israelite Cities, p. 65. It is true that the few fragments of inscription found by Professor Naville at Belbes did mention the goddess Bast and places belonging to the Bubastite nome; but this does not prove that Brst is Belb8s. 7 The fictitious writer of the satirical letter contained in Pap. Anastasi I is of mythological name and parentage; he was called Hori son of Onnophris of Abydos " born of Tewosret (" the powerful " i e Isis) goddess Bast and places belonging to the Bubastite nome; but this does not prove that Brst is Belb8s. 7 The fictitious writer of the satirical letter contained in Pap. 1 Latest and best edition, MAX MULLER, Egyptological Researches, vol. I, Pls. 17-32. 2 Skn is probably a derivative of Hebrew 1:r in spite of the determinatives of water; these may be borrowed fiom 'Ity by a kind of anticipatory attraction; so BURCHARDT, Die altkanaandischen Fremnd- worte, no. 885. 3 Probably so to be read, though the bird-sign actually employed is / and not tlw. 4 See above, p. 249. Since the first part of this article was in print, I have noticed that the inscrip- tion on the Carnarvon scarab, p. 131, under E, ought probably to be translated: " The-Castle-of-Ramesses- Beloved-of-Amrtln-Beloved-like-T7mr on the west of the 'Ity-waters." Where this temple was situated is un- known. There is as yet no reason to think that a town of Ramesses was built around it. 5 BRUGSCH, Dictionnaire Geographique, p. 197. 6 NAVILLE, Goshen, p. 26; ID., Mound of the Jew, pp. 24-5; PETRIE, Hyksos and Israelite Cities, p. 65. It is true that the few fragments of inscription found by Professor Naville at Belbes did mention the goddess Bast and places belonging to the Bubastite nome; but this does not prove that Brst is Belb8s. 7 The fictitious writer of the satirical letter contained in Pap. Anastasi I is of mythological name and parentage; he was called Hori, son of Onnophris of Abydos, " born of Tewosret (" the powerful," i.e. Isis) in the district of Brst, the chantress of Bast in Field-of-the-God." See GARDINER, Egyptian Hieratic Texts, vol. I, p. 5 and p. 7, notes 2 and 3. 8 BRUGSCH, Dictionnaire Geographique, p. 1328. 9 BENEDITE, Le Tombeaut de Ne/erhotpou, P1. III, in Memoires de la Mission Francaise, vol. v. THE DELTA RESIDENCE OF THE RAMESSIDES Such at least seems the natural way of interpreting the text; but Professor Breasted attaches the supposed local indications to the names themselves, thus obtaining in the case that concerns us "Herd (called) Usimare¢- Miamibn,-L-P-H,- is- the- conTqueror - of- the- Meshwesh - at- the- Waters-of- the-Sun4." Since, however, the victory of Ramesses III over the Meshwesh here alluded to certainly took place in the west of the Delta5, it would necessarily follow that "The- Waters- of-the-Sun" was the name of the western branch of the Nile. This agrees with nothing else that we know about this name; moreover, we have seen that the westernmost branch of the Nile was called " The- Western- Waters," "The- Western-River" (above, p. 130, under 0). he Agathodaemon river in Ptolemy. t., nos. 143, 231, 276; for no. 264, which may have named Pi-RaCmesse, see no. 37 of last 4 This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms INER a passage now to be portant local deities b ced <=> 01 ~ . J l of Brst, in The-Wat again in the great K reign invaders had I I , ,I eir) habitation2 in th water, and "the tra / 'Ity which, as w " river " of the XII s plausible to suppose adjacent to those two or Pr-Brst is the m te unmerited degree ALAN H. GARDINER Professor Breasted is obviously in error, as also a passage now to be quoted tends to show. In the list of dedications made to the less important local deities by Ramesses III are named 169 serfs who were placed <=> 01 ~ . J f ® l~ OS I l X"iln the House of Bast, lady of Brst, in The-Waters-of-the-Sun" (Pap. Harris, 62a, 2). The locality Brst is referred to again in the great Karnak inscrip- tion of Meneptahl, where (1. THE DELTA RESIDENCE OF THE RAMESSIDES 7) it is said that foreign invaders had pitched p , 1 , show. In the list of dedications made to the less important local deities by Ramesses III are named 169 serfs who were placed <=> 01 ~ . J f ® l~ OS I l X"iln the House of Bast, lady of Brst, in The-Waters-of-the-Sun" (Pap. Harris, 62a, 2). The locality Brst is referred to again in the great Karnak inscrip- tion of Meneptahl, where (1. 7) it is said that foreign invaders had pitched p , 1 , I!IlI r-^-\ I _IA I I , ,II "tents in front of Pr-Brst and had made (their) habitation2 in the tract of 'Ity3." Now the name 'Ity is determined with the signs for water, and "the tract of 'Ity" must therefore be the region watered by that r k ^ / 'Ity which, as we have seen, is described by the great Edfu geographical text as the " river " of the XIIIth (Heliopolitan) and the pehu of the XXth (Arabian) nome4; hence it is plausible to suppose that 'Ity was the name given to the portion of the Bubastite Nile-arm adjacent to those two nomes. Brugsch bears the responsibility for the conjecture that Brst or Pr-Brst is the modern Belbes (Coptic teX2ec5), a conjecture that has received a quite unmerited degree of acceptance6, seeing that it rests on nothing more than a superficial consonantal resemblance. There is no evidence that the Bubastite branch ran so far east as Belbes, and it may be guessed that Brst and Pr-Brst are but variant names, due to some cause unknown, of Bst and - r( Pr-B?st respectively. At all events it is a fact that in two of the three7 instances where Brst, Pr-Brst occurs it is in connection with the goddess of Bubastus. Brugsch and others have quoted a goddess j% <> nj Brst8; I am unable to lay my hands on the evidence for her, but if such exists, she is probably only Bast with the name abnormally spelt. This view is confirmed by the occurrence of a writing \|\| \1 -. j that is obviously intermediate between Brst and B?st9. p p , 3 BRUGSCH, Dictionnaire Geographique, pp. 439-40, 1247-8. For " The-Western-Border," see MARIETTE, Monuments Divers, 277, and several other passages that all throw light on its extension. From Pap. Harris 77, 1 it is clear that it extended from Memphis in a north-westerly direction. In the accounts of Meneptah's defeat of the Libyans it is twice said that this occurred in " The-Western-Border " viz., on the Athribis stele, Zeitschrift fur dgyptische Sprache, vol. xxi [1883], p. 66, and in a new fragment of the Karnak text, Recueil de Travaux, vol. xxxi, p. 179; and it is clear that it is in this sense that p/ rd is to be understood in 1. 30 of the Karnak inscription, where the locality is more definitely specified under the name Pr-lrw. In Pap. Bologna 1094, 7, 3 the writer of a letter addresses his prayers to the gods of "The-Western-Border," and later on (7, 9), when narrating events of local interest, mentions Pr-'Irw. No doubt Golenischeff is wrong in identifying this Pr-irw with a place south of Herakleopolis Magna mentioned in his hieratic glossary (Zeitschrift fur dgyptische Sprache, vol. XL [1902-3], p. 102); nor is the name to be read Pr-ir-sps, and Brugsch's identification with Prosopis therefore falls to the ground. pp 2 It had military officers of its own, who were apparently subordinated to the commandant of Theku in the Wady Tfimilat, Pap. Anastasi V, 25, 3. 4; their province of action must have been the nome of Arabia and the parts east of it. See also Inscriptions of Sinai, no.'295. ame to be read Pr ir sps, and Brugsch s identification with Prosopis t 4 Pap Harris 500 verso 5 2; SPIEGELBERG Rechnungen p 44 This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 1 Annales du Service, vol. xi, pp. 142-4. 2 It had military officers of its own, who were apparently subordinated to the commandant of Theku in the Wady Tfimilat, Pap. Anastasi V, 25, 3. 4; their province of action must have been the nome of Arabia and the parts east of it. See also Inscriptions of Sinai, no.'295. 3 BRUGSCH, Dictionnaire Geographique, pp. 439-40, 1247-8. For " The-Western-Border," see MARIETTE, Monuments Divers, 277, and several other passages that all throw light on its extension. From Pap. Harris 77, 1 it is clear that it extended from Memphis in a north-westerly direction. In the accounts of Meneptah's defeat of the Libyans it is twice said that this occurred in " The-Western-Border " viz., on the Athribis stele, Zeitschrift fur dgyptische Sprache, vol. xxi [1883], p. 66, and in a new fragment of the Karnak text, Recueil de Travaux, vol. xxxi, p. 179; and it is clear that it is in this sense that p/ rd is to be understood in 1. 30 of the Karnak inscription, where the locality is more definitely specified under the name Pr-lrw. In Pap. Bologna 1094, 7, 3 the writer of a letter addresses his prayers to the gods of "The-Western-Border," and later on (7, 9), when narrating events of local interest, mentions Pr-'Irw. No doubt Golenischeff is wrong in identifying this Pr-irw with a place south of Herakleopolis Magna mentioned in his hieratic glossary (Zeitschrift fur dgyptische Sprache, vol. XL [1902-3], p. 102); nor is the name to be read Pr-ir-sps, and Brugsch's identification with Prosopis therefore falls to the ground. 4 Pap. Harris 500, verso 5, 2; SPIEGELBERG, Rechnungen, p. 44. 5 Pap. Anastasi 1, 22, 2; 27, 5. 6 Recueil de Travaux, vol. xviii, p. 53. 34 2 5 Pap. Anastasi 1, 22, 2; 27, 5. 6 Recueil de Travaux, vol. xviii, p. 53. 4 Pap. Harris 500, verso 5, 2; SPIE 5 Pap. Anastasi 1, 22, 2; 27, 5. THE DELTA RESIDENCE OF THE RAMESSIDES Anastasi I is of mythological name and parentage; he was called Hori, son of Onnophris of Abydos, " born of Tewosret (" the powerful," i.e. Isis) in the district of Brst, the chantress of Bast in Field-of-the-God." See GARDINER, Egyptian Hieratic Texts, vol I, p 5 and p 7, notes 2 and 3 8 BRUGSCH, Dictionnaire Geographique, p 1328 7 The fictitious writer of the satirical letter contained in Pap. Anastasi I is of mythological name and parentage; he was called Hori, son of Onnophris of Abydos, " born of Tewosret (" the powerful," i.e. Isis) in the district of Brst, the chantress of Bast in Field-of-the-God." See GARDINER, Egyptian Hieratic Texts, vol I p 5 and p 7* notes 2 and 3 8 BRUGSCH Dictionnaire Geographique p 1328 vol. I, p. 5 and p. 7 , notes 2 and 3. 8 BRUGSCH, Dictionnaire Geographique, p. 1328. 9 BENEDITE, Le Tombeaut de Ne/erhotpou, P1. III, in Memoires de la Mission Francaise, vol. v. THE DELTA RESIDENCE OF THE RAMESSIDES 259 from the passage in Pap. Harris which gave rise to this discussion of Brst, Pr-Brst is that "The- Waters-of-the-Sun" was the name of the Nile-arm that ran past Bubastus. The designation is a natural one, for the Bubastite branch left the main river (the "Great River") near Heliopolis, to whose god it appears to owe its name. We have only to suppose that "The- Waters-of-the-Sun" was the term applied to the entire Bubastite branch in order to understand how that term could be used in connection with Pi-RaCmesse. Pi-RaCmesse was near the sea, and accordingly lay near the mouth of the Bubastite Nile-arm. But the Bubastite Nile-arm debouched in the Pelusiac mouth, and consequently we are again led back to the conclusion that Pi-RaCmesse is Pelusium. With this view of "The- Waters-of-the-Sun" agrees the fact that the entire region lying along this branch seems to have been called 2x, ^Si PI-rd-n-p?-RC, "The-Border-of-the-Sun"; a prince of this region has left a statue at Saft el- Henneh1. Brugsch rightly conjectured that "The-Border-of-the-Sun" represented the eastern margin of the Delta2 just as XI 16 E I P?-rd-tnity," The- Western- Border," was the name given to its western margin3; in two places & f jI P?-rd-'lbty, "The-Eastern-Border," is mentioned4, and either is identical with "The- Border-of-the-Sun" or else has a wider extension but includes it. ame to be read Pr ir sps, and Brugsch s identification with Prosopis 4 Pap. Harris 500, verso 5, 2; SPIEGELBERG, Rechnungen, p. 44. 5 Pap. Anastasi 1, 22, 2; 27, 5. 6 Recueil de Travaux, vol. xviii, p. 53. 1 Annales du Service, vol. xi, pp. 142-4. THE DELTA RESIDENCE OF THE RAMESSIDES Other place-names in the same general region into the composition of which RC, P]-RC enters are sc t- I j d ~ .fJ ~ W]ht-R¢-m-Pr-B?st, "Settlement-of-ReC-in-Bubastus," which clearly cannot have been far from Bubastus5, and , A -D P?-grg-p?-RB, " The-Foundation- of-the-Sun," attested by an inscription of Takelothis II as being somewhere in this vicinity6 y Thus the identification of Pi-RaCmesse with Pelusium is further suggested by its situation on the banks of " The- Waters-of-the-Sun," a good wine-growing district, as the 34-2 This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms ATLAN H. GARDINER 260 potsherds from the Ramesseum indicate. Pelusium is not mentioned by the classical writers as especially famous for its wines, but as we have seen, the wine of SinW, Swn enjoyed a high repute in Pharaonic Egypt from the very earliest times. That Pi-RaCmesse was in the centre of a great wine-growing region is proved by the praise bestowed upon the famous vineyards of Kenkeme; for these see nos. 15, 26 and perhaps no. 37, together with the notes thereupon. Pelusium was also a great place in Graeco-Roman times for the drying and salting of fish, and for the cultivation of lentils; in the Revenue papyrus it is a commercial port for oil rivalling even Alexandria. These conditions generally agree well with the account of Pi-RaCmesse given in Pap. Anastasi III (no. 15). g p Strabo (c. 803) says that Pelusiuin was more than twenty stades from the sea, i.e. some 21 miles, a distance which corresponds pretty well to modern conditions. Lying beside the Bubastite Nile-arm it was to all intents and purposes a sea-port. It seems not impossible-though for this we have no very tangible evidence-that the actual harbour of Pi-Racmesse was known as "[] (f r lt "the Port of Usimare¢-setpenr¢ " or simply X '~2~ i "the Port." In a model letter its g Strabo (c. 803) says that Pelusiuin was more than twenty stades from the sea, i.e. some 21 miles, a distance which corresponds pretty well to modern conditions. Lying beside the Bubastite Nile-arm it was to all intents and purposes a sea-port. THE DELTA RESIDENCE OF THE RAMESSIDES It seems not impossible-though for this we have no very tangible evidence-that the actual harbour of Pi-Racmesse was known as "[] (f r lt "the Port of Usimare¢-setpenr¢ " or simply X '~2~ i "the Port." In a model letter its writer describes how he sought two boats: "I weent southwards upon the edge of the water (the sea-shore ?) and found them in the Port of Usimaric-setpenriC in charge of the Fan- bearer Rekhyl." On several wine-jars discovered in the Raimesseum mention is made of "wine of the vineyard of the Castle-of- Usimarec-setpenre¢-in-the-House-of-Am n (i.e. the Ramesseum) on the west of the Port2," and once the entry "much oil from the Port" occurs in a list of imported oils and the like3. Whether this guess (for it is nothing more) be true or not, it is now familiar to us that Pi-Racmesse was considered as a port (see nos. 15, 16), and may therefore well have been Pelusium. y The last reason to be invoked in favour of the position here accorded to Pi-RaCmesse is its suitability from a historical point of view. The Hyksos had established themselves in a fortress-town midway between Palestine and the main valley of the Nile, and had thus created a precedent which Ramesses II may well have copied. In the flourishing period of the Eighteenth Dynasty the Pharaohs were powerful enough, and sufficiently little apprehensive of Eastern aggression, to rule their Syrian Empire from Thebes and Memphis. In the Nineteenth Dynasty, however, that Empire was no longer secure, but required careful watching fromn a central positionl; therefore Ramesses II, or it may even have been his grandfather Ramesses I4, founded a new Residence near Pelusium, much as 1 Pap. Leyden 348, 8, 6-7, cf. also 9, 2; " edge " (spt) here is an emendation, but a practically cer- tain one. 4 In 1913 M. Legrain discovered near the pylon of Hareinhab at Karnak two statues dating fromn that reign and belonging to a Vizier PraCmesse son of Setoy (Annales du Service, vol. xiv, pp, 29 38); in publishing the find M. Legrain makes the very probable suggestion that this Vizier was none other than Ramesses I. Recently (op. cit., vol. xvii, p. 168) M. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms 3 A nastasi IV, 14, 4. 2 SPIEGELBERG, Hieratic Ostraka, nos. 239, 262, 299. 1 Pap. Leyden 348, 8, 6-7, cf. also 9, 2; " edge " (spt) here is an emendation, but a practically cer- tain one. 1 Pap. Leyden 348, 8, 6-7, cf. also 9, 2; " edge " (spt) here is an emendation, but a practically cer- tain one. 2 SPIEGELBERG, Hieratic Ostraka, nos. 239, 262, 299. 3 A nastasi IV, 14, 4. 4 In 1913 M. Legrain discovered near the pylon of Hareinhab at Karnak two statues dating fromn that reign and belonging to a Vizier PraCmesse son of Setoy (Annales du Service, vol. xiv, pp, 29 38); in publishing the find M. Legrain makes the very probable suggestion that this Vizier was none other than Ramesses I. Recently (op. cit., vol. xvii, p. 168) M. Daressy has called attention to the title borne by him "overseer of the river-mouths " and also to the name of his father, which is simply that of the god Setekh found on the monuments of Tanis. Hence M. Daressy concludes that the family of the Rames- sides originated in these parts. This notion appears to me by no means improbable, though, from what we have seen, all that M. Daressy says about the connection between Setekh and Tanis must be trans- ferred to the site of Avaris and Pi-RaCmesse near Pelusium. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms THE DELTA RESIDENCE OF THE RAMESSIDES Daressy has called attention to the title borne by him "overseer of the river-mouths " and also to the name of his father, which is simply that of the god Setekh found on the monuments of Tanis. Hence M. Daressy concludes that the family of the Rames- sides originated in these parts. This notion appears to me by no means improbable, though, from what we have seen, all that M. Daressy says about the connection between Setekh and Tanis must be trans- ferred to the site of Avaris and Pi-RaCmesse near Pelusium. THE DELTA RESIDENCE OF THE RAMESSIDES 261 Peter the Great, needing to guard his Baltic provinces, removed his capital from Moscow to St Petersburg. Later, the Egyptian hold over Syria became so insecure that the Delta capital was withdrawn to Tanis. Such appears to have been the general sequence of events. From the number of serfs of Amun resident in Pi-RaCmesse under Ramesses III (no. 27) we learn that it must have been a town of considerable size. The officials and domestics of the Court will certainly have been numerous, and since the place became a great adminis- trative centre it must undoubtedly have ranked among the larger cities of Egypt, though never rivalling with Thebes, Memphis or Heliopolis. It owed its prominence to the favour bestowed upon it by all the Pharaohs from Ramesses II to Ramesses III, as well as to its strategic importance. 1 There is no evidence whatever that the temple " T/he-Castle-of-Ramesses-Beloved-of-A m Sn-Beloved- like-Tum-on-the-west-of-the-'Ity-waters" on the Carnarvon scarab (p. 131, E and p. 258, footnote 4) represents a town of Ramesses, so that this alternative may be left out of account. (a) Genesis xlvi, 28-29: " A nd he (Jacob) sent Judah before him unto Joseph, to shew the way before him unto Goshen; and they came into the land of Goshen. And Joseph made ready his chariot, and went up to meet Israel his father, to Goshen." The Hebrew text does not mention the name of Rameses; but the LXX presents the version : "And he sent Judah before them to Joseph to come to meet him at Heroonpolis into the land of Ramesses (avvavreo-at avrTo KaO' 'Hpwowv roXLtv elv y~v 'Patsec-r'r). And Joseph, having yoked his chariot, went up to V GARDINER 262 meet Israel his father at Heroonpolis (eis rvvavrrtilv 'Io-paw)X rTO rarpl avrov KaO' 'Hpccov irOXtv)." Whereas the Sahidic version' retains the name Heroonpolis in both places (KTua, 2,pwon Tuno3lc), the Boheiric version has p. noe-toJa fiKi _Sen HIR K&i npajuaccH "to the town of Pithom in the land of Ramasse" and enc c Aue¢wtxo ti&KI "towards the town of Pithom." meet Israel his father at Heroonpolis (eis rvvavrrtilv 'Io-paw)X rTO rarpl avrov KaO' 'Hpccov irOXtv)." Whereas the Sahidic version' retains the name Heroonpolis in both places (KTua, 2,pwon Tuno3lc), the Boheiric version has p. noe-toJa fiKi _Sen HIR K&i npajuaccH "to the town of Pithom in the land of Ramasse" and enc c Aue¢wtxo ti&KI "towards the town of Pithom." ¢ The LXX presents a twofold expansion of the phrase "into the land of Goshen": first the words "at Heroonpolis" are of the nature of a topographical gloss based on Exodus i, 11 (below c), and second, "into the land of Raresses" is a harmonistic rendering derived from xlvii, 11 (below b). g , ( ) Josephus, Arch. Jud., ed. NABER, II, 184, says that the meeting-place of Jacob and Joseph was Heroonpolis. g , Josephus, Arch. Jud., ed. NABER, II, 184, says that the meeting-place of Jacob and Joseph was Heroonpolis. (b) Genesis xlvii, 11: " And Joseph placed his father and his brethren, and gave them a possession in the land of Egypt, in the best of the land, in the land of Rameses, as Pharaoh had commanded." (b) Genesis xlvii, 11: " And Joseph placed his father and his brethren, and gave them a possession in the land of Egypt, in the best of the land, in the land of Rameses, as Pharaoh had commanded." gyp , , , The command of Pharaoh (xlvii, 6) actually was: "in the land of Goshen let them dwell"; clearly "the land of Rameses " here is synonymous with " the land of Goshen," a standpoint that was in the mind of the LXX translators of xlvi, 28-29 (above a). See Mission Archeologique Francaise, vol. vi, p. 26. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms V , p ( , & / q Q , The Targum of Palestine paraphrases: " And they builded walled cities to become Phar places, Tanis2 and Pilusin "; the Targumn of Jerusalem gives the names in the same form. (d) Exodus xii, 37: " And the children of Israel journeyed from Rameses to Succoth." This follows immediately upon the smiting of the first-born, upon the injunction to depart given by night to Moses and Aaron, and upon the spoiling of the Egyptians by the taking of their gold and silver. , p p g gyp y g g Targum of Palestine: "And the sons of Israel moved forth from Pilusin towards Succoth." g (e) Numbers xxxiii, 3-6: " And they journeyed from Rameses in the first month...on the morrow after the passover And the children of Israel journeyed from Rameses and pitched in &tccoth " g (e) Numbers xxxiii, 3-6: " And they journeyed from Rameses in the first month...on the morrow after the passover... .And the children of Israel journeyed from Rameses, and pitched in &tccoth." (e) Numbers xxxiii, 3-6: " And they journeyed from Rameses in the first month...on the morrow af the passover... .And the children of Israel journeyed from Rameses, and pitched in &tccoth." Targum of Palestine: " Pelusin " for Rameses in both cases; so too Targum of Jerusalem. y j y the passover... .And the children of Israel journeyed from Rameses, and pitched in &tccoth." Targum of Palestine: " Pelusin " for Rameses in both cases; so too Targum of Jerusalem. the passover... .And the children of Israel journeyed from Rameses, and pitched in &tccoth. Targum of Palestine: " Pelusin " for Rameses in both cases; so too Targum of Jerusalem. p j y , p & Targum of Palestine: " Pelusin " for Rameses in both cases; so too Targum of Jerusalem. To these passages must be added from the Apocrypha: (f) Judith i, 7-10: "And Nebuchadnezzar king of the Assyrians sent unto all that dwelt in Persia, .., and to all that were in Samaria and the cities thereof, and beyond Jordan unto Jerusalem, and Betane, and Chellus, and Kadesh, and the river of Egypt, and Tahpanhes, and Rameses, and all the land of Goshen, until thou comest above Tanis and Memphis, and to all that dwelt in Egypt, until thou comest to the borders of Ethiopia " iopia. See Mission Archeologique Francaise, vol. vi, p. 26. 2 It is curious that the Targum of Palestine consistently calls Poti-phera " the rabba of Tanis," while the Hebrew original has " priest of On " (Gen. xlii, 45, 50; xlvi, 20). See below, p. 265, footnote 2. V Biblical chronology assigns the king under whom Joseph lived to the Hyksos period; be this as it may, the phrase "tht land of Ramieses" must in any case be a crass anachronism, or, as the commentators more politely say, is used " proleptically." gyp , , , The command of Pharaoh (xlvii, 6) actually was: "in the land of Goshen let them dwell"; clearly "the land of Rameses " here is synonymous with " the land of Goshen," a standpoint that was in the mind of the LXX translators of xlvi, 28-29 (above a). Biblical chronology assigns the king under whom Joseph lived to the Hyksos period; be this as it may, the phrase "tht land of Ramieses" must in any case be a crass anachronism, or, as the commentators more politely say, is used " proleptically." anachronism, or, as the commentators more politely say, is used proleptically. The Targum of Palestine has: " And Joseph brought his father and brethren to dwell, and gave them a possession in the land of Mizraim, in a goodly part of the country, in the country of Pilusin, as Pharaoh had commanded." Targum Jerushalmi: " Pelusim"; it is quite evident that Pelusium is intended. , , p y y, p p y The Targum of Palestine has: " And Joseph brought his father and brethren to dwell, and gave them a possession in the land of Mizraim, in a goodly part of the country, in the country of Pilusin, as Pharaoh had commanded." Targum Jerushalmi: " Pelusim"; it is quite evident that Pelusium is intended. had commanded. Targum Jerushalmi Pelusim ; it is quite evident that Pelusium is intended. Josephus, Arch. Jud., ed. NABER, nI, 188, states that the place where Jacob was given leave to dwell was Heliopolis. had commanded. Targum Jerushalmi Pelusim ; it is quite evident that Pelusium is intended. Josephus, Arch. Jud., ed. NABER, nI, 188, states that the place where Jacob was given leave to dwell was Heliopolis. (c) Exodus i, 11: And they built for Pharaoh store cities, Pithom and Raamses. LXX has "strong cities" (7roXet o3Xvpas) for nrbl3D=) "store-cities"; the Hebrew term is explained to mean cities for provisions, materials of war, etc., possibly also cities used as trade emporia. LXX further adds "and On, which is Heliopolis" (r,>v Tr IIEtO& Kat 'Pa/eacraq Kat "Qv, oritv 'HXiov ordX-t). V So far as the Biblical city of Raamses-Rameses is concerned, the plan of this essay has been less to solve the problem than to provide the material for its solution. An estimate of the historical value of the Exodus narrative obviously lies outside the province of the Egyptologist; nor indeed can our quest be so extended as to include an exhaustive revision of the geography of the Exodus-route in the light of the Egyptian sources. Nevertheless, the conclusions here reached do seem to provide a basis for further deductions. These will be suggested in a tentative way, and it will remain for Biblical students to reject or to confirm them. Of the towns deriving their name from the Pharaoh Ramesses the foregoing investiga- tions have elicited only two which could with any plausibility be claimed as the original of the store-city mentioned in Exodus i, 11. One is the Residence-city at or near Pelusium, and the other the station named "The-Dwelling-of-Ramesses-Beloved-of-Amun" on the high-road from Thel (Sile) to Raphia (above p. 132, under H)1. The latter has been up to the present without an advocate, and is likely to remain so; it seems to have been a place of no great size or importance, and its name contains in its composition an unusual and characteristic element, "The-Dwelling" (t? t), the omission of which to yield the shortened place-name " Raamses" is highly improbable. On the other hand there is no difficulty in identifying the name of the Biblical Raamses with that of the Ramesside Delta Capital (see above, p. 138). The issue before us may, therefore, be defined: either Raamnses-Rameses of the Bible is the Residence-city of the Ramnessides near Pelusiumn, or else it is a town unknown to the Egyptian monuments, the existence of which is merely postulated. gyp y p In order that the problem may be the more clearly focussed, the various O.T. passages in which the name of Rameses or Raamses occurs are here set forth seriatim, with a few supplementary and explicative notes. 1 There is no evidence whatever that the temple " T/he-Castle-of-Ramesses-Beloved-of-A m Sn-Beloved- like-Tum-on-the-west-of-the-'Ity-waters" on the Carnarvon scarab (p. 131, E and p. 258, footnote 4) represents a town of Ramesses, so that this alternative may be left out of account. ALAN H. V The land of Goshen " here evidently means the Delta, and is opposed to Egypt, i.e. Upper Egypt. No great attention need be paid to the passage in Judith, the writer of which seems simply to have chosen some of the commoner Egyptian place-names familiar to him from the canonical scriptures; we may note, however, in passing, that Rameses is distinguished from Tanis. It is upon the other five passages that tradition and modern exegesis alike have based their hypothesis of a town of Ramesses in the Wady Timilat. So far as tradition is concerned, the only quite definite evidence of the existence of such a view is This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms THE DELTA RESIDENCE OF THE RAMESSIDES 263 a passage in the narrative of the Abbess Aetheria, the pilgrim lady from Gallia Narbonensis, the account of whose travels in Biblical lands (533-540 A.D.) is preserved in the Library of Arezzo. On her return journey from Sinai to Egypt via Clysma (Suez) and the Wady Tumllat she visits in turn the supposed sites of Migdol, Baal-zephon, Etham, Succoth, Pithom, Heroonpolis, Ramineses and Arabia, the last of which she says is a townii in the land of Goshen1. The two places Heroonpolis and Arabia are not alluded to in the Hebrew text of the Bible, but are derived from the Septuagint, on which, consequently, Aetheria's narrative is largely dependent. In both cases Aetheria, or the authorities upon whom she relies, has gone astray in her interpretation; for she distinguishes Heroonpolis from Pithom, whereas it was precisely the identity of those two places, as we shall see, which induced the LXX translator to substitute mcau' 'Hpcwv 7ro\Xtv for the Hebrew "Goshen" in Genesis xlvi, 28-9 (above a); and again, she inverts the relationship of the terms in the expressioun " Geshem of Arabia " used by the LXX for Hebrew " Goshen " in Gen. xlv, 10 (cf. xlvi, 34), interpreting Arabia as a city and Geshem as a land, the exact antithesis of the Greek translator's intention. Now both the city of Heroonpolis and that of Arabia were in Aetheria's day populous places, bearing just those names; Heroonpolis she describes as a large village, with a church, saints' tombs and many monasteries2, and Arabia we know to have been the seat of a bishopric. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms V Excavation has shown that Heroonpolis was, at all events, not very far from Tell el-Maskhuteh; and it is practically certain that Pithom and Heroonpolis are identical3. The town of Arabia is stated by the Oxford list of bishoprics4 to be the same as Fakus, otherwise one would have sought for it a position nearer, like Saft el-Henneh, to the mouth of the Wady Timilat. These two towns, ac- cordingly, are the fixed points in Aetheria's narrative. She may have concluded from Exodus i, 11 (c), that Pithom and Rameses were towns not very far apart from one another; she will certainly have argued from the words " at Heroonpolis into the land of Ramesses " in Genesis xlvi, 28 (LXX) that Rameses and Heroonpolis were close lose together; finally, since Rameses was the starting-point of the Exodus (passages d, e), this will have been in the land of Goshen, i.e. in the neighbourhood of the town of Arabia. Such considerations are enough to account for all Aetheria's learning on the subject of the town of Rameses. What she has to tell us about it is as follows: "But from the town of Arabia it is four miles to Ramesses. We, in order to arrive at Arabia, our stopping-place, had to pass through the midst of Harnesses, which town of arnamesses is now fields, so much so that it does not possess a single habitation. It is true that it is visible, since it both was huge in circuit and had many buildings; for its ruins, however tumble-down they may be, appear endless even to this day. But now there is nothing there saving only one huge Thebaii stone, in which two statues have been carved out, huge ones, which they say belonged to the holy me, i.e. to Moses and Aaron, for they say the children of Israel placed them there in their honour5." 1 " Ac sic ergo exeuntes de Hero pervenirnus ad civitatem, quae appellatur Arabia, quae est civitas in terra Jesse." I quote from the extracts of the pilgrimage printed in KUTHMANN, Die Ostgrenze Agyptens, pp. 6--10. eroum autem civitas...nunc est come, sed grandis, quod nos dicimus vicus. Namn ipse vicus habet et martyria et monasteria plurima sanctorum monachorum." pp 2 "Heroum autem civitas...nunc est come, sed grandis, quod nos dicimus vicus. 3 See the appended Note on Pithom and Heroonpolis, below, p. 268. 4 See DE ROUG, Ge'ographie de la Basse Iigypte, p. 155. On this question and on Goshenr generally, see further, Journal of Egyptian Archaeology, vol. v, pp. 218-223. see further, Journal of Egyptian Archaeology, vol. v, pp. 218 223. 5 " De Arabia autem civitate quattuor milia passus sunt in Ramessen. Nos autem, ut veniremus ad mansionem Arabiae, per media Ramesse transivimus, quae Ramessen civitas nunc campus est, ita, ut nec unam habitationem habeat. Paret sane, quoniam et ingens fuit per girum et multas fabricas habuit; pp 2 "Heroum autem civitas...nunc est come, sed grandis, quod nos dicimus vicus. Nam ecclesiam habet et martyria et monasteria plurima sanctorum monachorum." V Namn ecclesiam habet et martyria et monasteria plurima sanctorum monachorum." y p 3 See the appended " Note on Pithom and Heroonpolis," below, p. 268. pp p , , p 4 See DE ROUG, Ge'ographie de la Basse Iigypte, p. 155. On this question and on Goshenr generally, see further, Journal of Egyptian Archaeology, vol. v, pp. 218-223. pp p p 4 See DE ROUG*, Ge'ographie de la Basse Iigypte, p. 155. On this question and on Goshenr generally, see further, Journal of Egyptian Archaeology, vol. v, pp. 218-223. , gyp gy, , pp 5 " De Arabia autem civitate quattuor milia passus sunt in Ramessen. Nos autem, ut veniremus ad mansionem Arabiae, per media Ramesse transivimus, quae Ramessen civitas nunc campus est, ita, ut nec unam habitationem habeat. Paret sane, quoniam et ingens fuit per girum et multas fabricas habuit; , gyp gy, , pp 5 " De Arabia autem civitate quattuor milia passus sunt in Ramessen. Nos autem, ut veniremus ad mansionem Arabiae, per media Ramesse transivimus, quae Ramessen civitas nunc campus est, ita, ut nec unam habitationem habeat. Paret sane, quoniam et ingens fuit per girum et multas fabricas habuit; ALAN H. GARDINER 264 Not much importance need be attached to this passage, which, be it said in passing, is a very fair sample of the pious lady's literary style. Its historical value is no greater than that of the early Jewish tradition which, as has been seen in the comments on passages c, d, e, identified Raamses-Rameses with Pelusium. The latter identification may perhaps seem a strange coincidence, in view of the fact that the Ramesside capital of Pi-RaCmesse has been located at or near Pelusium. Nor, indeed, is it quite impossible that there may be some causal connection here, but its roots are hidden and can no longer be unearthed. For the present, at least, we must measure the value of this Targumic tradition by that of the parallel identification of Pithom with Tanis (see under c). p Going back some centuries to the time of the Septuagint translation, we learn from the variation between the Hebrew and Greek texts in (a) that the translators had made an effort to interpret the Exodus narrative as a consistent whole, and had formed at least some sort of conception of the Exodus geography. V It is impossible to account for the replacing of " Goshen " in the Hebrew text by the Greek "at Heroonpolis" except by regarding this as due to the influence of Exodus i, 11 (c), and to the knowledge that Heroonpolis was merely the Greek name of Pithom. Similarly, the addition "into the land of Ramesses ' resulted from the desire to connect the reference to Goshen in this passage with that in Genesis xlvii, 6, the latter being replaced in the Hebrew text a few verses later (xlvii, 11) by "in the land of Rameses" (b). Since the LXX translator probably understood this phrase to mean "in the land of (the town of) Rameses," as modern commentators, whether rightly or wrongly, have for the most part done', he is likely to have interpreted his own combination of words " at Heroonpolis into the land of Rameses " to mean that the town of Raamses-Raineses was not far from Pithom. But, as we see, this conclusion was the out- come of a harmonistic combination of the data of the Hebrew text, and represented no independent tradition demanding to be taken into account by ourselves. p g y A still earlier tradition, which is persistently ignored by students of the Exodus times, is that of the Psalmist who sang of the " marvellous things " which God did "in the land of Egypt, in the field of Zoan" (lxxviii, 12), "how he set his signs in Egypt, and his wonders in the field of Zoan " (lxxviii, 43). Here we have a quite definite view, at all events, that the scene of Moses' activities was the extreme north of the Delta. Are we to regard the reference to " the field of Zoan," on which see above, pp. 200, 248, as a reminiscence of the times of the Twenty-first Dynasty, when Tanis was the seat of the Lower Egyptian govern- ment ? Or is the phrase a genuine recollection of the fact that the Ramesside Pharaohs, under whom Moses lived, made their Delta residence near Pelusiuin, a place situated in the nome with which the region called " the field of Zoan" was intimately connected ? This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms posuerint. 1 Max Miiller (s.v. "Rameses" in Encyclopaedia Biblica, col. 4012) prefers "to understand Rameses here as having preserved the original sense, namely, that of a royal name." ruinae enim ipsius, quem ad modum collapsae sunt, in hodie infinitae parent. Nunc autem ibi nichil aliud est nIisi tantum unus lapis ingens thebaeus, in quo sunt duae statuae excisae, ingentes, quas dicunt esse sanctorum hominum, id est Moysi et Aaron, nam dicent, eo quod filii Israhel in honore ipsorum eas posuerint " V g y To turn now to modern views: the prevalent notion of a city of Ramesses within and near the mouth of the Wady Tumilat owes its existence to the same kind of standpoint as that which the LXX translator held, and partly indeed to his specific contributions to the problem, namely, the introduction of the names " Heroonpolis " and " Geshem of Arabia." The fixed point around which the whole theory circles is the known position of Pithom- Heroonpolis in the Wady Tumilat (this is now sufficiently confirmed by the excavations at ruinae enim ipsius, quem ad modum collapsae sunt, in hodie infinitae parent. Nunc autem ibi nichil aliud est nIisi tantum unus lapis ingens thebaeus, in quo sunt duae statuae excisae, ingentes, quas dicunt esse sanctorum hominum, id est Moysi et Aaron, nam dicent, eo quod filii Israhel in honore ipsorum eas posuerint " THE DELTA RESIDENCE OF THE RAMESSID 265 Tell el-Maskhiuteh). It is probably tacitly assumned that, since the Israelites, being confined to the land of Goshen, cannot have built cities outside it, the land of Goshen must have included the Wady Tumilat, where Pithom was situated (c); and Raamses, which is coupled with Pithom, is accordingly in the same region too. This is supported by (b), where " in the land of Rameses " corresponds to " in the land of Goshen " a few verses earlier, as well as by the LXX version in (a) already sufficiently discussed. Lastly, when the Egyptians set out upon their journey to Canaan, their starting-point is Rameses (d, e) which must, accordingly, have lain quite at the Egyptian end of the Wady Tumilat. g y q gyp y All this is good logic, and would be valid argument if the narrative of the sojourning in and departure from Egypt were a single homogeneous account, to be regarded as sound historical evidence. It is easy to show, however, that the entire story is clothed in a legendary form, and legend has no care for strict topographical exactitude. Modern critics have pointed out that two irreconcilable views underlie the earlier chapters of Exodus: in some places it is assumed, as throughout Genesis, that the Israelites were living apart in the land of Goshen; elsewhere, on the other hand, it is unmistakably implied that they were dwelling in the midst of the Egyptians'. V If Moses and the Israelites were separated off in the Wady Tumilat, how could Moses " rise up early in the morning and stand before Pharaoh " (Exodus viii, 20) ? Or how could his cradle have been deposited among the flags in the river, to be found by Pharaoh's daughter (Exodus ii, 1-10)? , y g ( , ) It having been established by our researches that the Delta capital of the Ramessides was at Pelusium, or at all events nowhere near the Wady TAmilat, we may feel convinced that there, if anywhere, must have occurred the finding of Moses as a babe and his later controversies with Pharaoh. Thus, those who view the Exodus narrative as authentic history can hardly refuse to deduce from the passages just quoted that at least a part of the Israelites, and among them Moses, dwelt at Pi-RaCmesse. Nor indeed is there in the Biblical passages referring to Raamses-Rameses anything seriously out of keeping with this conclusion. The passage Exodus i, 11 (c) does not necessarily imply that Raamses was near Pithom, and indeed the LXX translator, whose tendencies elsewhere are all in favour of harmonizing the Exodus geography, here adds a third town Heliopolis that was far away from Pithom2. The phrase "the land of Rameses " in a (LXX) and b is puzzling, and in g 1 See DRIVER on Exodus viii, 22, in the Cambridge Bible for Schools. For the view that the Israelites were still in Goshen he quotes viii, 22: " And I will sever in that day the land of Goshen, in which my people dwell, that no swarms of flies shall be there "; also ix, 26: " Only in the land of Goshen, where the children of Israel were, was there no hail." For the opposite view, cf. iii, 21-22: "And it shall come to pass, that, when ye go, ye shall not go empty: but every woman shall ask of her neighbour, and of her that sojourueth in her house, jewels of silver, and jewels of gold, and raiment"; and the similar passage, xi, 2. Driver represents the view of the Israelites' confinement to Goshen as belonging only to J, while E pictures them as living side by side with the Egyptians. But e.g. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms Journ. V xii, 23 (" For the Lord will pass through to smite the Egyptians; and when he seeth the blood upon the lintel, and on the two side posts, the Lord will pass over the door") presumes the standpoint attributed to E, but nevertheless is assigned to J p ) p p , g 2 On the origin of this addition a guess may be hazarded. Heliopolis was the chief home of the deity Tuim, and was therefore a Pi-tfim " House-of-Tum." The mention of Heliopolis will have arisen, accord- ingly, as a gloss on Pithom. Some confirmation of this view may be found in the coincidence that the Targum gives Tanis as the rendering, not only of Pithom here, but also of On in the phrase " Potiphar, priest of On," see above, p. 262, footnote 2; how the Targumic commentator came by the identification of these places with Tanis is unknown, but it is significant that they were associated in his mind. As for Josephus' identification of Goshen with Heliopolis (see above on b), it was probably due to the considera- tions (1) that Jacob would wish to live near Joseph, and (2) that Joseph, being married to the daughter of a Heliopolitan priest, was presumably domiciled at Heliopolis. 5 Journ. ALAN H. GARDINER 266 any case contains a grave anachronism; in Genesis xlvii, 11 it clearly corresponds to "the land of Goshen " in v. 6, but the variation of that name at such close quarters is so unnatural and incongruous, that it is difficult to do otherwise than infer that two discrepant sources have here been clumsily combined, in which case, of course, the term " land of Rameses " would not necessarily be co-extensive with " land of Goshen." As regards d and e, it would now become clear why the children of Israel, starting out on their journey, are said to have journeyed from Rameses-it is because that was the place where Pharaoh dwelt and persecuted them; the only difficulty in these passages is that, if Succoth is ,_(51 Theku towards the eastern end of the Wady Tumilat', this is more than one day's march from the Delta Capital. 1 The identity of riDD. Succoth with Tkw (see below p. 265, in the appended note) was first proposed by Brugsch (Zeitschrift fir dgyptische Sprache, vol. xiii (1875), p. 8); the rendering of the first two consonants is correct, but as regards the termination of the Hebrew name it has to be assumed that this has been altered in accordance with a popular etymology sukkoth =" huts." The identification is now pretty gener- ally accepted and may stand; but there is no longer much reason for it beyond the general correspondence of sound, and the fact that Theku was situated between Egypt and the desert and consequently corresponds passably well with the Exodus data. It may be here noted that in the hieroglyphic lists of nomes Theku is the capital of the VIIIth nome of the Eastern Harpoon. In Greek times this nome is merged in that of Arabia, except in the case of Strabo 805, who places a nome called Phagroriopolites in this region. On the VIIIth Lower Egyptian nome in general see KfUTHMANN op cit pp 27 foll distance as shown by the Egyptian Survey map 1: 50,000 is 8½ miles. 3 See PETRIE, Hykcsos and Jsraelite Cities, Pls. XXVIII-XXXVI c, with pp. 28--34. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms the VIIIth Lower Egyptian nome in general see KfUTHMANN, op. cit., pp. 27 foll. 2 Op. cit., p. 28, Professor Petrie says eight miles, but in Egypt and Israel, p. 33, he distance as shown by the Egyptian Survey map 1: 50,000 is 8½ miles. V p If we take the opposite point of view, namely that the Exodus story, whatever nucleus of truth it may contain, has come down to us in purely legendary garb, we shall not feel disposed to insist upon the distance betwoen Rameses and Succoth, nor to equate " the land of Rameses " exactly with " the land of Goshen." On the contrary, our verdict will be that the Biblical town of Raamses-Rameses keeps alive a dim recollection of the very city where the Pharaohs of the Oppression and of the Exodus actually resided. pp y To sum up: whether or no the Bible narrative be strict history, there is not the least reason for assuming that any other city of Ramesses existed in the Delta besides those elicited from the Egyptian monuments. In other words, the Biblical Raamses-Rameses is identical with the Residence-city of Pi-RaCmesse near Pelusium. And at this point we shall remember that there are two traditions, though both of uncertain value, which are in accord, or can be construed as in accord, with our conclusion: these are the traditions of the Psalmist and of the Targum, both discussed above. Psalmist and of the Targum, both discussed above. It may be objected: this fine-spun argument is all very well in its way, but has not the Biblical town of Raamses-Rameses, in point of fact, been discovered by Professor Petrie at Tell er-Retabeh, eight and a half miles westward of Tell el-Maskhuteh2? The evidence for the identification proves, on close examination, to be more than scanty3. A temple-wall was found on which Ramesses II is seen slaughtering prisoners before u " TAm, lord of Thu (sic)"; "Theku " ought here to be read, as Professor Petrie has himself seen, for among the few other Ramesside temple-fragments discovered there was one mentioning Theku, as well as part of a door-jamb from the tomb of the contemporary com- mandant of Theku. The remaining finds were a stele of Ramesses II recording the defeats 1 The identity of riDD. Succoth with Tkw (see below p. 265, in the appended note) was first proposed by Brugsch (Zeitschrift fir dgyptische Sprache, vol. xiii (1875), p. V 8); the rendering of the first two consonants is correct, but as regards the termination of the Hebrew name it has to be assumed that this has been altered in accordance with a popular etymology sukkoth =" huts." The identification is now pretty gener- ally accepted and may stand; but there is no longer much reason for it beyond the general correspondence of sound, and the fact that Theku was situated between Egypt and the desert and consequently corresponds passably well with the Exodus data. It may be here noted that in the hieroglyphic lists of nomes Theku is the capital of the VIIIth nome of the Eastern Harpoon. In Greek times this nome is merged in that of Arabia, except in the case of Strabo 805, who places a nome called Phagroriopolites in this region. On the VIIIth Lower Egyptian nome in general see KfUTHMANN op cit pp 27 foll the VIIIth Lower Egyptian nome in general see KfUTHMANN, op. cit., pp. 27 foll. 2 Op. cit., p. 28, Professor Petrie says eight miles, but in Egypt and Israel, p. 33, he says ten; the distance as shown by the Egyptian Survey map 1: 50,000 is 8½ miles. THE DELTA RESIDENCE OF THE RAMESSID 267 mflicted on the Shosu (Asiatic Beduins)'; a dyad in red granite representing Ramesses II and the god Tum; foundation deposits of Ramesses III; and various scarabs and minor objects from burials of the Ninth Dynasty onwards. There is nothing here to suggest that the place was called Raamses, and indeed Professor Petrie's commentary shows that the identification is based on erroneous assumptions. He writes2: "All of these discoveries exactly accord with the requirements of the city of Raamses, where both the second and third kings of that name are stated to have worked, and where a store-city was built by the Israelites along with that of Pithom, which is only eight miles distant. The absence of any other Egyptian site suitable to these conditions, which are all fulfilled here, makes it practically certain that this was the city of Raamses named in Exodus." In the first sentences Professor Petrie is obviously referring to the Residence-city, which we now know to have been situated elsewhere; and further on it is unjustifiably concluded from Exodus i, 11 that Raamses and Pithom were close to one another. NOTE ON PITHOM AND HEROONPOLIS. The positions of Pithom, Heroonpolis and Theku (the supposed prototype of Succoth, see above, p. 266, footnote 1) are sufficiently fixed for the purposes of this article, but I am the more desirous to review the problems connected with them as I can no longer adhere to my statement above, p. 128, that the excavations of Professor Naville at Tell el-Maskhfteh finally settled the question of Pithom, which was found to be identical with that site. Most of the issues are discussed elaborately, though not always con- vincingly, in NAVILLE, The Store-city of Pithom, pp. 4 foll. The old view of Lepsius, reiterated Zeitschrift fuiir agyptische Sprache, vol. xxi (1883), pp. 41-53, to the effect that Pithom lay near Tell Abu Suleiman, while Heroonpolis-Raamses was to be sought at or near Tell el-MaskhfAtteh, reposed (1) on the identification of Pithom with Thou in the Antonine Itinerary, the place where the road via Daphnae to Pelusium 1 This stele, as is not unusual in the Ramesside period, consists merely of royal names and epithets, of which the concluding words are very interesting. They run: -> n > T~TJ^ A> ll X A A A Ai , , n Hy1Y/vt I)rnIITjT7AelIp2 I I A/V (Z ')>l"". In Professor Petrie's text this is translated: "(of the) great enemies in the land of the 1 This stele, as is not unusual in the Ramesside period, consists merely of royal names and epithets, of which the concluding words are very interesting. They run: -> n > T~TJ^ A> ll X A A A Ai , , n Hy1Y/vt I)rnIITjT7AelIp2 I I A/V (Z ')>l"". In Professor Petrie's text this is translated: "(of the) great enemies in the land of the 1 This stele, as is not unusual in the Ramesside period, consists merely of royal names and epithets, of which the concluding words are very interesting. They run: -> n > T~TJ^ A> ll X A A A Ai , , n Hy1Y/vt I)rnIITjT7AelIp2 I I A/V (Z ')>l"". In Professor Petrie's text this is translated: "(of the) great enemies in the land of the j p rofessor Petrie's text this is translated: "(of the) great enemies in the land of th y j p (Z ')>l"". V No additional argument is offered beyond the daring suggestion that the dyad of Ramesses II and Tuim is none other than Aetheria's "lapis ingens thebaeus, in quo sunt duae statuae excisae, ingentes, quas dicunt esse sanctorum hominum, id est Moysi et Aaron3." y We may thus feel certain that Tell er-Retabeh is not Raamses, since there is no serious evidence that a town of Ramesses ever existed in this region. Its ancient name is not known to us, unless it, rather than Tell el-Maskhuteh, be the true site of Pithom4; but our incertitude on this point need not trouble us, for there are several places in the Wady Tumil&t still to be identified, and the topography of the whole region is still regrettably obscure. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms , 2 Op. cit., This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms NOTE ON PITHOM AND HEROONPOLIS. Anastasi V, v, 20,1, where the words " the fortress " appear to refer to Theku in 19, 8. Also Theku possessed an 'dnw, the title usually borne by the "commandant" of a military station, cf. op. cit., 25, 2 ; 26, 1. Turning now branched off from the Heliopolis-Hero-Serapiu-Clysma (Suez) road, and (2) upon Herodotus ii, 158 KTraL 8E a7ro rov NE'Xov TO vacop Es avrr,v (scil. TR?v &(opvKa), ?'KTC 8e KaTrv7repOe o'Xiyov Bovf3iomo0 7roAotLs0 7rapa HaTrovpAov T77v 'ApafIr'v IrOXLv. As to (1), the suggestion would hardly have arisen but for the ill-supported various reading Tlioum, which is certainly to be rejected; as to (2), this much-disputed passage (see NAVILLE, op. cit., pp. 34-9) is quite plausibly taken by Dillmann and others to mean that the water of the canal was drawn away from the Nile not at, but alongside, Pithom, which may thus be at some distance from the point of junction. The view of Brugsch that Pithom was near Lake Menzaleh hangs together with his whole Exodus theory, some portion of which (the identity of Tanis and Raamses) has been disproved in the present article. At the present time the only scholar who appears to disagree with Prof. Naville's con- clusions is M. Daressy, see Bulletin de l'Institut .gyptien, vol. 5 (1911), p. 3. The ground may be cleared by considering the relations of Pithom and Heroonpolis; the identity of these two places may be proved both (1) on traditional and (2) on linguistic grounds. (1) The Boheiric version of Genesis xlvi, 28 has Pethomn for LXX Heroonpolis, see passage a on p. 261; and as I have shownl (p. 264), Heroonpolis in the LXX trans- lation was due simply to the presence of Pithom in Exodus i, 11. (2) The variants Eron (Geogr. Rav.), Ero (inscriptions from Maskhfiteh), Ero and Hero (Aetheria) and 'Hpco (Steph. Byz.) suggest that the original Greek name of the city was not Heroonpolis, but Heropolis, being derived from the name of an obscure deity 'Hpcov of whom there was a temple at Magdola (see GRENFELL-HUNT, Tebtunis Papyri, no. 80, intro- duction); and this is confirmed by the existence, in the translation of the (Flaminian ?) obelisk preserved by Ammianus Marcellinus, of a royal epithet 'Hpovos vios, which, in spite of the caveat entered by Sethe apud ERMAN, Die Obeliskeniibersetzung des Hernapion, p. 253, footnote 3, in Sitzb. d. NOTE ON PITHOM AND HEROONPOLIS. Berliner Akad., 1914, ix, must surely correspond to sl Tm " son of Tfm " on the Flaminian obelisk. Thus the name Heropolis, later misunderstood as Heroonpolis, would originally have been a simple translation of Pithom. The ex- planations of the name Hero given by Lepsius (op. cit., p. 52) and Naville (op. cit., p. 10) are quite un- satisfactory. Now it cannot be denied that the evidence for the identification of Pithom-Heroonpolis with Maskhlfteh provided by Prof. Naville's excavations there is very formidable, seeing that they yielded not only hieroglyphic inscriptions mentioning Pithom, but also Latin inscriptions mentioning Ero. None the less, I incline to think that a still more likely view is that Pithom-Heroonpolis is identical with Tell er- Ret..beh, 8 English miles further westward. As has been already seen (p. 266), " Tum lord of Theku " was here worshipped, as at Maskhftteh, so that theoretically, at least, Tell er-Retabeh can be regarded as a Pi-Tfim, Het-Tfim, "House of Tulm." The testimony of the hieroglyphic inscriptions from Tell el-Maskhfteh shows indisputably that the commonest name of the place was Theku; Theku is mentioned there again and again, whereas Pithom is named only in two documents, namely the Bubastite statue, op. cit., P1. 4, A, D, and the great stele of Ptolemy Philadelphus. Particularly clear evidence that Maskhfteh was Theku is the invocation on the statue, op. cit., P1. 5, A, to the priests " who enter into the temple of Tuim, the great living god in the midst of Theku" and the statement on the great Ptolemaic stele (the so-called Pithom stele, latest edition SETHE, Urkunden, II, 81-105) that this was erected " in front of Tiim, the great living god of Theku." On the same stele (op. cit., 93), when the return of the gods from Persia is being described, it is said that " the gods of Pithom and Theku (one can hardly translate " the gods of Pithom of Theku" as Prof. Naville does) came to rest there" (i.e. in Egypt), and this expression seems to imply that Pithom and Theku were distinguished. For the rest, the Pithom stele affords no evidence that Maskhciteh was Pithom, for it describes the events that took place throughout the entire region, and if it records the establishment of offerings for Pithom (op. cit., 98), it does as much also for another place called Pr-Krht (op. cit., 104). NOTE ON PITHOM AND HEROONPOLIS. In Professor Petrie's text this is translated: "(of the) great enemies in the land of the Bedawyn he plundered their hill fortresses, slaughtering their faces, and building in cities upon which his name is to eternity"; the comment is added that "this allusion to building in the various cities called after Ramesses suggests that this city was one of such-that is, Raamses." The first phrases are simple, but are mistranslated by Professor Petrie; at the end it seems necessary to bring the words kd mn dmnlw somehow into connection with the Asiatics (Shosu), and it is therefore likely that -sn "their" has been omitted. Accordingly I translate: " making a great massacre in the land of the Shosu; he plunders their hills, and slaughters upon them; building in (their) cities in his (own) name for ever." The reference, then, would be to the annexation of towns in Palestine, not to the building of towns of Ramesses in Egypt. 2 Op cit p 28 3 Op cit p 30 4 See below the appended note then, would be to the annexation of towns in Palestine, not to the building of 2 Op. cit., p. 28. 3 Op. cit., p. 30. 4 See below, the , 2 Op. cit., te. 35-2 te. 35-2 H. GARDINER 26 branched off from the Heliopolis-Hero-Serapiu-Clysma (Suez) road, and (2) upon Herodotus ii, 158 KTraL 8E a7ro rov NE'Xov TO vacop Es avrr,v (scil. TR?v &(opvKa), ?'KTC 8e KaTrv7repOe o'Xiyov Bovf3iomo0 7roAotLs0 7rapa HaTrovpAov T77v 'ApafIr'v IrOXLv. As to (1), the suggestion would hardly have arisen but for the ill-supported various reading Tlioum, which is certainly to be rejected; as to (2), this much-disputed passage (see NAVILLE, op. cit., pp. 34-9) is quite plausibly taken by Dillmann and others to mean that the water of the canal was drawn away from the Nile not at, but alongside, Pithom, which may thus be at some distance from the point of junction. The view of Brugsch that Pithom was near Lake Menzaleh hangs together with his whole Exodus theory, some portion of which (the identity of Tanis and Raamses) has been disproved in the present article. At the present time the only scholar who appears to disagree with Prof. Naville's con- clusions is M. Daressy, see Bulletin de l'Institut .gyptien, vol. 5 (1911), p. 3. NOTE ON PITHOM AND HEROONPOLIS. The ground may be cleared by considering the relations of Pithom and Heroonpolis; the identity of these two places may be proved both (1) on traditional and (2) on linguistic grounds. (1) The Boheiric version of Genesis xlvi, 28 has Pethomn for LXX Heroonpolis, see passage a on p. 261; and as I have shownl (p. 264), Heroonpolis in the LXX trans- lation was due simply to the presence of Pithom in Exodus i, 11. (2) The variants Eron (Geogr. Rav.), Ero (inscriptions from Maskhfiteh), Ero and Hero (Aetheria) and 'Hpco (Steph. Byz.) suggest that the original Greek name of the city was not Heroonpolis, but Heropolis, being derived from the name of an obscure deity 'Hpcov of whom there was a temple at Magdola (see GRENFELL-HUNT, Tebtunis Papyri, no. 80, intro- duction); and this is confirmed by the existence, in the translation of the (Flaminian ?) obelisk preserved by Ammianus Marcellinus, of a royal epithet 'Hpovos vios, which, in spite of the caveat entered by Sethe apud ERMAN, Die Obeliskeniibersetzung des Hernapion, p. 253, footnote 3, in Sitzb. d. Berliner Akad., 1914, ix, must surely correspond to sl Tm " son of Tfm " on the Flaminian obelisk. Thus the name Heropolis, later misunderstood as Heroonpolis, would originally have been a simple translation of Pithom. The ex- planations of the name Hero given by Lepsius (op. cit., p. 52) and Naville (op. cit., p. 10) are quite un- satisfactory. Now it cannot be denied that the evidence for the identification of Pithom-Heroonpolis with Maskhlfteh provided by Prof. Naville's excavations there is very formidable, seeing that they yielded not only hieroglyphic inscriptions mentioning Pithom, but also Latin inscriptions mentioning Ero. None the less, I incline to think that a still more likely view is that Pithom-Heroonpolis is identical with Tell er- Ret..beh, 8 English miles further westward. As has been already seen (p. 266), " Tum lord of Theku " was here worshipped, as at Maskhftteh, so that theoretically, at least, Tell er-Retabeh can be regarded as a Pi-Tfim, Het-Tfim, "House of Tulm." The testimony of the hieroglyphic inscriptions from Tell el-Maskhfteh shows indisputably that the commonest name of the place was Theku; Theku is mentioned there again and again, whereas Pithom is named only in two documents, namely the Bubastite statue, op. cit., P1. 4, A, D, and the great stele of Ptolemy Philadelphus. NOTE ON PITHOM AND HEROONPOLIS. Particularly clear evidence that Maskhfteh was Theku is the invocation on the statue, op. cit., P1. 5, A, to the priests " who enter into the temple of Tuim, the great living god in the midst of Theku" and the statement on the great Ptolemaic stele (the so-called Pithom stele, latest edition SETHE, Urkunden, II, 81-105) that this was erected " in front of Tiim, the great living god of Theku." On the same stele (op. cit., 93), when the return of the gods from Persia is being described, it is said that " the gods of Pithom and Theku (one can hardly translate " the gods of Pithom of Theku" as Prof. Naville does) came to rest there" (i.e. in Egypt), and this expression seems to imply that Pithom and Theku were distinguished. For the rest, the Pithom stele affords no evidence that Maskhciteh was Pithom, for it describes the events that took place throughout the entire region, and if it records the establishment of offerings for Pithom (op. cit., 98), it does as much also for another place called Pr-Krht (op. cit., 104). Unfortunately the fact that Maskhtiteh can be shown to be Theku (or, as we had better say, the very heart of Theku, for we shall soon see that the term had a wider, as well as a narrower, significance) does not exclude the possibility that Maskhiteh was also called Pithom; and this, indeed, is the standpoint that Prof. Naville takes. Still, there is evidence that " the fortress of Theku " was to the east of Pithom, for the famous passage Pap. Anastasi VI, 11. 54-7 states " we have finished causing the Beduin tribes of Edom to pass the fortress of Meneptah belonging to Theku towards the pools of Pithom [of] Meneptah belonging to Theku, in order to feed themselves and to feed their flocks." Here Theku appears as a wider term including Pithom, and with this fact we may compare the other that at Tell er-Retabeh the deity worshipped is "Tfum, lord of Theku." Obscure as are the facts, it appears, then, that the most likely con- clusion from the hieroglyhic data is that Maskhfiteh represents the "fortress of Theku" and that Tell er-Retabeh represents Pithom. For further evidence of a fortress at Theku see the passage Pap. NOTE ON PITHOM AND HEROONPOLIS. 10) do suggest that this is "the fortress of Theku." NOTE ON PITHOM AND HEROONPOLIS. Unfortunately the fact that Maskhtiteh can be shown to be Theku (or, as we had better say, the very heart of Theku, for we shall soon see that the term had a wider, as well as a narrower, significance) does not exclude the possibility that Maskhiteh was also called Pithom; and this, indeed, is the standpoint that Prof. Naville takes. Still, there is evidence that " the fortress of Theku " was to the east of Pithom, for the famous passage Pap. Anastasi VI, 11. 54-7 states " we have finished causing the Beduin tribes of Edom to pass the fortress of Meneptah belonging to Theku towards the pools of Pithom [of] Meneptah belonging to Theku, in order to feed themselves and to feed their flocks." Here Theku appears as a wider term including Pithom, and with this fact we may compare the other that at Tell er-Retabeh the deity worshipped is "Tfum, lord of Theku." Obscure as are the facts, it appears, then, that the most likely con- clusion from the hieroglyhic data is that Maskhfiteh represents the "fortress of Theku" and that Tell er-Retabeh represents Pithom. For further evidence of a fortress at Theku see the passage Pap. Anastasi V, v, 20,1, where the words " the fortress " appear to refer to Theku in 19, 8. Also Theku possessed an 'dnw, h l ll b b h " d " f l f 25 2 26 1 T THE DELTA RESIDENCE OF THE RAMESSID 269 to the Latin inscriptions discovered at Maskhuteh, it is, indeed, awkward that two, and not merely one, should mention Ero; but on the other hand the simplest way of interpreting the obscure words on the milestone AB ERO IN CLVSMA vimIII is to understand this to signify " nine miles on the road from Ero to Clysma (Suez)," which would bring Ero westward within a few hundred yards of Tell er-Retibeh; it is curious that scholars should have failed to notice this easy way of reconciling Mommsen's rendering of the Latin words with Prof. Naville's protests against supposing that the milestone had been brought to Maskhfiteh from elsewhere (see on the whole question, NAVILLE, op. cit., pp. 22-24). NOTE ON PITHOM AND HEROONPOLIS. Turning now to the Antonine Itinerary, it will be seen that its data are at least as favourable to the view that Ero is Tell er-Retabeh as they are to the accepted' view that Ero is Maskhfiteh. Unhappily neither of the two places, Thou and Serapiu, with which Hero is there placed in direct relation, has as yet been certainly identified. Thou is placed by Prof. Naville (op. cit., p. 36) at Shugaflyeh near Tell el-Kebir; being, however, the meeting-point of the Pelusium-Daphnae-Heliopolis and the Clysma-Heliopolis roads, this is far too much to the east. Even (Abbaseh is too far eastward, and Saft el-Henneh would be a more suitable position. Now Saft is 30 English miles, as the crow flies, from Tell el-Maskhftteh (the figures here given have been verified on the 1 :50,000 Survey maps), while the Itinerary gives only 24 Roman miles as the distance between Thou and Hero. If, then, Thou was at or near Saft, Tell er-Retabeh (21- English miles) would be a most suitable position for Hero. The situation of Serapiu is even more doubtful than that of Thou; but if it is to be sought, as Prof. Naville supposes (op. cit., p. 25), at the foot of Gebel Maryam, where there are Roman ruins, this is only 12 English miles from Tell el-Masklhateh, whereas the Itinerary gives 18 Roman miles between Hero and Serapiu. In this case Tell er-Retabeh is three to four miles too far away, but is, nevertheless more suitable than Maskhfiteh. It is true that the distances indicated by the Itinerary between Sile and Serapiu, on the one hand, and Serapiu and Clysma (Suez) on the other, seem rather to point to a position near the modern railway-station Serapeum (the identification has been inherited from the authors of the Description de 7l'Eqypte); but against this must be set the fact that Serapiu was the place where the Pelusium-Clysma road parted from the road from Clysma via Hero and Thou to Heliopolis, and that from this standpoint a position near Gebel Maryam would be much more appropriate. In conclusion, we have seen (p. 263) that in Aetheria's time Ero had many monasteries and saints' tombs, besides a church. None of these seems to have been found either at Maskhftteh or at Tell er-Retabeh, so that archaeology here fails us; but the thick surrounding walls of Maskhfteh (op. cit., p. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms VI In conclusion, it will be well to pass in review the results that have been attained. In the first section the cities of Ramesses other than the Delta Capital were enumerated and discussed. There were found to exist towns of Ramesses II near Naucratis (the modern Ramsis), at Abu Simbel and at Derr, besides a small station on the Syrian road east of Kantareh known as " The-Dwelling-of-Ramesses"; a town called after Ramesses III was situated in the north-western Delta. After a careful consideration of the name of the Delta Residence, which was " Pi-RaCmesse," often with the epithet " Great-of- Victories" or, after the death of Ramesses II, " The-Great-Soul-of-the-Sun-Horus-of-the-Horizon" (§ II), the passages where this is mentioned were set forth in chronological order (§ III). It was seen that Pi-RaCmesse was the regular northern abode of the Pharaohs from the reign of Ramesses II to Ramesses III, and was the seat of their Delta government. Hither the taxes were brought, and here were the great public offices. Pi-RaCmesse possessed temples of Amufin, ReC, Ptah and Setekh, besides shrines of other lesser deities. The population was large, though doubtless it could not vie with that of Thebes or Memphis. The surrounding country appears to have been rich in produce of all kinds; in the neighbourhood of Pi- RaCmesse were the celebrated vineyards of Kenkeme. In the fourth section the geographical position of Pi-RaCmesse was discussed. The problem was seen to turn largely upon the relations of the XIVth and XIXth Lower Egyptian nomes, and it was shown that Brugsch, ALAN H. GARDINER 270 in identifying the XIVth nome with the Tanites and the XIXth with the Sethroites, exactly reversed the truth. The association of Pi-RaCmesse with a stretch of water called Sz-Hr "The-Waters-of-Horus" proved that Pi-RaCmesse was connected with the XIVth (Sethroite) nome, of which " Waters-of-Horus " is said to be the " river." From the identi- fication of the Egyptian " Waters-of-Horus " with the Biblical Shihor it became evident that these names could only refer to the lower reaches of the Bubastite or Pelusiac branch of the Nile. But since some quite explicit passages showed that Pi-RaCmesse lay near the sea, the obvious conclusion seemed to be that it was situated either at or near Pelusium, where the said river-arm debouched. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms VI The Egyptian evidence in connection with Pelusium was next discussed, and it was shown that tradition associated Avaris with that town. On the other hand, it appeared likely that the god Seth of Avaris and the god Seth of Pi- Racmesse were identical, so that here again would be evidence in favour of the location of Pi-RaCmesse at Pelusium. Further consideration of the deities of Pi-RaCmesse led to the same result; and in particular the mention, on a monument from Bubastus, of " the temple of PreC of Ramesses of The- Waters-of-the-Sun" recalled the fact that several slightly doubtful passages connected Pi-Racmesse with " The- Waters-of-the-Sun"; the latter term was found to be a name of the Pelusiac Nile-arm, and this, once more, pointed to the identity of Pi-RaCmesse with Pelusiuin. Finally, the choice of Pelusium for the Delta Residence was seen to have been politically opportune, its situation being such that the Ramesside Pharaohs could thence control their ever increasingly restive tributaries in Palestine and Syria. y From the Egyptian city of Residence we passed on to the consideration of the Biblical Raamses-Rameses. It was first pointed out that either the Raamses of Exodus was Pi- Racmesse of the Pharaohs, or else it was another city of which the very existence had to be postulated. A consideration of the Old Testament passages and of their traditional inter- pretation showed that there was no real evidence that a town of Ramesses ever existed in or near the Wady Tumilat; and it was further shown that, whether the Bible narrative be regarded as sound historical evidence or whether it be looked upon as purely legendary, in either case the scene of Moses' birth and struggles with the Pharaoh of the Oppression could only be located at the Delta Residence of the Ramesside Pharaohs, i.e. at Pi-Rarmesse at or near Pelusium. This conclusion proved quite reconcilable with the Biblical passages naming Raamses-Rameses. Lastly, examination showed that Professor Petrie's claim to have discovered Raamses at Tell er-Retabeh lacked justification. ADDITIONS AND CORRECTIONS. It was unfortunately necessary to print a small portion of this essay before the whole investigation was completed, so that a few minor inconsistencies have found their way into the text. It ought to have been stated, at the outset, that the name of Ramses formerly given to the railway-station at Maskhuteh owed its existence to Lepsius' erroneous conclusions con- cerning the topography of the WVady Tuimilat and its neighbourhood. Similarly, a doubtful inference on the part of the topographers of the Description de Vl'gypte forms the basis of the name borne by the modern railway-station of Serapeum. the name borne by the modern railway station of Serapeum. On p. 128, the statements with regard to Pithom and Succoth should be modified in accordance with the conclusions reached in the note on pp. 267-9. THE DELTA RESIDENCE OF THE RAMESSIDES 271 P. 129. A reference in AMfLINEAU, La Geographie de l'Egypte a l'epoque Copte, p. 402, was overlooked. In the first of the stories preserved in the Arabic book called "Forty edifying stories" (Ms. Bibl. Nat., no. 155, fol. 50), one monk says to another: " 0 my father, I am of a village of the land of Alexandria, which is called Ramsls." g , P. 131. The name of the temple mentioned on the Carnarvon scarab is probably to be translated " The-Castle-of-Ramesses-Beloved-of-Amrin-Beloved-like-Tam on the west of the (waters called) 'Ity." See p. 258, footnote 4. g , P. 131. The name of the temple mentioned on the Carnarvon scarab is probably to be translated " The-Castle-of-Ramesses-Beloved-of-Amrin-Beloved-like-Tam on the west of the (waters called) 'Ity." See p. 258, footnote 4. the (waters called) Ity. See p. 258, footnote 4. P. 133. A kneeling statue of the " Royal Son of Kush, PesiAr," a contemp the (waters called) Ity. See p. 258, footnote 4. P. 133. A kneeling statue of the " Royal Son of Kush, PesiAr," a contemp Ramesses II, has an invocation to 0I (- . - 1 "Amen-ReC-in- the-House-of-Ramesses-Beloved-of-Am in"; the owner's title renders it likely that Abu Simbel is here meant, rather than the Delta Residence. The statue is preserved in the British Museum (Exhib. no., 604; Invent, no., 1376); it is figured ARUNDALE-BONOMI, Gallery of Antiquities, P1. This content downloaded from 128.255.6.125 on Thu, 26 May 2016 10:11:29 UTC All use subject to http://about.jstor.org/terms ADDITIONS AND CORRECTIONS. 51, and again in the official Guide to the Egyptian Galleries (1909) p 197 ( ), p The sketch-map of the Eastern Delta accompanying this article (Plate XXXV) has been very carefully drawn by Messrs Emery Walker's skilled map-maker Mr Staton from the latest maps of the Egyptian Survey.
https://openalex.org/W4293213647	https://research-information.bris.ac.uk/files/342177363/Final_Version_IJTM_Shin_Stylos_et_al._2022.pdf	English	null	How does the blockchain find its way in the UAE The blockchain as a sociotechnical system	International journal of technology management	2,022	public-domain	11,559	Link to published version (if available): 10.1504/IJTM.2022.124616 Link to publication record on the Bristol Research Portal PDF-document This is the accepted author manuscript (AAM). The final published version (version of record) is available online via Inderscience at https://doi.org/10.1504/IJTM.2022.124616. Please refer to any applicable terms of use of the publisher. Shin, D., Stylos, N., Asim, M., Park, Y., & Williams, R. (2022). How does the blockchain find its way in the UAE? The blockchain as a sociotechnical system. International Journal of Technology Management, 90(1-2), 122-140. https://doi.org/10.1504/IJTM.2022.124616 Peer reviewed version Shin, D., Stylos, N., Asim, M., Park, Y., & Williams, R. (2022). How does the blockchain find its way in the UAE? The blockchain as a sociotechnical system. International Journal of Technology Management, 90(1-2), 122-140. https://doi.org/10.1504/IJTM.2022.124616 Shin, D., Stylos, N., Asim, M., Park, Y., & Williams, R. (2022). How does the blockchain find its way in the UAE? The blockchain as a sociotechnical system. International Journal of Technology Management, 90(1-2), 122-140. https://doi.org/10.1504/IJTM.2022.124616 University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ SOCIOTECHNICAL BLOCKCHAIN How Does the Blockchain Find Its Way in the UAE? The Blockchain as a Sociotechnical System How Does the Blockchain Find Its Way in the UAE? The Blockchain as a Sociotechnical System Donghee Shin, Ph.D. (Corresponding Author) Professor College of Communication and Media Sciences Zayed University P.O. Box 144534 Abu Dhabi, United Arab Emirates, Office: +971-2-599-3488 email: dshin1030@gmail.com; donghee.shin@zu.ac.ae Abstract Blockchain is emerging as a powerful technological force that promises to change society’s status quo. The UAE has taken a proactive approach to blockchain innovation and development. With the framing of blockchain as a sociotechnical system, blockchain initiatives in the UAE are critically examined in terms of prospects, challenges, and implications for future digital societies. Blockchain developmental trajectories are analyzed from a sociotechnical lens, i.e., societal, technological, industrial, and economic trends that characterize the interfaces and interaction among people, organizations, technology, and society, eliciting underlying potential influences of cultural, ethical, and legal rhetoric. Despite strong initiatives, a series of challenges, such as trust, privacy, and transparency, have emerged as blockchain begins to develop its full potential. With these issues, sociotechnical concerns have increased regarding how to respond to the possible consequences that such a policy choice would entail, how to govern blockchains, and how to create a sustainable blockchain ecosystem. The sociopolitical ramifications of UAE blockchains are critically assessed to establish key features and conceptualize issues to unlock opportunities to guarantee a sociotechnical approach to blockchains. Keywords: Blockchain; trust; sociotechnical perspective; sociotechnical blockchain SOCIOTECHNICAL BLOCKCHAIN How Does the Blockchain Find Its Way in the UAE? The Blockchain as a Sociotechnical System The Blockchain as a Sociotechnical System lockchains have flourished with an amazing growth across the globe (Duan et al., Blockchains have flourished with an amazing growth across the globe (Duan et al., 2020). Blockchain technology is increasingly considered an innovative enabler for addressing social and technical challenges in today’s society (Wamba & Queiroz, 2020). Blockchains have evolved as a vibrant and innovative technology, which proves to be effective and powerful in a number of critical services. Numerous leading industrial firms have been keenly investing in blockchain utilization in order to enhance their portfolio of products. A majority of practitioners believe that blockchain technology will drastically transform the manner we use online applications today (Yang, 2019). The positive promises of a blockchain society are widely recognized, and there is mounting pressure to achieve the dream of a full blockchain-based era (Marsal-Llacuna, 2018). Blockchain will trigger a fundamental paradigm change to a wide range of products and will certainly influence the digital ecology in the world, but it also creates a series of challenges and issues that need to be considered when such a large-scale system is widely integrated into societies (Casino, Macrinici, Cartofeanu & Gao, 2018; Shin & Hwang, 2020). There are mounting concerns as to social, ethical and political concerns (Yeh et al., 2020). One concern is that both public and private sectors have faced glitches in determining justifiable governance and applicable management with regard to blockchain drives (Lim et al., 2021). In the UAE, for instance, while the technical capability of blockchains is rapidly progressing, the related social progress is sluggish, mainly due to complicated concerns and issues. Blockchains have emerged as a new distributed ledger platform that securely saves transactions, and any other data, and confirms them without the need of any centralized SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN mediation (Ghosh, 2019). The innovative features of blockchain, such as immutability, smart contract, security, consensus, and transparency, create negative side effects, namely, privacy, accountability, and fairness (Shin & Hwang, 2020). Security concerns are the most salient ones as most active blockchains are vulnerable to security attacks (Casino et al., 2018; Chang, Chen, & Lu, 2019). The key to sustainable development is tackling the security concerns in blockchains (Joshi, Han, & Wang, 2018). Addressing the privacy and security provision in blockchains has been a key task to make the technology everyday useable and familiarize users with the possibilities it offers (Shin & Hwang, 2020). While these concerns continue to trigger further debates and actions, recent blockchain designs have been industry-leading and technology-driven, leaning toward technical performance at the expense of user rights and societal benefits (Upadhyay, 2020). Thus far, most blockchain development has focused on the supply aspect, or on the overly technical nitty-gritty While these concerns continue to trigger further debates and actions, recent blockchain designs have been industry-leading and technology-driven, leaning toward technical performance at the expense of user rights and societal benefits (Upadhyay, 2020). Thus far, most blockchain development has focused on the supply aspect, or on the overly technical nitty-gritty implementation of distributed ledger systems and commercializing token-based processes (Du et al., 2019). Little effort has been spent on in-depth contextual considerations of the social, ethical, cultural, and legal aspects essential to blockchain design, deployment, and optimization (Shin & Ibarahim, 2020). The ongoing concerns over blockchains such as data integrity, privacy provisions, transparency, and governance have rarely been tackled, in contrast to the proactive technical focus (Duan et al., 2020). The question of how we operationally manage and strategically maneuver the evolution of blockchains will be key to its success and growth, both in the UAE and worldwide. development has focused on the supply aspect, or on the overly technical nitty-gritty implementation of distributed ledger systems and commercializing token-based processes (Du et al., 2019). Little effort has been spent on in-depth contextual considerations of the social, ethical, cultural, and legal aspects essential to blockchain design, deployment, and optimization (Shin & Ibarahim, 2020). The ongoing concerns over blockchains such as data integrity, privacy provisions, transparency, and governance have rarely been tackled, in contrast to the proactive implementation of distributed ledger systems and commercializing token-based processes (Du et al., 2019). SOCIOTECHNICAL BLOCKCHAIN Little effort has been spent on in-depth contextual considerations of the social, ethical, cultural, and legal aspects essential to blockchain design, deployment, and optimization (Shin & Ibarahim, 2020). The ongoing concerns over blockchains such as data integrity, privacy provisions, transparency, and governance have rarely been tackled, in contrast to the proactive technical focus (Duan et al., 2020). The question of how we operationally manage and strategically maneuver the evolution of blockchains will be key to its success and growth, both in the UAE and worldwide. This study views blockchain as a sociotechnical artifact and contextually translates the technical attributes to its ongoing local context. This perspective is to critically assess the vital issue of whether blockchains are a society-wide innovation or a mere political slogan or SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN marketing blurb. Blockchain technologies are a part of the human condition and the wider social realities, and so, their design and development process should be based on user-centered design principles and and wider social impacts within a political, economic, and societal milieu (Shin, 2019). This point is consistent with the sociotechnical systems emphasizing the need for the integration of human and technical factors and concurrent design of their social and technical sub-systems (Shin & Bianco, 2020). Social issues regarding blockchains, such as trust, sub systems (Shin & Bianco, 2020). Social issues regarding blockchains, such as trust, transparency, security, and accountability, have emerged, posing potential threats to blockchain deployment and ecosystem (De Domenico & Baronchelli, 2019; Lemieux, 2016). Such issues are in situ since they are not mechanically resolved with technical fixes, as the issues are inherently intertwined with the locality of a socio-political economy context (Macrinici, Cartofeanu, & Gao, 2018). These social issues become even more complicated in a large-scale system where numerous sub-systems interact and often clash, and there is no one-size-fits-all solution (Kumar Liu & Shan 2019) Such issues can be further complicated by interactions transparency, security, and accountability, have emerged, posing potential threats to blockchain deployment and ecosystem (De Domenico & Baronchelli, 2019; Lemieux, 2016). Such issues are in situ since they are not mechanically resolved with technical fixes, as the issues are inherently intertwined with the locality of a socio-political economy context (Macrinici, transparency, security, and accountability, have emerged, posing potential threats to blockchain deployment and ecosystem (De Domenico & Baronchelli, 2019; Lemieux, 2016). Such issues are in situ since they are not mechanically resolved with technical fixes, as the issues are inherently intertwined with the locality of a socio-political economy context (Macrinici, Cartofeanu, & Gao, 2018). These social issues become even more complicated in a large-scale system where numerous sub-systems interact and often clash, and there is no one-size-fits-all solution (Kumar, Liu, & Shan, 2019). Such issues can be further complicated by interactions with heterogeneous constituents in sociotechnical blockchain platforms and systems. A high level of complexity and a multitude of internal and external systems along with related social implications entail the idea of a blockchain ecosystem, in which firms, regulators, policy makers, and users converge with relevant roles in the distributed ledger technology (Yang, 2019). Cartofeanu, & Gao, 2018). SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN and technical artifacts. As the UAE takes on deploying a full-scale blockchain, the social ramifications of the blockchain could be considerable and challenging. It is socially meaningful and technologically relevant to frame the UAE initiatives with a sociotechnical perspective to develop strategic optimization of people and technology with each other. An examination of blockchains from a sociotechnical lens has brought up the following questions: Research Question 1: What challenges and issues have been encountered with state initiatives to transform into a digital blockchain society? Research Question 2: How do sociotechnical factors affect the development of blockchains in the UAE? What are the social, technical, and environmental factors associated with blockchain services in the country? Research Question 3: What critical success factors for blockchain provisioning in the UAE or what factors associated with failure in service provisioning are inhibiting the successful deployment of a nationwide blockchain? Blockchain technology promises to transform every sector and industry, but design of blockchain has focused on technical performance or isolated platforms whose stakeholders contribute mechanically by operating algorithmically automated transactions (Shin & Ibarahim, 2020). We highlight the significance of sociotechnical dimensions within a broader ecosystem that encompasses a wide range of technical and non-technical conditions. We argue that blockchain should be designed by the people, for the people, and of the people such that it develops in a user-oriented and socially sustainable way to contribute to a more visible and more trusted development, which leads to a significant, positive impact with clear responsibility. SOCIOTECHNICAL BLOCKCHAIN These social issues become even more complicated in a large-scale system where numerous sub-systems interact and often clash, and there is no one-size-fits-all solution (Kumar, Liu, & Shan, 2019). Such issues can be further complicated by interactions with heterogeneous constituents in sociotechnical blockchain platforms and systems. A high level of complexity and a multitude of internal and external systems along with related social implications entail the idea of a blockchain ecosystem, in which firms, regulators, policy makers, and users converge with relevant roles in the distributed ledger technology (Yang, 2019). The idea of a blockchain ecosystem has been applied to the UAE case of blockchain development, where the government has been proactively expanding infrastructure and investing significant capital and resources in technology deployment over recent years. The UAE’s blockchain trajectories offer insightful models in regard to trustless platform development, regulatory systems, organizational innovation, and industry mobilization within the broader ecosystem that contribute to shaping outcomes, including the role of social, cultural, political, SOCIOTECHNICAL BLOCKCHAIN Contextualized design and assessment for the desired outcome are key in blockchain development (Casino et al., 2018). The idea of an ecosystem is quite useful for this task as the blockchain consists of ecological components: infrastructure, existing social relations, users, markets, and technological innovations (Kshetri, 2018). Performing a contextual evaluation is akin to sociotechnical assessment since it analyzes the mechanisms that each of these factors exert to contribute to forging a large-scale human system (Shin, 2019). Sociotechnical designs are a method and practice that consider human, societal, organizational, and cultural dimensions, as well as technological capability in the design of human organizations. The development of blockchain essentially involves a systematic consideration of sociotechnical factors because blockchains are sociotechnical systems that require social, organizational, and technological conditions to work properly (De Domenico & Baronchelli, 2019). From a network point of view, blockchain systems comprise a set of a distributed network of computers, appliances, and nodes linked through the internet (Lim et al., 2021). From a people’s perspective, blockchains can be viewed as a trust-based cooperative platform including end-users, designers, providers, minders, and operators. From a functional perspective, it can be considered as an ecosystem involving a distributed ledger system on top of which operates highly integrated interconnected software and tools, each playing a separate but complementary function in the whole network (Casino et al., 2019). What characterizes a blockchain as a sociotechnical innovation is that it is established by or related to a system that is adjusted and consumed by users in societies. Within a sociotechnical systems perspective, any institution, or part of it, is made up of a set of interacting sub-systems. Sociotechnical lenses to blockchain development address the connection and liaison between humans, technology, and the social milieu. A sociotechnical perspective helps us SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN practice. The social aspect considers how blockchain integrates into the real milieu, represents the social structure where benefits of blockchain implementation will occur, and characterizes the stakeholders who will benefit from and influence its development. The political aspect analyses the legal and regulatory issues regarding the introduction of blockchains in the UAE. The technical frame assesses the representation and materialization of the technical provisions and architecture of blockchains. Many technical configurations are mapped out and designed at this phase in the technology’s development (e.g., infrastructure, hardware, middleware, software, applications, services, and content) and are likely to reappear over time with either updates of existing infrastructure or the development of new services. Hardware and software blockchain infrastructure refer to equipment and software used to power, support, and operate blockchain applications and services. The sociotechnical ensemble framework evaluates the alignment between the social context where transformation is carried out and the blockchain technology designed to improve the transformation, as well as the contextual impact – both between society and within – at organizational and individual levels. These dimensions are not detached, linear, or independent, but rather are symbiotic and co-evolving concepts similar to structures of complex adaptive systems where a one-dimensional problem is frequently interrelated to other dimensions. When a technological change is introduced, system components find it complex to deal with the change mainly due to their symbiotically interdependent relations with the existing context. It is necessary to restructure them by engaging social components with the new context to decrease or minimize the inertia against the change. SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN to consider blockchains, not only as technical artifacts, but also as humanizing devices that can help us to reconsider entrenched premises as to privacy, security, transparency, distributed decision, and integrity (Shin & Hwang, 2020). These are intertwined and complex, and differ in their relative focus based on design and operations. The transparency of personal data could put people at risk of abuse, while transparency of the demographic information, personal to consider blockchains, not only as technical artifacts, but also as humanizing devices that can help us to reconsider entrenched premises as to privacy, security, transparency, distributed decision, and integrity (Shin & Hwang, 2020). These are intertwined and complex, and differ in their relative focus based on design and operations. The transparency of personal data could put people at risk of abuse, while transparency of the demographic information, personal preferences, or other personal data could put a someone at risk for discrimination. In addition, the immutability of data saved on blockchains conflicts with the legal right to be forgotten. When a personal record is mistakenly stored within the blockchain, deleting or modifying it is almost impossible. preferences, or other personal data could put a someone at risk for discrimination. In addition, Concern has risen in recent years regarding the trust and robustness of blockchain systems (Shin, 2019). The user's blockchain account can be manipulated if the private key is obtained by criminals. Since there are no centralized controls that manage the blockchain, it is problematic to trace the criminal's behaviors and recover the rigged blockchain data. Ironically, blockchain’s premise is to confirm trust but the biggest threat of blockchains is a lack of trust. If not addressed properly, these security and trust concerns can seriously weaken a blockchain society by generating a number of negative and even lethal problems in distributed environments (De Domenico & Baronchelli, 2019). Task on how to optimize and maximize the technical features in blockchains will affect the feasibility of its services and form potentially critical consequences on everyday lives. The sociotechnical framework ensures that both social and technical dimensions are thoroughly considered. The social framework evaluates the context in which the system is designed, developed, and deployed; it is far-reaching in terms of the related players, which involve user groups, online communities, industrial firms, market players, and communities of SOCIOTECHNICAL BLOCKCHAIN The UAE’s interest in blockchain technology shows how and why blockchain has a role to play in the future of society. In late 2015, a group of blockchain experts and policy makers met at the World Government Summit in Dubai to plan a new technology with potential to disrupt the digital landscape of the society. With government leadership, the Global Blockchain Council was formed, which then launched the Dubai Blockchain Strategy with the goal of fostering a local blockchain industry with global reach. The Smart Dubai Office manages and oversees Dubai’s blockchain strategies, with the goal of making Dubai the blockchain capital of the world. Abu Dhabi has positioned itself as the testbed for blockchain experiments. The Ministry of Artificial Intelligence has held an annual blockchain event since 2018. The minster has issued a challenge to move all viable government transactions to blockchain by 2025, effectively turning the government paperless. Meanwhile, the Abu Dhabi Blockchain Strategy aims to establish more than 2,000 new businesses and opportunities utilizing the technology, thereby cementing Abu Dhabi’s status as a global hub. The UAE Government initiated the UAE Blockchain Strategy 2021 in 2018, which aims to move 60% of public transactions onto a blockchain platform by 2025, which will save 3 billion USD in transaction costs, 100 million paper documents, and 55 million operating hours annually. -Blockchain in the financial sector One of the country’s largest banks, Abu Dhabi Commercial Bank runs an end-to-end blockchain trade transaction with automated tracking. The bank’s internal network produces accurate credentials of the transaction cycle with reliable and fully digitized documents at every stage. Abu Dhabi Global Financing tokenizes traditional financial assets and enhances investor -Blockchain in the government sector The public and government sectors overall have also shown significant interest in blockchain technology (Ølnes, Ubacht, & Janssen, 2017). Since 2017, the UAE government has started a series of blockchain strategies to spur exploration and adoption of the technology in the society. Numerous blockchain projects have emerged nationwide that have yielded many learning opportunities for participating stakeholders. The government aims to use blockchain systems as a tool to provide a secure and efficient platform that achieves the potential of transparent and fair e-government. SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN transparency, streamlines regulatory compliance, and improves liquidity by operating a simplified platform to purchase, trade, and transact startup equity. transparency, streamlines regulatory compliance, and improves liquidity by operating a simplified platform to purchase, trade, and transact startup equity. -The blockchain-based smart identity system The UAE Ministry of Finance has connected an identity card to individuals as a digital identification token. This system utilizes encryption and smart functions offered by the ID to validate information, including the individual’s biometric data. This saves cost, effort, and time and facilitates the digital transformation. - Blockchain in the media sector Recently, blockchain technology has been applied in the media sector (Shin & Bianco, 2020). Blockchains help media and entertainment companies rethink their business models. The Gulf News, one of the largest newspapers in the country, has partnered with Civil, a blockchain startup, to provide new services. The proposed services attempt to uproot the long-standing advertising-reliant revenue mechanism and provide reporters/writers with rights to their articles. These blockchain services intend to improve reliability and reestablish trust in the journalism industry, in which fake news has been prevailing. The new services use a new platform built on Ethereum-enabled tokens that would support and fund worthy, independent newsrooms. Blockchains can establish a more efficient medium by which several completely anonymous or semi-anonymous parties can complete different sorts of transactions at a reasonably cheap price. Because blockchains are decentralized peer-to-peer networks, there is no single breakdown point and no single access point for malicious hackers. Hence, it enhances security and safety for data. This ability to keep a fully auditable and unchallengeable database that is available to all participants of the blockchain provides another level of enhanced SOCIOTECHNICAL BLOCKCHAIN transparency and trust that is not always available within traditional media and communication processes. transparency and trust that is not always available within traditional media and communication processes. -Blockchain in the government sector SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN in the development of blockchain in the UAE were interviewed by video meeting mode, face-to- face meeting, phone, and email. A total of 32 respondents were recruited (Table 1; 9 from the public sector, 10 from the blockchain industry, and 13 from research communities). These first- hand methods were used as a complementary tool to verify the archival data (such as industry reports, government white papers, public records, and newspapers regarding the planning, p y g , face meeting, phone, and email. A total of 32 respondents were recruited (Table 1; 9 from the public sector, 10 from the blockchain industry, and 13 from research communities). These first- hand methods were used as a complementary tool to verify the archival data (such as industry reports, government white papers, public records, and newspapers regarding the planning, design, and deployment of cryptocurrency and blockchains). We used content analysis methods to probe the secondary data collected from the literature (print media, scholarly journals, technical reports, public documents, and databases). Since content analysis is context-sensitive, this enabled us to analyze the symbolic features of the blockchain era. With a triangulation method, we validated the qualitative data and refined the findings by mapping the sociotechnical design, and deployment of cryptocurrency and blockchains). We used content analysis methods to probe the secondary data collected from the literature (print media, scholarly journals, technical reports, public documents, and databases). Since content analysis is context-sensitive, this enabled us to analyze the symbolic features of the blockchain era. With a triangulation method, we validated the qualitative data and refined the findings by mapping the sociotechnical trajectory in blockchain project development, the roles of governments and firms, the demand and supply roles in the ecology model, and player involvement in blockchain development. Table 1. Interpretive Data Collected Sectors Venues Participants Government/public sector Interviews 3 9 Online meeting 3 Email 3 Blockchain industry (Practitioners) Meeting 3 10 Online meeting 3 Email 4 Blockchain researcher group Focus group 5 13 Online meeting 4 Email 5 Total 32 3. Methods An in-depth case study was performed to analyze blockchain from a sociotechnical perspective. Blockchain systems are complicated and multifaceted, and approaches to understanding their essence can be diverse and multidimensional. The multimethod approach to blockchain evaluation is one which views first-hand data and second-hand data as complementary. This combination of methods allows exploration of the what, how, and why of a social phenomenon that first-hand data can address, and the impact, extent, or influence of blockchain phenomena that second-hand methods establish. As the data were collected with diverse methods from diverse sources, cross analysis and triangulation methods were used to analyze the data from multiple sources. An in-depth case study was performed to analyze blockchain from a sociotechnical perspective. Blockchain systems are complicated and multifaceted, and approaches to understanding their essence can be diverse and multidimensional. The multimethod approach to blockchain evaluation is one which views first-hand data and second-hand data as complementary. This combination of methods allows exploration of the what, how, and why of a social phenomenon that first-hand data can address, and the impact, extent, or influence of blockchain phenomena that second-hand methods establish. As the data were collected with diverse methods from diverse sources, cross analysis and triangulation methods were used to analyze the data from multiple sources. The main sociotechnical method is ethnographic and is based on interviews with people. Such an interpretative approach was useful as it allowed us to consider the interactions of diverse actors and their diverse interpretations. Using snowball sampling methods, relevant stakeholders SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN components: the features and functions that each of these core components exert in contributing to a system evolution. The functions of these components are closely related to a web of interdependent interactions, and these functions normally evolve over time. As per components: the features and functions that each of these core components exert in contributing to a system evolution. The functions of these components are closely related to a web of interdependent interactions, and these functions normally evolve over time. As per sociotechnical systems theory, blockchain development can be dismembered into parts of the systematic ecosystem, i.e. operating platforms, data repository, industry, governance, practice, and users. This perspective examines the ongoing dynamics within the wider ecology of societies, communities, institutions, markets and the practice and production of services. Framing blockchain with a sociotechnical lens is useful since it contextualizes blockchains within the scope of their ongoing practice in the society (Shin & Ibahrine, 2020). sociotechnical systems theory, blockchain development can be dismembered into parts of the systematic ecosystem, i.e. operating platforms, data repository, industry, governance, practice, and users. This perspective examines the ongoing dynamics within the wider ecology of societies, communities, institutions, markets and the practice and production of services. Framing blockchain with a sociotechnical lens is useful since it contextualizes blockchains within the scope of their ongoing practice in the society (Shin & Ibahrine, 2020). 4. The Blockchain as a Sociotechnical Ecosystem A broad spectrum of technical and non-technical issues will need to be considered with the introduction of blockchain technology. From the perspective of blockchain as a sociotechnical ecosystem, it is important to scrutinize the interaction among ecosystem -Technological factors in blockchain growth -Technological factors in blockchain growth Although a general high aspiration toward blockchain has been projected in the UAE, the country’s blockchains bear inherent technological limitations that inhibit blockchain from evolving sociotechnical ensembles. First, one technological issue regarding the UAE blockchains is scalability, which remains a key hurdle for governments and companies. Creating scalable blockchain platforms that can adapt to the changing external dynamics of a government or firm can pose difficulties regarding operation, expenses, and change management. It was realized during early experiments with UAE blockchains that scalability is an essential technological challenge. With every transaction, the blockchain adds one more block to its ledger of transactions, and every block adds to the chain size with data since the chain keeps track of the blocks before it. As more users participate and the individual transaction histories increase, the system is at risk of collapsing. Numerous solutions are being examined and tested to solve this Although a general high aspiration toward blockchain has been projected in the UAE, the country’s blockchains bear inherent technological limitations that inhibit blockchain from evolving sociotechnical ensembles. First, one technological issue regarding the UAE blockchains is scalability, which remains a key hurdle for governments and companies. Creating scalable blockchain platforms that can adapt to the changing external dynamics of a government or firm can pose difficulties regarding operation, expenses, and change management. It was realized during early experiments with UAE blockchains that scalability is an essential technological challenge. With every transaction, the blockchain adds one more block to its ledger of transactions, and every block adds to the chain size with data since the chain keeps track of the blocks before it. As more users participate and the individual transaction histories increase, the system is at risk of collapsing. Numerous solutions are being examined and tested to solve this SOCIOTECHNICAL BLOCKCHAIN The UAE has long been a frontrunner in the development and deployment of digital innovations. Developing blockchain technology in the UAE has been no different. The country has a huge crypto market, which holds 29 percent of the global cryptocurrency exchange, as well as a very large financial market. The UAE accommodates resourceful and experienced industries that could take blockchain innovation to the next level of excellence. The UAE’s establishment of its information technology (IT) and the competitive IT industry has led to its global recognition as a technology powerhouse. This momentum has continued in the initiatives of blockchains and digital transformations. Major firms are raising investments in their blockchain systems and commercializing services as they seek potential market opportunities. General views from the industry respondents consistently show high enthusiasm about blockchains. Many respondents saw blockchain can help solve specific business problems. One respondent from a financial firm stated, “I think blockchain is transformative. The potential of blockchain in the financial industry as well as other overall industry could be transformative and technically innovative.” One of the blockchain services that health industries are actively implementing is a blockchain-based digital health data storage platform. This platform will serve as a streamlined tool to improve the search for medical information, health facilities, licensed health practitioners, and health information. Health practitioners, pharmaceutical companies, and medical facilities will all benefit from the blockchain platform. The improvements can also help encourage medical tourism and supply information that can save and improve the well-being of UAE citizens. SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN problem, such as off-chain transaction or Sharding, but they are realizing that scalability becomes an even more serious problem as the blockchain keeps growing. problem, such as off-chain transaction or Sharding, but they are realizing that scalability becomes an even more serious problem as the blockchain keeps growing. Second, as different kinds of blockchains quickly develop in the UAE, concern about interoperability is rising. Thus far, there is no standard in blockchain services to ensure that different types of blockchains will be compatible with each other and work in harmony. There are more than 5000 active blockchain projects with different platforms and varied parameters such as different protocol schemes, different consensus models, and different smart contract functionality. As the blockchains have been developed for more autonomy and freedom, this causes interoperability and communication issues. The lack of interoperability between different blockchains has added to the confusion and uncertainty in development and diffusion of blockchains. Third, the high complexity embedded in blockchains creates a number of problems. Blockchain transactions can take a longer time to process than traditional payment systems due to their complexity and the encrypted, distributed feature. Related to the complexity, users find it difficult to figure out, use, and fully adopt blockchain services. Most active blockchain services in the country are not user-friendly in that the mechanism of transporting tokens and coins in a decentralized manner is fairly complex and requires specialized knowledge. As blockchain uses encryption to warrant its security as well as create consensus over different networks, users need to write to the chain and run complex algorithms, which involves large amounts of computing power. This creates a huge environmental cost in that the computing power required to keep the system running is fairly high. -The Technical Industry Social Dimensions -Social issues with blockchain development SOCIOTECHNICAL BLOCKCHAIN In many cases, social dimensions are more critical than technical ones in the process of sociotechnical system development. The social system and its’ innovations in general have been underdeveloped in the UAE blockchain case as the country tends to consider blockchain as a magic wand, an all-powerful game changer that would bring some kind of benefit to the country. Under this rosy blueprint development, blockchain weaknesses have rarely been discussed or have been overridden by overhyped technical terminology. Critical social issues related to blockchain have been underestimated or overlooked. One respondent from an academic research group noted, “Social issues related blockchain are seriously underdeveloped at present. Other than technical blockchains, the government and industry do not care or don’t have understanding about such issues.” Another respondent from a university agreed with this sentiment, stating “The country traditionally has followed technology universalism. Due to the traditionally technology-driven nature of blockchain and cryptocurrency, which does not look at design from a user-centered perspective.” Ironically, one of the main social issues comes from the most salient technical features. By design, blockchain architectures are vulnerable to attacks. A blockchain has its solid security features from the decentralized system of its nodes. Since the trace of transactions is publicly open, no one can covertly make alterations. When a modification is recorded, the other nodes on the platform approve the change before the record is stored. Owing to the design of blockchain technology, all public blockchains are vulnerable to 51 percent attacks, which occur when hackers make up more than half of a blockchain’s computing power (Pazaitis et al., 2017). They become the network majority and can manipulate the whole blockchain, permitting the hackers to prevent other users from producing blocks, double-spend coins, and blocking transactions entirely. The vulnerable security could be deteriorating in the UAE, considering rather SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN blockchain hinges on transparency, a lack of transparency in data collection and process may pose a challenge. The privacy governance needs to address the blockchain case. blockchain hinges on transparency, a lack of transparency in data collection and process may pose a challenge. The privacy governance needs to address the blockchain case. Third, from a sociotechnical system perspective, a lack of awareness by the public can be significant obstacles to effective development. Many people in the country do not fundamentally understand how blockchain operates or the overarching effects blockchain can have in everyday life. One respondent from a user group said, “I heard about blockchain, but most of my friends never heard about it. Even myself, I do not know specific details of blockchain. I think this is true to most people here in this country.” This lack of awareness even extends to most policymakers and related stakeholders, which is problematic since blockchain as a disruptive technology needs flexible regulations to ensure that its capability is utilized in a constructive manner to build a sustainable society. Because blockchain is a new set of systems that change the rule of the game, only those who understand how it functions differentiate it from cryptocurrencies. There is a doubt of acceptance and diffusion in the blockchain as the lack of appropriate social structure and supporting programs. The growing doubt of acceptance and diffusion of blockchain is fueled by a lack of appropriate training and social awareness programs. SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN vulnerable environments to security. Numerous security challenges have been identified in the UAE, such as online fraud, system hackers, and frequent mobile security issues. While the country has numerous prospects regarding its blockchain sector, a misuse of customer data from financial firms illustrates how the online environment in the UAE is vulnerable with regard to security. These incidents hurt the trustworthiness of the whole market and critically undermine blockchain technology development in the UAE. Second, the weak privacy features and vague privacy regulations of the blockchain platform could be the weakest point for the UAE. Although privacy is another technical attribute of blockchain that is widely praised, it is also one of the key loopholes in blockchain. The way shared ledgers are designed makes it almost impossible to hide a transaction on blockchains. Second, the weak privacy features and vague privacy regulations of the blockchain platform could be the weakest point for the UAE. Although privacy is another technical attribute of blockchain that is widely praised, it is also one of the key loopholes in blockchain. The way shared ledgers are designed makes it almost impossible to hide a transaction on blockchains. Every transaction ever stored on the blockchain is accessible for anyone to see. When personal information is available in the public domain, it poses challenges in the healthcare, financial, and legal sectors, where robust privacy requirements are necessary. One respondent from the technology industry expressed the concern over privacy, stating, “I do not believe blockchain is good at privacy. I know it has critical privacy loopholes due to its design.” Another respondent from a user group shared the concern, saying, “I have a serious concern over privacy and security in general because the distributed nature of blockchains means that blockchain is publicly available and every transaction can be shared by anonymous public.” Every transaction that happens is verified publicly, logged publicly, and kept in an openly confirmable ledger maintained by public consensus, with an immutable digital certificate. This privacy concern may create conflicts within the UAE where there are no privacy guidelines for blockchains. While the UAE’s Data and Privacy Protection Act (Article 378) governs privacy and data protection, it does not specifically include blockchain. Also, as the privacy of a SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN government can potentially reduce paperwork by 200 million and work hours by 60 million per year. It expects to save about 11 billion dirhams ($3 billion) in transactions and document processing. “These visions will also help UAE develop the correct governance for blockchain, based on the specific needs from those deploying the technology,” said an official of the Dubai blockchain group. Traditionally, the UAE has been highly innovative regarding new technology and initiatives in general. Blockchain has been a core national technology agenda. Since 2016, the UAE has spearheaded a large number of blockchain projects. The government formed a “Digital Transformation Committee” that directly reports to the President and is authorized to manage important policy issues pertaining to the design and development of blockchain technology. The UAE has taken its positive stand toward blockchains and the innovation it creates for financial technology by giving tariff reliefs and relaxing regulations for blockchain projects. Traditionally, the UAE has been highly innovative regarding new technology and initiatives in general. Blockchain has been a core national technology agenda. Since 2016, the UAE has spearheaded a large number of blockchain projects. The government formed a “Digital Transformation Committee” that directly reports to the President and is authorized to manage important policy issues pertaining to the design and development of blockchain technology. The UAE has taken its positive stand toward blockchains and the innovation it creates for financial technology by giving tariff reliefs and relaxing regulations for blockchain projects. While the government does not approve Bitcoin as an official form of payment, trading Bitcoin is widely accepted in the market. Considered the most liquid cryptocurrency, Bitcoin involves accessing a cryptocurrency exchange that allows trading and investing. The government has designated blockchains as a key strategic R&D area and has offered a stimulus and tax-relief measure. This support is an effort to boost awareness and drive in the blockchain field, while exploring possibilities for the future open innovation it could offer the financial sector as well as other vital sectors. While the government does not approve Bitcoin as an official form of payment, trading Bitcoin is widely accepted in the market. Considered the most liquid cryptocurrency, Bitcoin involves accessing a cryptocurrency exchange that allows trading and investing. The government has designated blockchains as a key strategic R&D area and has offered a stimulus and tax-relief measure. -The UAE’s Blockchain Initiative -The UAE’s Blockchain Initiative The UAE has been taking numerous measures to roll out blockchain into the nation’s economy and governance. In 2017, the government introduced its Emirates Blockchain Strategy to strengthen blockchain adoption in government offices. It expects 50% of transactions to be completed using blockchain by 2021. Also, the Dubai Blockchain Strategy was launched in 2017 to explore and evaluate blockchain applications. Leveraging blockchain technology, the SOCIOTECHNICAL BLOCKCHAIN This support is an effort to boost awareness and drive in the blockchain field, while exploring possibilities for the future open innovation it could offer the financial sector as well as other vital sectors. The UAE government holds a strategic assumption that blockchains will be one of the key enabling momentums in bringing about new growth engines and vitalizing existing industries. Blockchains have been chosen by the Ministry of Artificial Intelligence as a core field for the country to emphasize as a part of the digital transformation initiatives (Zhang, 2016). The SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN Ministry of Industry and Advanced Technology has started to support blockchain by approving over 19 major blockchain companies and the development of up to 5,000 professionals in order to establish the UAE’s strategic position as a spearhead in financial technologies. The ministry intends to capitalize 350 million USD in the emerging market through 2026. The government plans to establish a 10 billion USD blockchain subsidy to support new start-ups in blockchains. To advance the UAE’s competitive edge in the blockchain sector, the ministry, with teamwork from the Ministry of Energy and Infrastructure, has been exploring blockchain platform architectures that incorporate different sources of data with feasible services (Zhang, 2016). The government’s proactive approach has led to increased industry involvement. Together, the government and firms have rapidly launched a series of blockchain programs to raise awareness, and a few blockchain firms have started to provide useful services accessible by users. The UAE’s big firms (First Abu Dhabi Bank, Emirates Telecommunications Group, and ADNOC) have been actively developing blockchain services. -Regulation: Governance and policy Blockchain technology in the UAE has advanced way ahead of regulations. Despite the burgeoning blockchains, lack of consistent regulatory clarity and differences between jurisdictions pose a challenge for emerging blockchains. Although the UAE presents blockchain as a government priority and has introduced various blockchain-related initiatives, regulation of cryptocurrencies in the onshore UAE and the Dubai International Financial Centre remains limited. Although coins and tokens are not prohibited, the Securities and Commodities Authority has issued circulars to caution investors on cryptocurrencies, without, however, taking a firm regulatory position. Both the onshore UAE and the Dubai International Financial Centre have adopted a wait-and-see approach; however, in SOCIOTECHNICAL BLOCKCHAIN Currently, blockchain is a key agenda in the UAE as the digital transformation initiatives progress toward achieving development goals. The UAE has one of the most promising environments for blockchains to prosper due to its well-implemented network infrastructures and the tremendous volume of data produced, processed, and analyzed in real-time. The UAE continues to enhance the ICT momentum to blockchain initiatives with a vision that digital transformation through a token-based society can bring immense social benefits (through social- good applications) to emerging economies. Such vision, however, is not likely to be easily realized without careful and proactive sociotechnical provisions and preparations. With the extremely favorable conditions for the blockchain ecology, this enthusiasm comes at the price of intertwined sociotechnical complexity. Blockchain in the UAE has not yet realized a tangible service as it confronts a series of technical, social, political, and operational challenges. A lingering question in the technology-oriented initiatives is how to integrate or align the technical features of the blockchains to existing social progress as well as other ongoing technology projects, which is fundamentally related to sociotechnical issues. Blockchains, as a tool for securing privacy and offering even greater system performance, are undoubtedly a common interest that government and industry share in promoting digital transformation. With this shared interest, however, the ongoing implementation has faced substantial complications to appropriate and successful economic deployment, as attempts have been focused only to advance blockchain technical performance. The question lingers concerning how to carry on the momentum for social innovation and the future sustainability of blockchains over the long-term. With continued developments in overlapping projects, how can blockchain transformation be justified societally? Will blockchain technologies mature against the rising public concerns over security, privacy, transparency, and sustainability? These concerns warn against the old-fashioned belief that Currently, blockchain is a key agenda in the UAE as the digital transformation initiatives progress toward achieving development goals. The UAE has one of the most promising environments for blockchains to prosper due to its well-implemented network infrastructures and the tremendous volume of data produced, processed, and analyzed in real-time. The UAE continues to enhance the ICT momentum to blockchain initiatives with a vision that digital transformation through a token-based society can bring immense social benefits (through social- good applications) to emerging economies. Such vision, however, is not likely to be easily realized without careful and proactive sociotechnical provisions and preparations. SOCIOTECHNICAL BLOCKCHAIN October 2019, the Commission issued a draft regulation on virtual assets with a request for public commentary. As of 2021, no regulation has come into force. October 2019, the Commission issued a draft regulation on virtual assets with a request for public commentary. As of 2021, no regulation has come into force. Currently, there is no statute or guidance from the UAE regulatory bodies that provides coherent oversights on how blockchains should be regulated under the UAE law. One government official noted, “Currently, no regulation regarding blockchain has come into place in the UAE. We adopt a wait-and-see posture and try to learn from other cases as they make the first mover’s lessons for us.” Relevant regulators lack the necessary understanding of blockchain and cryptocurrencies and remain unprepared to apply a consistent approach towards coping with regulatory concerns. The present regulations over cryptocurrency are unsettled and confusing. Cryptocurrency in the UAE has yet to be defined as a currency or controlled by the Regulatory Framework for Electronic Payment System. In some places, cryptocurrencies are forbidden completely, while others regulate their use by regulating exchanges. Owing to a lack of legislation at the federal level, there are no specific regulations in place for the misuse of virtual coins or tokens in the country. There are no unified international standards, and this will continue to be a challenge for the UAE case. There are additional concerns about how to best regulate data and data ownership, but there are no internationally endorsed standards or codes of conduct so far. The efforts of the UAE to establish global standards for blockchain are a positive move and signal how the technology is maturing. Yet, the government has not provided clear standards for domestic blockchain cases. This can be alarming considering the enthusiasm expressed by the government, but the UAE’s banking laws in 2019 do not explicitly include the state of cryptocurrency in the jurisdiction. SOCIOTECHNICAL BLOCKCHAIN With the extremely favorable conditions for the blockchain ecology, this enthusiasm comes at the price of intertwined sociotechnical complexity. Blockchain in the UAE has not yet realized a tangible service as it confronts a series of technical, social, political, and operational challenges. A lingering question in the technology-oriented initiatives is how to integrate or align the technical features of the blockchains to existing social progress as well as other ongoing technology projects, which is fundamentally related to sociotechnical issues. Blockchains, as a tool for securing privacy and offering even greater system performance, are undoubtedly a common interest that government and industry share in promoting digital transformation. With this shared interest, however, the ongoing implementation has faced substantial complications to appropriate and successful economic deployment, as attempts have been focused only to advance blockchain technical performance. The question lingers concerning how to carry on the momentum for social innovation and the future sustainability of blockchains over the long-term. With continued developments in overlapping projects, how can blockchain transformation be justified societally? Will blockchain technologies mature against the rising public concerns over security, privacy, transparenc and s stainabilit ? These concerns arn against the old fashioned belief that SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN highly complex technology and most users do not even understand what it is, much less how to use it. The concepts of distributed ledgering and encryption in blockchains are difficult to comprehend for end-users. In addition, users cannot immediately see the result of cryptocurrency transactions as they take several hours to finalize, which creates another level of uncertainty and inconvenience. Third, security issues present critical sociotechnical questions to the UAE blockchains. People tend to consider blockchains as entirely safe and have a tendency to undervalue the key conditions for blockchain security. People tend to take security in blockchains for granted as they assume that blockchains are completely safe and secure. It may be a sociotechnical realization that blockchain can be highly vulnerable to risks, just as any other technical system no matter how robust. Recently, numerous security incidents have been occurred related to smart contracts and Ethereum. Additionally, trust issues have become a key factor in UAE blockchains. If the blockchain is a mecca of transactions and file sharing, the question arises as to how we can trust this technology. For blockchain to be a socially acceptable technology in a wide society, it should be trustable and reliable for the users. At present, there is a low level of trust, which is due to a prevailing perception that cryptocurrency is a scam or online fraud. A recent series of security breaches in Bitcoin transactions have contributed to this perception. SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN systems can be best developed from a centralized top-down approach in a sheer predetermined, deliberately planned, and entirely controlled manner. In fact, a centralized top-down design directly contradicts the core principles of decentralized and distributed contribution blockchains. systems can be best developed from a centralized top-down approach in a sheer predetermined, deliberately planned, and entirely controlled manner. In fact, a centralized top-down design directly contradicts the core principles of decentralized and distributed contribution blockchains. The development of blockchains should heavily include a sociotechnical dimension within which a blockchain is assessed and designed for its societal value rather than its technical utility, and it should be visible and transparent to the citizens, eventual benefactors of blockchain (Shin & Bianco, 2020). Related to this point, the real issues of blockchains in the UAE are less about technological advancement, but more about the way society is arranged and restructured with blockchain deployment and implementation. It also relates how blockchains and policy are harmoniously aligned and construed. There is currently a strategic misalignment between industry and government in the UAE as they have different perspectives about blockchain, particularly in security and privacy. From a sociotechnical viewpoint, this kind of misalignment is a big challenge for blockchain deployment in the UAE. and government in the UAE as they have different perspectives about blockchain, First, a lack of regulatory clarity can create a hazardous environment for blockchains. The absence of regulatory oversight has led to increasing data manipulation and scam. Financial sectors are facing challenges of transparency, accountability, privacy, and data security. An overarching governing framework is urgently needed to safeguard that fairness, accountability, and transparency requirements are ensured (Shin & Hwang, 2020). Related to this, a lack of industry standard for blockchain hinders the diffusion of such technologies into the wide society, resulting in the absence of interoperable system. First, a lack of regulatory clarity can create a hazardous environment for blockchains. Second, since the beginning of its innovation, it has been criticized for the difficulties in using blockchain properly. The limited usability issues pose potential obstacles and hinder its wide-scale adoption. The findings reveal that blockchain in the UAE has been perceived as a SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN involved, and onboarding stakeholders with the best governance practices and standards in place. The response to these key questions for the UAE can be that the government performs a key role in managing disruptive innovations and emphasizing the value of digital transformation, involved, and onboarding stakeholders with the best governance practices and standards in place. The response to these key questions for the UAE can be that the government performs a key role in managing disruptive innovations and emphasizing the value of digital transformation, supporting both the private and public sectors in discussing, planning, and adjusting blockchain technologies. Thus, the government and industry work together for the formation of a cooperative setting and put in place processes for both sectors to stabilize blockchain technology at scale. It may be useful for the UAE to recognize that blockchain is not the magic cure-all some people believe it to be. It can be a beginning point of a sociotechnical transition to sustainable blockchains. From a sociotechnical systems perspective, practical suggestions can be made for ongoing blockchain initiatives in the country. involved, and onboarding stakeholders with the best governance practices and standards in place. The response to these key questions for the UAE can be that the government performs a key role in managing disruptive innovations and emphasizing the value of digital transformation, supporting both the private and public sectors in discussing, planning, and adjusting blockchain technologies. Thus, the government and industry work together for the formation of a cooperative setting and put in place processes for both sectors to stabilize blockchain technology at scale. It may be useful for the UAE to recognize that blockchain is not the magic cure-all some people believe it to be. It can be a beginning point of a sociotechnical transition to sustainable blockchains. From a sociotechnical systems perspective, practical suggestions can be made for ongoing blockchain initiatives in the country. First, blockchain use and application in the UAE reveal notable obstacles: the social practices of technology adjustment, calibration, and integration. From a sociotechnical angle, how technology is adjusted to fit a new social milieu and how technology is integrated into everyday life are important questions to examine (Shin, 2014). The UAE blockchain case shows little trajectories of these technological adjustment and integration into society. 6. Practical Suggestions: A Sociotechnical Approach to Blockchains The results imply that the key questions of blockchain development remain tied to regulatory, societal, ethical, and legal questions. While government authorities, firms, and service providers share the concerns over blockchain, core questions involve developing society- wide beneficial blockchain programs, providing training and awareness for stakeholders SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN blockchain use applies predominantly to limited applications in financial services or narrow commercial sectors (e.g., cryptocurrency trading) rather than to integration for wider societal usages and public services. The blockchain design process should constructively incorporate ethical considerations and societal issues as well as the cultural and contextual factors that can affect the deployment of technical blockchain (Shin & Bianco, 2020). Although the technical dimensions of blockchain obviously can be essential in developing initiatives, they also inherently carry critical risks that should be addressed before its full-fledged implementation (Marsal-Llacuna, 2018). In the due process of aligning blockchain with social values, it is key that fundamental user rights and human wellbeing/values should be considered in a meaningful way. Perhaps the most important strategy for the UAE is the proper usage, rather than a one-size- fits-all solution of blockchain systems, which are not a silver bullet capable of accomplishing any goal (Kumar et al., 2019). Although digitalization, encryption, transparency, and accessibility are underlying features in blockchain platforms, blockchain usability is perhaps the most important as it fosters the digital ecosystem. Although the government promotes blockchain applications and services, people should gladly accept blockchains and digital transformation as part of their lives. Citizens should be able to accept the innovations into the core of their social practices and the inclusion of blockchain technology in the different aspect of their lives. Users can apply blockchain services in such a way that it makes sense to them in their own frames of existing social practices. The success of integrating blockchain into the existing social system hinges on the trust and opinions of the public toward the blockchain system. The country is attempting this the other way around, trying to frame existing societies into the proposed blockchain scheme. This creates a lot of confusion as a new set of technical features of blockchain clashes with existing social values and user practices in the society. The blockchain use applies predominantly to limited applications in financial services or narrow commercial sectors (e.g., cryptocurrency trading) rather than to integration for wider societal usages and public services. The blockchain design process should constructively incorporate ethical considerations and societal issues as well as the cultural and contextual factors that can affect the deployment of technical blockchain (Shin & Bianco, 2020). SOCIOTECHNICAL BLOCKCHAIN There are a well- planned vision and technical roadmap, robust platforms for accommodating distributed ledgers, a strong network infrastructure, and well-funded blockchain firms. Currently, however, there is no single usable service or visible application that is used or even proposed in the near future for everyday life. The findings serve to raise awareness of the common challenges and inhibiting factors encountered in blockchain deployment. While both the federal and local governments have taken active roles in developing blockchain, the use of blockchain remains underdeveloped. Education and awareness of the related stakeholders have not done well and also identifying and understanding the most relevant applications of blockchain have been neglected. So far, SOCIOTECHNICAL BLOCKCHAIN Although the technical dimensions of blockchain obviously can be essential in developing initiatives, they also inherently carry critical risks that should be addressed before its full-fledged implementation (Marsal-Llacuna, 2018). In the due process of aligning blockchain with social values, it is key that fundamental user rights and human wellbeing/values should be considered in a meaningful way. Perhaps the most important strategy for the UAE is the proper usage, rather than a one-size- fits-all solution of blockchain systems, which are not a silver bullet capable of accomplishing any goal (Kumar et al., 2019). Although digitalization, encryption, transparency, and SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN government’s plan of Digital Transformation should be reconsidered as the plan is to make a total overhaul of existing society into digital society without considering a smooth transition or sociotechnical adjustments. Second, blockchain initiatives should be continued as a constant effort rather than a one- off event or one-time policy by political slogan. Interview respondents shared the key concern that the core challenges in blockchain implementation remain in the operational and regulatory domain rather than on the technical matter. Bringing stakeholders to the table, alignment of diverse interests, and coordination between different stakeholders were viewed as top challenges for blockchain projects in the UAE. The public sector considered public awareness and coordination of different players as the most urgent task, whereas the private sector’s main issue was regulatory ambiguity. In the trajectory of technology development in the UAE, a government-driven top-down approach continues to play a dominant role. There has been a negative side effect of such technology-oriented plans, and blockchain development seems to follow the same track. Creating infrastructure and shaping an ecology normally involves considerable effort and cost. A blockchain is more than a set of ready-made encrypted codes; rather, it is a sophisticated social reengineering approach to model a contextually identified set of social factors that could contribute to ongoing social practices and embedded cultural values in the society. The government should additionally focus on the societal and ethical effects of blockchains and move away from technological determinism. Third, as a sociotechnical evaluation method, the government may shift its current focus on technical mentality to cultural and ethical aspects of blockchains. Blockchain cannot replace all existing systems and the government must consider very carefully the pros and cons of a blockchain system. Although there are numerous blockchain initiatives nationwide, few SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN entrepreneurs or academics have researched the ethical aspects of blockchains. Like the potential positive impacts of blockchain, the ethical consequences of blockchain can be just as diverse and wide ranging. The ethics of blockchain are essential for technology adoption and diffusion. Blockchain’s potential for social impact spans a wide spectrum, as it can disrupt different types of organizations and social systems across the country. Thus, policymakers need to understand the ethical approaches used in designing blockchain technology, especially how they influence marginalized and underserved social groups. A clear guideline or framework (something like blockchain ethical design framework) should be introduced as a tool to create a sociotechnical design that integrates key ethical issues for the design and use of blockchain. The main goals of the ethical guideline should provide user-centric tools to evaluate the context-specific consequences and ethical ramifications of their blockchain design and deployment options and to achieve better social outcomes. The design process should include anyone who is influencing a social-impact solution that may involve the design and implementation of a blockchain. Blockchain’s potential for social impact spans a wide spectrum, as it can disrupt different types of organizations and social systems across the country. Thus, policymakers need to understand the ethical approaches used in designing blockchain technology, especially how they influence marginalized and underserved social groups. A clear guideline or framework (something like blockchain ethical design framework) should be introduced as a tool to create a sociotechnical design that integrates key ethical issues for the design and use of blockchain. The main goals of the ethical guideline should provide user-centric tools to evaluate the context-specific consequences and ethical ramifications of their blockchain design and deployment options and to achieve better social outcomes. The design process should include anyone who is influencing a social-impact solution that may involve the design and implementation of a blockchain. 7. Conclusions The promise that blockchain will have a significant impact on the UAE is genuine and has actually been happening. By enhancing the security, trust, and verifiability of the way organizations operate and how access to services is delivered, data are measured and processed, and transactions are traced, blockchain’s possibilities can transform society and nation to a higher level of the digital era. Achieving this potential, however, requires a careful sociotechnical approach that aligns and integrates the relationship between design and human outcomes. This study seeks to resolve blockchain problems and unlock opportunities in the country to guarantee a sociotechnical approach to blockchains and consider the needs of all players in the SOCIOTECHNICAL BLOCKCHAIN SOCIOTECHNICAL BLOCKCHAIN ecosystem. The findings of this study contribute to the theoretical principles and practical application of how to govern blockchain systems effectively, how to make sense of blockchains within a social setting, and how to design blockchains that are user-oriented and fulfill the needs of the society and its citizens. These sociotechnical insights will be constructive in transforming blockchain promise into a sociotechnical setting. The ability of blockchains to have an immense repercussion on the UAE is positive and promising. Its key features, platforms, and financial impact have the power to move ahead with momentum. Yet these revolutionary possibilities will not be realized unless governments strategically design systems to redistribute power from the elites to the users in the country who would take the form of public good provision. The realization of the potential depends on a thorough sociotechnical design that aligns technological blockchain systems with human meaning of the country. 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W2597241575.txt	https://periodicos.pucpr.br/pistispraxis/article/download/1369/1300	de		Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau	Revista Pistis & Praxis	2,015	cc-by	17,626	doi: 10.7213/revistapistispraxis.07.002.DS04 ISSN 1984-3755 Licenciado sob uma Licença Creative Commons [T] Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau The 2 Vatican Council, the threefold conception of Holy Orders and the perspectives on female Diaconate Karl-Heinz Menke* Universität Bonn, Katholisch-Theologische Fakultät, Bonn, Deutschland Abstrakt Ist ein sakramental verstandener Diakonat der Frau möglich, ohne zugleich ihre Zulassung zur Priester- und Bischofsweihe auszusprechen? Theologinnen und Theologen, die diese Frage positiv beantworten, erklären, dass es zwar nur ein Sakrament des Ordo gebe, dass dessen Gestaltung (die Trias von Bischof, Priestern und Diakonen) aber veränderlich bis hin zur Schaffung eines Frauendiakonates sei. Die Gegenthese behauptet die Sakramentalität von Einheit und Trias des Ordo. Beide Parteien berufen sich auf dieselben Texte des Zweiten Vatikanischen Konzils. Deshalb blickt die vorliegende Abhandlung zurück in die Geschichte der Verhältnisbestimmung von Episkopat, Presbyterat und Diakonat, um so zu erweisen, dass die Väter des Zweiten Vatikanums eine jahrhundertelang ungeklärte Frage endgültig wie folgt entschieden haben: Die sakramentale * KHM: Doktor der Theologie, e-mail: k.menke@uni-bonn.de Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 350 MENKE, K.-H. Repräsentation des ‚Voraus‘ Christi gegenüber der Kirche geschieht durch die drei Ämter von Bischof, Presbyter und Diakon. Wenn die Frau nicht zur Bischofs- und Priesterweihe zugelassen ist, dann auch nicht zum Diakonat des sakramentalen Ordo. Schlüsselwörter: Diakonat. Kirche. Vatikanische Konzil 2. Abstract Is it possible to consider the diaconate of women as a sacrament without addressing the question of women’s admission to the ordination as a priest and bishop? Male and female theologians who answer this question in the affirmative declare that though there is only one sacrament of Holy Orders, its forms (i.e. its three degrees the episcopate, the presbyterate and the diaconate) can vary and even lead to the creation of a diaconate of women. The counter thesis asserts the sacramentality of the unity and the three degrees of ordination. Both sides refer to the same documents of the Second Vatican Council. This is why this treatise looks back on the history of the definition of the relation between the episcopate, the presbyterate and the diaconate in order to show that the Fathers of Vatican II once and for all have settled a question which had remained unresolved for centuries: The sacramental representation of Christ’s going ahead of the Church is realized in the three ministries of the bishop, the priest and the deacon. If women are not admitted to episcopal and priestly ordination, then they are neither admitted to the diaconate being a sacrament of Holy Orders. Keywords: Diaconate. Church. II Vatican Council. Vorwort Die Erinnerung an den fünfzigsten Jahrestag der Eröffnung des Zweiten Vatikanischen Konzils ist weithin geprägt von Debatten über die hermeneutischen Voraussetzungen1 der Interpretation. Einig 1 Dazu: KOCH, K. Das Zweite Vatikanische Konzil: Die Hermeneutik der Reform. Augsburg, 2012; TÜCK, J.-H. Die Verbindlichkeit des Konzils: Die Hermeneutik der Reform als Interpretationsschlüssel, in: DERS (Hg.). Erinnerung an die Zukunft. Das Zweite Vatikanische Konzil. Freiburg i. Br., 2012, 85–104. Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau ist man sich in der Einbeziehung der sogenannten Praeparatoria und Antipraeparatoria. Doch diese Einbeziehung geschieht meistens selektiv; die jeweilige Selektion folgt einem bestimmten Interesse. Exemplarisch für dieses Phänomen sind die oft konträren Schlussfolgerungen, zu denen zwei italienische Schulen — die eine in Bologna, die andere in Rom2 — gelangen. Man kann sich des Eindrucks nicht erwehren, dass jede Partei in den Konzilstexten das wiederfindet, was sie dort finden will. Jedenfalls ist die Rückfrage nach den Aussagen des Konzils oft von Interessen geleitet, die mit der Entstehung der jeweiligen Texte nichts zu tun haben. Ein beredtes Beispiel ist die Befragung bestimmter Abschnitte der Kirchenkonstitution (LG 18-29) durch Befürworter und Gegner eines Diakonates der Frau. Im Folgenden soll der Versuch unternommen werden, die Intention der besagten Texte aus ihrer Vorgeschichte zu erheben. Denn erst eine umfassende Sichtung der Problemgeschichte, an deren Ende die besagten Antipraeparatoria und Praeparatoria des Konzils stehen, lässt erkennen, was die Konzilsväter warum so und nicht anders gesagt und beschlossen haben. Diachrone Betrachtung der Lehrentwicklung hinsichtlich: Verhältnisbestimmung Episkopat/Presbyterat/Diakonat In der Regel geht die Forschung3 von zwei bis in die apostolische Zeit zu ückreichenden Verfassungen der ersten christlichen Gemeinden aus. Da ist einerseits die im Judenchristentum verortete 2 3 Für die “Bologneser Schule”: ALBERIGO, G. (Hg.). Geschichte des Zweiten Vatikanischen Konzils 1959–1965, 5 Bände, deutsche Ausgabe herausgegeben von K. Wittstadt und G. Wassilowski, Mainz/Leuven 1997– 2008. Für die “Römische Schule”, wenn nicht repräsentativ, so doch bezeichnend: GHERARDINI, B. Concilio ecumenico Vaticano II: Un discorso da fare. Torino 2009 (deutsche Ausgabe: Das Zweite Vatikanische Konzil: Ein ausstehender Diskurs. Fohren-Linden, 2010); ders., Concilio Vaticano II. Il discorso mancato, Torino 2011; ders., Valore magistrale del Concilio Vaticano II, Rom 2012; R. de Mattei, Das Zweite Vatikanische Konzil. Eine bislang ungeschriebene Geschichte (ital. Originalausgabe: Il Concilio Vaticano II: Una storia mai scritta. Torino, 2010; deutsche Ausgabe Stuttgart 2012). Vgl. von CAMPENHAUSEN, H. Kirchliches Amt und geistliche Vollmacht in den ersten drei Jahrhunderten. Tübingen, 1963. DASSMANN, E. Ämter und Dienste in den frühchristlichen Gemeinden. Bonn, 1994. WAGENR, J. Die Anfänge des Amtes in der Kirche: Presbyter und Episkopen in der frühchristlichen Literatur. Tübingen, 2011. Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 351 352 MENKE, K.-H. „Presbyterverfassung“ und andererseits die im „Heidenchristentum“ angesiedelte Leitung durch Episkopen und Diakone (z. B. Phil 1,1). Meistens wird diese Dualität kulturgeschichtlich aus Strukturen der Synagoge beziehungsweise der griechischen Polis erklärt4. Die Mehrzahl der Forscher vermutet ein allmähliches Zusammenwachsen beider Ordnungen. Diese zwei konkurrierenden Konzeptionen werden bereits von Lukas ineinander geblendet und harmonisierend miteinander identifiziert: In Apg 20,28 redet der lukanische Paulus die Ältesten von Milet als „vom heiligen Geist eingesetzte Episkopen“ an und betraut sie mit seinem Vermächtnis als Gemeindeleiter. Ähnliche Tendenzen finden sich in den Pastoralbriefen, die die Episkopenverfassung […] auch in presbyteral geleiteten Gemeinden durchsetzen wollen und zu diesem Zweck Episkopen mit Presbytern identifizieren. Tit 1,5f etwa lässt die Rede von „Ältesten“ in die von „Episko pen“ übergehen. Damit […] ist klar, dass in den Pastoralbriefen noch kein dreigliedriges Amt im späteren Sinne vorliegt und dass die Grenze zwischen den beiden Funktionen noch einigermaßen fließend war5. Man darf vermuten, dass in Städten, in denen es mehrere Hausgemeinden gab, die Leiter zunächst im Plural entweder Presbyter oder auch Episkopen (Phil 1,1) genannt wurden. Erst später ist der Leiter einer Ortskirche der eine Episkopos, dem eine Gemeinschaft von Presbytern und Diakonen zugeordnet ist. Wie es schließlich zur flächendeckenden Durchsetzung des Monepiskopats und der Ämtertrias von Bischof, Presbyterkollegium und Diakonen kam, ist auf Grund der 4 5 Dietrich-Axel Koch (cf. KOCH, D. A. Die Entwicklung der Ämter in frühchristlichen Gemeinden Kleinasiens, in: SCHMELLER, T.; EBNER, M.; HOPPE, R. [Hgg.]. Neutestamentliche Ämtermodelle im Kontext. Freiburg i. Br., 2010, p. 166–206 und p. 170–172) meint, dass die Ableitung der Presbyterverfassung aus dem zeitgenössischen Judentum auf schwachen Füßen stehe, weil sich dort kein entsprechendes Vorbild nachweisen lasse. TIWALD, M. Die vielfältigen Entwicklungslinien kirchlichen Amtes im Corpus Paulinum und ihre Relevanz für heutige Theologie, in: SCHMELLER, T.; EBNER, M.; HOPPE, R. (Hgg.), Neutestamentliche Ämtermodelle im Kontext, p. 101–128, 118. Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Quellenlage nicht hinreichend zu klären6. Selbst wenn man die Briefe des Ignatius von Antiochien nicht mehr in die Zeit des Kaisers Trajan datiert, dokumentieren sie eine erstaunlich rasche Entwicklung hin zur triadisch strukturierten Gemeindeleitung unter einem einzigen Bischof 7. Ignatius betont, dass nicht die Gemeinde, sondern Christus den Bischof und dessen Helfer, die Presbyter und Diakone, autorisiert8. In seinem Brief an die Ortskirche von Smyrna schreibt er: Folgt dem Bischof wie Jesus Christus dem Vater, und dem Presbyterium wie den Aposteln; die Diakone aber achtet wie Gottes Gebot! Keiner soll ohne Bischof etwas, was die Kirche betrifft, tun. Jene Eucharistiefeier gelte als zuverlässig, die unter dem Bischof oder einem von ihm Beauftragten stattfindet. Wo der Bischof erscheint, dort soll die Gemeinde sein, wie da, wo Christus Jesus ist, die katholische Kirche ist. Ohne Bischof darf man 6 7 8 Michael Theobald erklärt die Entwicklung wie folgt: „Während die kollegiale ‚Presbyter‘-Verfassung die Einheit der Ortsgemeinde gewährleistet, hängt am ‚Episkopen‘/‚Diakonen‘-Gespann eher ihre konkrete Verwirklichung in den Gemeindevollversammlungen, die das Herzstück einer so organisierten Ortsgemeinde sind. Als Gespann bezeugen sie eine Ausdifferenzierung des Amtes, die Ausdruck wachsender sozialer Komplexität in den Gemeinden ist. Wie die Termini ἐπίσκοποι und ἐπισκοπή schon sagen, ging es bei ihrem Amt um ‚Aufsicht‘ bzw. Verantwortlichkeit für das Ganze, also Gemeindeleitung, mit der sich im Laufe der Zeit auch der Vorsitz bei der eucharistischen Vollversammlung verband. Die ihnen in der Regel zugeordneten διάκονοι waren dann – salopp gesagt – die frühkirchlichen ‚Netzwerker‘ oder ‚Netzwerkerinnen‘“ (THEOBALD, M. Eucharistie als Quelle sozialen Handelns: Eine biblisch-frühkirchliche Besinnung. Neukirchen-Vluyn, 2012, p. 204). Aus der Sicht von J. Ysebaert (YSEBAERT, J. Die Amtsterminologie im Neuen Testament und in der Alten Kirche: Eine lexikographische Untersuchung, Breda, 1994, p. 100–104), kommt Ignatius von Antiochien nicht deshalb eine herausragende Bedeutung zu, weil er den Monepiskopat profiliert, sondern weil er für das eine und einzige Haupt der Gemeinde den Titel ἐπίσκοπος reserviert hat. Der Bischof ist in den Ignatianischen Sendschreiben sichtbarer Ausdruck für die Autorität desselben Vaters, von dem Christus sagen durfte: „Ich und der Vater sind eins“ (Joh 10,30). In seinem Brief an die Magnesier nennt Ignatius den göttlichen Vater „den Bischof aller“, der, weil unsichtbar, durch den sichtbaren Bischof repräsentiert wird: „Aber euch ziemt es, das jugendliche Alter des Bischofs nicht auszunützen, sondern entsprechend der Kraft Gottes des Vaters ihm alle Ehrfurcht zu erweisen, so wie ich erfahren habe, daß auch die heiligen Presbyter seine offensichtliche Jugend nicht missbraucht haben, sondern sich ihm als Verständige in Gott fügen – doch nicht ihm, sondern dem Vater Jesu Christi, dem Bischof aller. Zur Ehre dessen, der uns erwählt hat, ist es daher geziemend, ohne jede Heuchelei zu gehorchen, da man ja nicht diesen sichtbaren Bischof täuscht, sondern den unsichtbaren betrügt“ (IGNATIUS VON ANTIOCHIEN. An die Magnesier 3,1–2, in: FISCHER, J.A. Die Apostolischen Väter, griechisch und deutsch, eingeleitet, übersetzt und herausgegeben. Darmstadt, 1986, p. 163/165). Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 353 354 MENKE, K.-H. weder taufen, noch das Liebesmahl halten; was aber jener für gut findet, das ist auch Gott wohlgefällig9. Die neutestamentlichen Schriften bezeugen weder den Monepiskopat noch die Unterordnung des Presbyteramtes unter das Episkopenamt. Sie stehen, wie die folgenden Ausführungen zeigen werden, in latenter Spannung zur faktischen Ausbreitung der ignatianischen Hierarchie. Diese hatte sich schon Mitte des dritten Jahrhunderts flächendeckend durchgesetzt10. Im Osten wird diese Entwicklung dokumentiert durch die „syrische Didaskalie“ und die auf ihr basierenden „Apostolischen Konstitutionen“. In diesen kommt dem Bischof bereits das Recht zu, Presbyter und Diakone zu erwählen11. Etwa zur selben Zeit entsteht im Westen die dem hl. Hippolyt († 235) zugeschriebene „Traditio Apostolica“; sie unterscheidet zwischen den Riten der Bischofs-, der Presbyter- und der sie unterscheidet zwischen den Riten der Bischofs-, der Presbyterund der Diakonenweihe12. Diese Unterscheidung bezeichnet keineswegs IGNATIUS VON ANTIOCHIEN. An die Smyrnäer 8,1–2, in: FISCHER (Anmerkung 8), 211. Ähnliche Belege: “Denn wenn ihr euch dem Bischof wie Jesus Christus unterordnet, scheint ihr mir nicht nach Menschenart zu leben, sondern nach Jesus Christus” (IGNATIUS VON ANTIOCHIEN, An die Trallianer 2,1, in: FISCHER [Anmerkung 8] p. 173). – “Alle nämlich, die Gottes und Jesu Christi sind, diese sind mit dem Bischof; und alle, die reumütig zur Einheit der Kirche kommen, auch diese werden Gottes sein, auf daß sie nach Jesus Christus leben. Laßt euch nicht täuschen, meine Brüder! Wenn einer einem Schismatiker folgt, erbt er das Reich Gottes nicht; wenn einer in fremdartiger Lehre wandelt, der stimmt mit dem Leiden nicht überein. Seid deshalb bedacht, eine Eucharistie zu gebrauchen – denn eines ist das Fleisch unseres Herrn Jesus Christus und einer der Kelch zur Vereinigung mit seinem Blut, einer der Opferaltar, wie einer der Bischof zusammen mit dem Presbyterium und den Diakonen, meinen Mitknechten – damit ihr, was immer ihr tut, gottgemäß tut” (Idem, An die Philadelphier 3,2–3 und 4, in: FISCHER [Anmerkung 8] p. 197). 10 Vgl. COLSON, J. Les fonctions ecclésiales aux deux premiers siècles: Textes et Études théologiques. Paris, 1956, p. 283–316. ZOLLITSCH, R. Amt und Funktion des Priesters: Eine Untersuchung zum Ursprung und zur Gestalt des Presbyters in den ersten zwei Jahrhunderten. Freiburg i. Br., 1974. JAY, E. G. From PresbyterBishops to Bishops and Presbyters. Christian Ministry in the Second Century: A Survey, in: The Second Century 1 (1981), p. 125–162. SAXER, V. Die Organisation der nachapostolischen Gemeinden (70–180), in: PIETRI, L. (Hg.). Die Zeit des Anfangs bis 250. Freiburg i. Br., 2003, p. 269–339. 11 Vgl. Constitutiones Apostolorum II, 26,1–8 (Sources Chrétiennes vol. 320, p. 235–241). 12 Vgl. die konzise Analyse von W. Geerlings in der Einleitung zu der von ihm erstellten Edition und Übersetzung der „Traditio Apostolica“ in der Reihe Fontes Christiani, Band 1, Freiburg i. Br., 1991, p. 160–171. 9 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau drei unterschiedliche „Ordines“ bezie hungsweise „Ordo-Sakramente“13. Vielmehr vermittelt die Bischofsweihe die Gnadengabe der Leitung im umfassenden Sinn. Und die Presbyter erhalten in Unterordnung unter den Bischof daran Anteil14 Nicht so die Diakone: Sie haben keinen Sitz im Rat des Bischofs oder im Kreis der Konzelebranten. Sie bringen die Gaben herbei und dürfen die Eucharistie austeilen. Kurzum: Sie haben keinen Anteil am „sacerdotium“15 des Bischofs und der Presbyter und bilden dennoch eine Gestalt der besagten Trias. Mit der Zuschreibung von Landgemeinden zu einer von einem Bischof geleiteten Stadtgemeinde erhalten die Presbyter — anders als die Diakone — ein zunehmendes Eigengewicht. Doch bei Hippolyt16 oder Tertullian17 ist die Leitung der Eucharistiefeier durch Presbyter (im Falle der Verhinderung des zuständigen Bischofs!) noch nicht bezeugt. Obwohl erst die Theologie der Folgezeit einen klaren Sakramentsbegriff entwickelt, darf man doch behaupten, dass die im dritten Jahrhundert bezeugte Trias der Weihen keine Parataxe, sondern eine Einheit beschreibt. 14 Vgl. LÉCUYER, J. Épiscopat et Presbytérat dans les écrits d’Hippolyte de Rome, in: RSR n. 41, 1953, p. 30-50. 15 “Das Bischofsamt war das erste, das in priesterlichen Begriffen beschrieben wurde. Tertullian spricht nur einmal vom Bischof als Hohempriester (summus sacerdos) […]. Andere Äußerungen von ihm deuten aber darauf hin, dass sacerdos in der nordafrikanischen Kirche eine übliche Bezeichnung für den Bischof gewesen sein könnte (Tertullian, exh. Cast. 11,1 f.; mon. 12; pud. 20,10; 21,17); und ein halbes Jahrhundert später nennt Cyprian den Bischof regelmäßig sacerdos, während summus sacerdos für Christus vorbehalten bleibt (z. B. Cyprian, ep. 63,14). Diese Entwicklung war mitnichten auf Nordafrika beschränkt. Im Osten beschreibt Origenes die Bischöfe durchwegs als Priester (z. B. Origenes, or. 28), und auch die Kirchenordnung Hippolyts betrachtet das Bischofsamt als priesterliches. Hier jedoch zeigt sich ein bezeichnender terminologischer Unterschied: Der Bischof erscheint im Ordinationsgebet der Kirchenordnung Hippolyts nicht nur als ἱερεύ, sondern als einer, der das Hohepriestertum ausübt (ἀρχιερατεύειν) und Autorität hat durch den hohepriesterlichen Geist (τῷ πνεύματι τῷ ἀρχιερατικῷ). Dasselbe gilt für die syrische Didascalia Apostolorum aus dem 3. Jh. […], die zwar betont, dass Christus der wahre Hohepriester sei, aber zugleich nicht zögert, den Bischof als den ‚levitischen Hohenpriester‘ […] zu bezeichnen (Didascalia II,26,4)” (BRADSHAW, P. F. Priester/Priestertum III-1. Christliches iesteramt: Geschichtlich, in: TRE n. 27, 1997, p. 414–421, 414 f.). 16 “Die Presbyter sind sicherlich bei der Eucharistiefeier um den Bischof beim Altar versammelt, sie strecken die Hände ebenfalls über die Gaben aus, während der Bischof die Anaphora spricht […], aber erst später, in der Zeit nach Hippolytus, wurde den Presbytern auch die Zelebration gestattet, ohne dass dabei die Anwesenheit eines Bischofs erforderlich gewesen wäre” (BÂRLEA, O. Die Weihe der Bischöfe, Presbyter und Diakone in vornizänischer Zeit. München, 1969, p. 75). 17 ”Superest ad concludendam materiolam de observatione quoque dandi et accipiendi baptismi commonefacere. Dandi quidem habet ius summus sacerdos, qui est episcopus; dehinc presbyteri et diaconi, non tamen sine episcopi auctoritate, propter ecclesiae honorem, quo salvo salva pax est” (TERTULLIAN, De baptismo 17 [CSEL 20/I, 214]) . 13 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 355 356 MENKE, K.-H. Beide unterstellen die Presbyter so eindeutig der Autorität des Bischofs, dass sie ohne seine Erlaubnis nicht einmal taufen, geschweige denn firmen oder ordinieren dürfen. Noch Possidius berichtet in seiner „Vita Augustini“, dass Bischof Valerius sich heftigen Angriffen ausgesetzt sah, weil er Augustinus, als der noch Presbyter war, in seiner Anwesenheit predigen ließ, statt selbst die Predigt zu halten18. Bevor es zu einer relativen Verselbstständigung der Presbyter gegenüber dem Bischof kommt, steht Ende des 2. Jahrhunderts in Ost und West fest: Es gibt einen einzigen Ordo, und dieser bevollmächtigt den Empfänger einer entsprechenden Weihe zu einer gewissen Repräsentation Christi in einer bestimmten Ortskirche. Es gibt immer nur ein Oberhaupt einer Ortskirche, und dieses wird spätestens zu Beginn des dritten Jahrhunderts alsepiscopus beziehungsweise Bischof bezeichnet. Aber es gibt auch Empfänger ein und desselben Ordo-„Sakraments“, die in Unterordnung unter den jeweiligen Bischof lehren, heiligen und leiten, nämlich die Presbyter. Und es gibt mit den Diakonen Empfänger des Ordo-„Sakramentes“, die gleichsam den Boden bereiten für das von jeweils einem Bischof mit seinem Presbyterium gemeinsam ausgeübte Lehren, Heiligen und Leiten. Gregor Predel hat exemplarisch gezeigt, dass dort, wo die Menschen auf dem Land und nicht in der Stadt wohnten, eine Verselbstständigung der Presbyter gegenüber dem Bischof unvermeidlich war. Während der Diakonat an Bedeutung verlor19, avancierten die Presbyter zu den weithin 18 19 POSSIDIUS, Vita Augustini, V (in: PL vol. 32, c. 37). „Die hier vorliegende Entwicklung des Diakonates (wie auch die gleichzeitige Entwicklung des Presbyterates) erinnert, wenn auch auf einer völlig anderen Ebene, stark an einen natürlichen Evolutionsprozess durch Mutation und Selektion. Der zunächst von außen an die Kirche herangetragene (Umwelt-)einfluss einer sich radikal verändernden Gesellschaft erzwingt eine recht schnell verlaufende ‚Mutation‘ des Presbyterates, die dieser Situation besser angepasst ist als seine ursprüngliche Form. Der nachfolgende Selektionsprozess lässt den Diakonat, der sich kaum verändert hat, in der veränderten gesellschaftlichen Situation im Laufe der Zeit weitgehend verschwinden. Als Selektionsvorteil für das Erstarken des Presbyterates ist dabei zu sehen, dass er […] ein sehr viel breiteres Spektrum liturgischer, spiritueller und pastoraler Aufgaben abdecken konnte“ (PREDEL, G. Vom Presbyter zum Sacerdos: Historische und theologische Aspekte der Entwicklung der Leitungsverantwortung und Sacerdotalisierung des Presbyterates im spätantiken Gallien. Münster, 2005, 127 f.). Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau eigentlichen Repräsentanten des nachapostolischen Amtes20. Allerdings blieb stets bewusst, dass ein Presbyter sein Amt nur im Gehorsam gegenüber dem Bischof ausübt. Predel schreibt: Weil der Presbyter […] aus theologischen Gründen keine eigenständige, vom Bischof unabhängige, geistliche Leitung wahrnehmen kann, können auch die Kirchen und Gemeinden, die ihm unterstehen, nicht als eigenständige Ortskirchen definiert werden, selbst wenn diese Gemeinden finanziell und verwaltungsmäßig ein hohes Maß an Eigenständigkeit erreicht haben. Die geistliche Abhängigkeit der Presbyter von ihrem Bischof zeigt damit, dass Presbytern unterstehende Kirchen und Gemeinden als Teil oder vielleicht Erweiterung der bischöflichen Ortskirche zu verstehen sind, die auch weiterhin grundsätzlich vollständig der geistlichen Leitung und Verantwortung des Bischofs selbst unterstehen21. Obwohl die Trias von Bischof, Presbyter und Diakon für die große Mehrheit der Gläubigen kaum noch als die eine Repräsentation Christi durch je einen Bischof mit Presbytern und Diakonen anschaulich blieb, war doch theologisch und rechtlich unbestritten: Das nachapostolische Amt ist kein Ordo, der beliebig gestaltet werden kann. Allerdings blieb über Jahrhunderte ungeklärt, ob nur die Einheit des Ordo oder auch der Unterschied zwischen Bischof, Presbyter und Diakon als sakramental betrachtet werden muss. Die der theologischen Reflexion aufgegebene Frage lautete: Erhält ein Mann, der das Sakrament des Ordo empfängt, mit der Weihe auch die Einweisung in einen der drei Ränge von Bischof, Presbyter und Diakon? Oder ist der Unterschied zwischen den drei Rängen kein im Sakrament des Ordo wurzelnder, sondern ein der rechtlichen Ordnung geschuldeter? „In dieser Veränderung dürfte sich einerseits eine zunehmende Verrechtlichung im Verständnis des Bischofsamtes zeigen, die durch die Wahrnehmung politischer Aufgaben der Bischöfe noch verstärkt wurde. Andererseits ist davon auszugehen, dass der Presbyter angesichts der zunehmenden Profilierung des Presbyterates durch Übernahme bis dahin genuin bischöflicher Aufgaben vermehrt als sacerdos erfahren wurde, während umgekehrt der Bischof mit seinem in den Gemeinden verringerten sacerdotalen Erscheinen langsam aus dem Blickfeld der Menschen verschwand“ (PREDEL [Anmerkung 19], 197). 21 PREDEL (Anmerkung 19), 167. 20 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 357 358 MENKE, K.-H. Es gibt — bezogen auf die gesamte Theologiegeschichte des Christentums — kaum eine These, die ähnlich erfolgreich war wie die des Kirchenvaters Hieronymus (347–420) über die sakramentale Gleichrangigkeit der Presbyter mit den Bischöfen. Während im Osten22 die sakramentale Bestimmung nicht nur der Einheit, sondern auch der Verschiedenheit von Bischof, Presbyter und Diakon ungebrochen weiter tradiert und durch die neuplatonische Denkweise (hierarchisch sich ausdifferenzierende Einheit) des Pseudo-Dionysius-Areopagita theologisch untermauert wurde, kam es im Westen auf Grund der exegetischen Autorität des Hieronymus (a) zu einer sakramentalen Identifikation von Bischof und Presbyter, (b) zu einer Ausgliederung des Diakonates aus dem einen Ordo und — damit verbunden — (c) zu einer verhängnisvollen Trennung der rechtlichen von der sakramentalen Ordnung — genauer gesagt, zu der Auffassung, dass nicht das Sakrament des Ordo, sondern der Papst als Inhaber aller Jurisdiktionsgewalt die Rangunterschiede der Empfänger des Ordo-Sakramentes bestimmt. Die These des Hieronymus wurzelt in einem Rangstreit zwischen Presbytern und Diakonen23, zu dem auch andere PaulusbriefKommentatoren — z. B. der durch Erasmus als Pseudo-Ambrosius enttarnte Ambrosiaster24 und Pelagius25 — Stellung genommen haben. In diesem Streit ging es um Diakone, die zwar dem einen Bischof, nicht aber auch den Presbytern untergeordnet sein wollten. In einem Brief26 Dazu: LUTTENBERGER, G. H. The Decline of Presbyterial Collegiality and the Growth of Individualization of the Priesthood (4th–5th centuries), in: RThAM n. 48, 1981, p. 14–58; besonders 49-56. 23 Dazu: PRAT, F. Les prétentions des diacres Romains au quatrième siècle, in: RSR n. 3, 1912, p. 463-475. 24 Vgl. AMBROSIUS [Ambrosiaster], In Ephes. 4,11 f. (in: PL vol. 17, c 410); In 1 Tim 3,8–10 (in: PL vol. 17, c. 470). – Ders. auch in den fälschlicherweise Augustinus zugeschriebenen „Quaestiones Veteris et Novi Testamenti“: q. 101 (in: PL vol. 35, c. 2305-2307). 25 PELAGIUS, In Phil. 1 (PL vol. 30, c. 879); In Tim 3 (PL vol. 30, c. 922); In Tit. 1 (PL vol. 30, c. 941). 26 HIERONYMUS, Ep. 146 (PL 22,1192–1195; CSEL 56/I,3, 308–312). – Dazu: BODIN, Y. Saint Jérôme et l’église (Théologie historique, 6), Paris, 1966. 22 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau an den römischen Presbyter Evangelus27 stellt sich Hieronymus auf die Seite der Presbyter. Er begründet seine Einschätzung ähnlich wie Ambrosiaster und Pelagius mit einem ausführlichen Hinweis auf die Bibelstellen, in denen die Bezeichnungen presbyter und episcopus als Synonyma erscheinen. Damit will er sagen: Diespätere Entwicklung zur Unterordnung einer Vielzahl von Presbytern unter jeweils einen Bischof28 ist der kirchlichen Ordnung geschuldet29. Vom Sakrament des Ordo her betrachtet ist dem Presbyter dieselbe Würde wie dem Bischof zuzusprechen. Mit anderen Worten: Bischof und Presbyter stehen, was die Weihegewalt betrifft, auf derselben Stufe; und nur damit es in der Kirche eine rechtlich geregelte Ordnung gibt, kann oder „Der römische Presbyter Evangelus schickte Hieronymus die anonymen Erörterungen zum Alten und Neuen Testament (Quaestiones veteris et novi testamenti), die unter den Werken Augustins überliefert sind und von der modernen Forschung dem sogenannten Ambrosiaster (vgl. LACL 18 f.), einem namentlich nicht bekannten Pauluskommentator in Rom zur Zeit des Damasus, zugeschrieben werden (CSEL 50), und fragte Hieronymus nach seiner Meinung zu der darin vorgenommenen Identifizierung des Melchisedek mit dem Heiligen Geist (vgl. quaest. test. 109,20 f.). Hieronymus antwortete im Frühjahr 398 mit einem kleinen Traktat zu diesem Thema (ep. 73). Zu einem nicht feststellbaren Zeitpunkt schrieb Hieronymus Evangelus einen weiteren Brief über die Rangordnung von Diakonat und Presbyterat (ep. 146). Evangelus ist wahrscheinlich mit demjenigen Evangelus zu identifizieren, dem der pelagianische Diakon Anianus (auch Annianus) von Celeda (um 420; vgl. LACL 37) seine Übersetzung der sieben Lobreden des Johannes Chrysostomus auf den Apostel Paulus (De laudibus sancti Pauli) widmete“ (FÜRST, A. Hieronymus: Askese und Wissenschaft in der Spätantike. Freiburg i. Br., 2003, 176 f.). In seinem 389 verfassten Kommentar zum Titusbrief formuliert Hieronymus die oft zitierte Sentenz: „Idem est ergo presbyter qui et episcopus […]“ (In Tit. 1,5 [PL 26,597]). Aus der Tatsache, dass in Phil 1,1 im Plural von Bischöfen und Presbytern derselben Gemeinde gesprochen wird, folgert er die Synonymität der beiden Amtsbezeichnungen. Zu Apg 20,28 bemerkt er, dass Paulus Presbyter aus Ephesus zu sich nach Milet kommen lässt und sie dort als Bischöfe anredet. Außerdem vermutet er, dass Hebr 13,17 und 1 Petr 5,1 f. eine nicht monepiskopale, sondern presbyteriale Gemeindeleitung spiegeln. 28 „[…] in toto orbe decretum est, ut unus de presbyteris electus superponeretur ceteris, ad quem omnis Ecclesiae cura pertineret et schismatum semina tollerentur“ (HIERONYMUS, In Tit. 1,5 [PL vol. 26, c. 597]). 29 „Sicut ergo presbyteri sciunt se ex Ecclesiae consuetudine ei qui sibi praepositus fuerit, esse subiectos: ita episcopi noverint se magis consuetudine, quam dispositionis Dominicae veritate, presbyteris esse maiores et in commune debere Ecclesiam regere, imitantes Moysen, qui cum haberet in potestate solum praeesse populo Israel, septuaginta elegit, cum quibus populum iudicaret (Num 11,16s). Videamus igitur qualis presbyter, sive episcopus ordinandus sit“ (HIERONYMUS, In Tit. 1,5 [PL vol. 26, c. 598]). 27 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 359 360 MENKE, K.-H. darf ein Presbyter nicht alles, was ein Bischof kann oder darf30. Die Diakonenweihe aber überträgt nicht dieselbe Gewalt, die der Bischof und der Presbyter empfangen. Daraus folgert Hieronymus: Der Diakon empfängt zwar eine Weihe, aber nicht das Sakrament des Ordo; deshalb ist die Unterordnung des Diakons unter den Presbyter — anders als die Unterordnung des Presbyters unter den Bischof — nicht nur eine der rechtlichen Ordnung geschuldete, sondern eine wesentliche. Viele Theologen des Mittelalters zählen die Diakonenweihe zu den Konsekrationsriten, die wie Lektorat, Akolythat oder Subdiakonat der Vorbereitung auf das Sakrament des Ordo dienen. Weil Hieronymus neben Augustinus das Mittelalter maßgeblich beeinflusst hat, wurden seine Invektiven gegen eine mehr als ordnungslogische oder juridische Vorordnung des Bischofs über den Presbyter nicht vergessen. Isidor von Sevilla († 636) bekräftigt die Grundthese des Hieronymus, erklärt den Unterschied zwischen Episkopat und Presbyterat rein kirchenrechtlich, will sich aber — um der Ordnung willen — dem Grundsatz fügen, nur der Bischof dürfe firmen und die Weihen spenden31. Ratramnus († 869) erklärt den Unterschied zwischen Bischof und Presbyter vor allem damit, dass Bischöfe Presbyter weihen, nie aber Die Reformatoren haben sich besonders gern auf jene Passage des Hieronymus-Briefes an Evangelus berufen, die von der alexandrinischen Ortskirche berichtet; dort sei es bis zu den Bischöfen Heraklas und Dionysius (3. Jahrhundert) üblich gewesen, dass die Presbyter einen aus ihrem Kreis — ähnlich wie ein Heer seinen Führer oder die Diakone einen Archidiakon — erwählten, damit er ihr Vorgesetzter sei: „Quod autem postea unus electus est, qui ceteris praeponeretur, in scismatis remedium factum est, ne unusquisque ad se trahens Christi ecclesiam rumperet. Nam et Alexandriae a Marco evangelista usque ad Heraclam et Dionysium episcopos presbyteri semper unum de se electum et in excelsiori gradu conlocatum episcopum nominabant, quomodo si exercitus imperatorem faciat aut diaconi eligant de se, quem industrium noverint, et archidiaconum vocent“ (HIERONYMUS, Ep. 146 [CSEL 56/I,3, 308–312; 310]). – Dazu: HENNINGS, R. Hieronymus zum Bischofsamt und seine Autorität in dieser Frage bei Luther, Melanchthon und Zwingli, in: GRANE, L; SCHINDLER, A.; WRIEDT, M. Auctoritas Patrum II. Neue Beiträge zur Rezeption der Kirchenväter im 15 und 16 Jahrhundert, herausgegeben. Mainz, 1998, p. 85-104. 31 „De presbyteris. […] Praesunt enim Ecclesiae Christi, et in confectione divini corporis et sanguinis consortes cum episcopis sunt, similiter et in doctrina populorum, et in officio praedicandi. Ac sola propter auctoritatem summo sacerdoti clericorum ordinatio et consecratio servata est, ne a multis Ecclesiae disciplina vendicata concordiam solveret, scandala generaret“ (ISIDORUS, De ecclesiasticis officiis II, 7 [PL vol. 83, c. 787]). 30 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau umgekehrt Presbyter Bischöfe32. Etwa zur selben Zeit, in der Ratramnus seine Traktate verfasst, erinnert Amalarius von Metz († 850) mit Berufung auf die biblische Ununterscheidbarkeit des Rangs der Bischöfe von dem der Presbyter an die Mahnung des Hieronymus, die Presbyter sollten sich den Bischöfen der Gewohnheit entsprechend unterordnen, die Bischöfe sollten aber auch wissen, dass sie eher auf Grund der Gewohnheit als auf Grund einer Anordnung Christi den Presbytern übergeordnet seien33. Auch Ivo von Chartres († 1116) sieht den Unterschied zwischen Bischof und Presbyter lediglich in Funktionen, die nur um der Disziplin willen dem Priester vorenthalten sind34. Besonders förderlich für den bestimmenden Einfluss der Hieronymus-These auf die Ekklesiologie des Mittelalters war die Aufnahme der entscheidenden Zitate seines Titus-Kommentars und seines Briefes an Evangelus in das Decretum Gratiani35 in jene Concordia discordantium canonum also, die zwischen 1125 und 1140 von dem Bologneser Mönch Gratian „Et revera inter sacerdotes et episcopos differentia non parva consistit. Sacerdotes namque, sicut reliqui gradus ecclesiastici, per episcoporum ministerium consecrantur; at episcopi non a sacerdotibus benedicuntur. Ipsi quoque sanctum chrisma sanctificant, et oleum consecrant: omnisque gradus ecclesiasticus illorum dispositione peragitur. Quod considerantes ecclesiastici viri, statuerunt ut frontes baptizatorum non a presbyteris, sed ab episcopis chrismate sancto linirentur“ (RATRAMNUS, Contra Graecorum opposita IV, 7 [PL vol. 121, c. 333]). 33 „Hieronimus exponit quid plus habeat episcopus quam presbyter, dicens in epistola ad Evangelum saepe repetitum: ‚Quid enim facit, excepta ordinatione, episcopus quod presbyter non facit?‘ et exponit cuius constitutione sit episcopus constitutus, in tractatu super Titum dicens: ‚Sicut ergo presbyteri sciunt se ex ecclesiae consuetudine ei qui sibi praepositus fuerit esse subiectos, ita episcopi noverint se magis consuetudine, quam dispositionis dominicae veritate presbyteris esse maiores, et in commune debere ecclesiam regere, […]‘“ (AMALARIUS, Liber officialis II,13,15 [ed. J. M. Hanssens. Rom, 1948, p. 232]). 34 Vgl. IVO VON CHARTRES, Panormia III, 5 (PL vol. 161, c. 1130). 35 Vgl. Decretum Gratiani I, 95,5 (PL vol. 187, c. 448–452). 32 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 361 362 MENKE, K.-H. grundgelegt, zur Basis der Kanonistik wurde36. In den einschlägigen kanonistischen Kommentaren wird die These des Hieronymus fortgeschrieben. Sicard von Cremona († 1215) zum Beispiel unterscheidet zwischen der Gleichrangigkeit von Bischof und Presbyter in der Sakramentenspendung (officium) bei gleichzeitiger Unterschiedenheit ihrer Leitungsaufgabe (administration)37. Huguccio († 1210) nimmt zwar an, dass die Überordnung des Bischofs über die Presbyter keine erst nachträglich eingeführte ist, betrachtet sie aber deshalb nicht als göttlichen Rechts38. Unter den Kanonisten wagt niemand, den Hieronymus-Zitaten des Decretum Gratiani zu widersprechen. In den Sentenzen, Summen und Quästionensammlungen der Theologen wird die Hieronymus-These nur beiläufig oder gar nicht behandelt. Eine Ausnahme bildet die wirkungsgeschichtlich kaum überschätzbare Summa de sacramentis christianae fidei des Hugo von St. Viktor († 1141). Er sieht Episkopat und Presbyterat auf der sakramentalen Ebene als Einheit unbeschadet unterschiedlicher Befugnisse und Funktionen (unus gradus […] in sacramento, non tamen una potestas in ministerio), wohingegen der Diakonat nicht nur funktional, „Für das Mittelalter und insbesondere die Frühscholastik war nicht ausschlaggebend, welche Lehre die authentische der Väter wäre, sondern vielmehr, welche Texte vom Fluß der Überlieferung an ihren Strand getrieben und dort aufgenommen als Grundstock der theologischen Arbeit Verwendung fanden. Unsere Untersuchung hat […] ergeben, daß dies fast ausschließlich die beiden Hieronymussentenzen waren, in denen man das Priestertum gleichrangig neben den Episkopat gestellt sah. Dazu kam, dass diese Sentenzen in die Paulinenliteratur und in das Dekret Gratians Eingang fanden und gerade hier zum Nährboden des frühscholastischen Denkens werden mussten. Wenn man nun bedenkt, daß die damalige Spekulation unser Problem für ein vornehmlich kanonistisches ansah und darum seine Behandlung den Dekretisten zuschieben wollte, so versteht man, daß bei dem Konservativismus der Kanonistik die von Hieronymus überkommene Lösung im Verlauf der frühscholastischen Periode sich behauptete […]“ (LANDGRAF, A. M. Dogmengeschichte der Frühscholastik. Dritter Teil: Die Lehre von den Sakramenten: Band II. Regensburg, 1955, 301 f.). 37 „Et attende, quod episcopi dicuntur tenere locum apostolorum et presbiteri LXXII discipulorum, quia sicut apostoli preerant discipulis in amministratione, sic episcopi sacerdotibus in amministratione et officiorum executione. Olim namque in amministratione dispares, in officio pares et quodcunque sacramentum dabat Petrus, quilibit sacerdos conferre poterat. Set hodie dispares in utroque, cum solis episcopis liceat confirmare et similia“ (SIKARD VON CREMONA, Cod. Bamberg, can.38, fol. 68; hier zitiert nach LANDGRAF [Anmerkung 37], p. 287). 38 Vgl. LANDGRAF (Anmerkung 37), 288. 36 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau sondern auchsakramental betrachtet etwas Eigenes ist39. Trotz der sakramentalen Geeintheit von Episkopat und Presbyterat bezeichnet Hugo nur die Bischöfe als Nachfolger der Apostel, die Presbyter hingegen als Nachfolger und Stellvertreter der 72 Schüler, aus denen der Herr die Zwölf ausgewählt hat40. Die Sentenzensammlung des Petrus Lombardus († 1160) — Pflichtlektüre für jeden Theologiestudenten des Mittelalters — verzichtet zwar auf die Wiederholung der besagten Hieronymus-Zitate, setzt aber voraus, „dass bei den Alten dieselben episcopi und presbyteri genannt wurden, dass aber die presbyteri als sacerdotes den Gipfel des Pontifikates nicht besitzen wie die episcopi, da sie die Stirne nicht mit Chrisam zeichnen, noch den Paraclitus geben, was nach Erweis der Apostelgeschichte einzig den episcopi zustehe“41. Doch der so bezeichnete Unterschied begründet keinen eigenen Ordo, sondern nur eine höhere Würde des Bischofs gegenüber dem Priester, wohingegen der Diakonat für einen eigenen Ordo steht42. Petrus Lombardus unterscheidet zwischen den officia, die durch das Sakrament der Bischofsbeziehungsweise Priesterweihe übertragen „De ordinibus hoc primum attendendum est, quod alii sunt secundum gradum differentem, sicut est diaconus et sacerdos; alii in eodem gradu secundum potestatem excellentem, sicut diaconus et archidiaconus. Unus gradus est in sacramento; non tamen una potestas in ministerio. Diaconus enim sacerdoti ministrat in sacramento corporis et sanguinis Christi. Archidiaconus autem hoc plus habet, quod praeter ministerium altaris sub episcopo et vice episcopi curam habet Ecclesiarum; et causas ecclesiasticas examinat et ministeria dispensat. Similiter sacerdos et pontifex sive summus sacerdos unus gradus est in sacramento, diversa tamen potestas in ministerio; quia cum utrisque corporis et sanguinis Christi consecrandi, baptizandi, catechizandi, praedicandi, ligandi, solvendi, una quodammodo sit dignitas, pontificibus tamen ecclesias dedicandi, ordines faciendi, manus imponendi, sacri chrismatis consecrandi, communem super populum benedictionem faciendi, singularis data est potestas. Sic HUGO VON ST. VICTOR, De sacramentis II, 2,5 [PL vol. 176, c. 419; Corpus Victorinum, herausgegeben von BERNDT, R. Band I. Münster, 2008, 339 f.]. 40 Vgl. HUGO VON ST. VICTOR, De sacramentis II, 3,12 (PL vol. 176, c. 428; Corpus Victorinum Band I, herausgegeben von BERNDT, R. Münster, 2008, 355 f.). 41 LANDGRAF (Anmerkung 37), p. 291. „Ideo autem et presbyteri sacerdotes vocantur, quia sacrum dant. Qui, licet sint sacerdotes, tamen pontificatus apicem non habent, sicut episcopi, quia nec chrismate frontem signant, nec Paraclitum dant: quod solis deberi episcopis, lectio Actuum Apostolorum demonstrat. Unde et apud veteres iidem episcopi et presbyteri fuerunt, quia illud nomen est dignitatis, non aetatis“ (PETRUS LOMBARDUS, Sent. IV, d. 24, c. 11). 42 „Cumque omnes spirituales sint et sacri, excellenter tamen canones duos tantum sacros ordines appellari censent, diaconatus scilicet et presbyteratus, quia hos solos primitiva Ecclesia legitur habuisse, et de his solis praeceptum Apostoli habemus“ (PETRUS LOMBARDUS, Sent. IV, d. 24, c. 12). 39 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 363 364 MENKE, K.-H. werden (z. B. Konsekrationsvollmacht), und den officia, die durch einen Rechtsakt verliehen werden (z. B. Leitungsvollmacht). So wird er zum Wegbereiter der später klassisch gewordenen Unterscheidung zwischen potestas ordinis und potestas iurisdictionis. Bonaventura († 1274) betont in seinem Kommentar zu den Sentenzen des Petrus Lombardus, dass der Bischof in Bezug auf die Feier der Eucharistie dem Presbyter nichts voraushat und dass deshalb seine Abgehobenheit gegenüber dem sogenannten einfachen Priester lediglich die einer höheren Würde und Rechtsstellung sei. Ausdrücklich vergleicht er den Unterschied zwischen Bischof und Priester mit dem Unterschied zwischen Erzbischof und Suffraganbischof, zwischen dem Papst und einem Patriarchen43. Auch Thomas von Aquin († 1274) urteilt zumindest in seinem Frühwerk ähnlich. Er unterscheidet zwischen der Gewalt über den eucharistischen und der Gewalt über den mystischen Leib Christi44. Was die letztere betrifft, so unterscheidet sich der Bischof erheblich vom Priester; was die erstere betrifft, in keiner Weise; denn die Bischofsweihe „Conclusio. Episcopatus proprie non est ordo, sed ordinis eminentia vel dignitas. […] Dicendum, quod, sicut tactum est, cum ordinis potestas principaliter sit ordinata ad dispensationem Sacramentorum et maxime illius Sacramenti nobilissimi, scilicet corporis Domini, quod ibi est status graduum et ordinum ascendentium, et ita ultra sacerdotium non est gradus ordinis. Sed tamen intra hunc gradum et ordinem contingit esse distinctionem dignitatum et officiorum, quae tamen novum gradum vel ordinem non constituunt, ut archipresbyter, episcopus, archiepiscopus, patriarcha, Pontifex Summus, quae ultra sacerdotium non addunt ordinem nec gradum novum, sed solum dignitatem et officium. Et ita episcopatus, prout concernit ordinem sacerdotii, bene potest dici ordo; sed prout distinguitur contra sacerdotium, dicit dignitatem quandam vel officium ipsi annexum, et non est proprie nomen ordinis, nec novus character imprimatur, nec novapotestas datur, sed potestas data ampliatur“ (BONAVENTURA, In IV Sent. d. 24, p. 2, a. 2, q. 3). 44 „Ad secundum quaestionem dicendum, quod ordo potest accipi dupliciter. Uno modo secundum quod est sacramentum; et sic, ut prius dictum est, ordinatur omnis ordo ad eucharistiae sacramentum; unde, cum Episcopus non habeat potestatem superiorem sacerdote quantum ad hoc, non erit episcopatus ordo. Alio modo potest considerari ordo secundum quod est officium quoddam respectu quarumdam actionum sacrarum; et sic, cum Episcopatus habeat potestatem in actionibus hierarchicis respectu corporis mystici supra sacerdotem, episcopatus erit ordo; […] ordo secundum quod est sacramentum imprimens characterem, ordinatur specialiter ad sacramentum eucharistiae, in quo ipse Christus continetur, quia per characterem ipsi Christo configuramur; et ideo licet detur aliqua potestas spiritualis Episcopo in sui promotione respectu aliquorum sacramentorum, non tamen illa potestas habet rationem characteris; et propter hoc episcopatus non est ordo, secundum quod ordo est quoddam sacramentum. Ad tertium dicendum, quod potestas episcopalis non est tantum iurisdictionis, sed etiam ordinis, ut ex dictis patet, secundum quod ordo communiter accipitur“ (THOMAS VON AQUIN, In IV Sent. d. 24, q. 3, a 2). 43 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau verleiht keinen eigenen sakramentalen Charakter. Allerdings schreibt Thomas schon in seinem Sentenzenkommentar, dass die Bischofsweihe doch mehr als ein rituell eingekleideter Rechtsakt sei45. Deshalb relativiert er die entsprechenden Ausführungen des Hieronymus über die sakramentale Identität des Priesters mit dem Bischof — in seinen theologischen Opuscula ebenso wie in seinen Paulusbrief-Kommentaren. Joseph Lécuyer hat gezeigt, dass man diachron betrachtet von einer zunehmend sakramentalen Bestimmung nicht nur der Einheit, sondern auch der Gestuftheit des Ordo im Werk des Aquinaten sprechen kann46. In der unvollendeten Summa theologica konfrontiert Thomas die Position des Hieronymus (sakramentale Identifizierung der Priester mit den Bischöfen) mit der ganz anderen Position (sakramentale Unterschiedenheit von Bischof, Presbyter und Diakon) des PseudoDionysius Areopagita47. Dabei orientiert er sich an der neuplatonischen Lehre, dass ein und dieselbe Wirklichkeit auf vollkommene oder weniger vollkommene Weise empfangen und verliehen werden kann48. Es gibt nur ein Sakrament des Ordo; aber eine unterschiedlich vollkommene Weise der Spendung und des Empfangs49. Für die Folgezeit kann man bilanzieren, dass die Franziskaner eher zu einer fortschreitenden Trennung zwischen potestas ordinis und potestas iurisdictionis neigen, wohingegen die Dominikaner die Tendenz fortschreiben, die sichim Spätwerk des Aquinaten abzeichnet. In den Sentenzenkommentaren der Dominikaner Durandus de San Porciano († 1334) und Petrus de Palude († 1342), aber auch des Augustiners Thomas von Straßburg († 1357) erscheint die Bischofsweihe als „Ad secundum dicendum, quod in promotione episcopi datur sibi potestas quae perpetuo manet in eo; quamvis dici non possit character, quia per eam non ordinatur homo directe ad Deum, sed ad Corpus Christi mysticum; et tamen indelebiliter manet sicut character, quia per consecrationem datur“ (THOMAS VON AQUIN, In IV Sent. d. 25, q. 1, a. 2). 46 Vgl. LÉCUYER, J. Les étapes de l‘enseignement thomiste sur l‘épiscopat, in: RThom n. 57, 1957, p. 29-52. – Dazu auch: SEITERICH, E. Ist der Episkopat ein Sakrament?, in: Schol. n. 18, 1943, p. 200-219. 47 Vgl. THOMAS VON AQUIN, S.th. II-II, q. 184, a. 6. 48 „Sed contra est quod Dionysius dicit, in Eccl. Hier. [cap. 5]: ‚Pontificum quidem ordo consummativus est et perfectivus; sacerdotum autem illuminativus et lucidativus; ministrantium vero purgativus et discretivus.‘ Ex quo patet quod perfectio solis episcopis attribuitur“ (THOMAS VON AQUIN, S.th. II-II, q. 184, a. 6,3). 49 Vgl. LÉCUYER (Anmerkung 47), p. 45–50. 45 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 365 366 MENKE, K.-H. sakramentale Vervollkommnung der Priesterweihe50. Von einem eigenen Sakrament der Bischofsweihe aber sprechen erst die Theologen der Schule von Salamanca. Francisco de Vitoria OP († 1546) wendet sich aus exegetischen und theologischen Gründen gegen die These, dass die Unterscheidung des Bischofs vom Priester nicht in der Bischofsweihe wurzele, sondern in einem Delegationsakt des Papstes51. Er beruft sich auf Apg 14,22, aber mit Thomas von Aquin auch auf die spekulativen Argumente des Areopagiten52. Der im spanischen Alcalá geborene Pedro De Soto († 1563) geht noch einen Schritt weiter. Er bezeichnet ausdrücklich alle Stufen (ordines) der Trias von Bischof, Priester und Diakon als „verissima sacramenta“ — dies allerdings ohne Klärung der Frage, wie diese These mit der Einheit des Ordo-Sakramentes zu Vgl. MÜLLER, H. Zum Verhältnis zwischen Episkopat und Presbyterat im Zweiten Vatikanischen Konzil. Eine rechtstheologische Untersuchung. Wien, 1971, p. 50-52. WALTER, P. Das Verhältnis von Episkopat und Presbyterat von der Alten Kirche bis zum Reformationsjahrhundert, in: Das kirchliche Amt in apostolischer Nachfolge, Band II: Ursprünge und Wandlungen. Herausgegeben von SATTLER, D; WENZ, G., Freiburg i. Br./ Göttingen, 2006, p. 39-96, p. 66–72. 51 „Tertia propositio: Tota potestas ordinis in ecclesia derivata est et pendet immediate ab episcopis.Volo dicere, quod sicut apostoli et illi soli habuerunt iure divino ordinare et consecrarepresbyteros et alios inferiores ministros, ita omnes et soli episcopi hoc habent etiam iure divino“ (FRANCISCO DE VITORIA, Vorlesungen I [Relectiones]. Völkerrecht – Politik – Kirche. Herausgegeben von HORST, U; JUSTENHOVEN, J.; STÜBEN, J. Stuttgart, 1995, p. 278–351; p. 332). 52 „Nec de hoc inter catholicos est dubitatio episcopos hanc habere potestatem. Et quod soli episcopi habeant, videtur, quia nunquam legimus ordinationes factas nisi ab apostolis vel ab episcopis aliis. Unde etiam Act 14,22 de Paulo et Barnaba legitur, quod constitutis Lystrae et Iconio et Antiochiae per singulas ecclesias prebyteris decesserunt. Et Dionysius apostolorum discipulus et synchronus libro De ecclesiastica hierarchia, ubi formam ecclesiasticam ab apostolis traditam diserte et copiose tractat, solis pontificibus ordinationem ministrorum tribuit. Immo nec oleum sanctum, quo sacrantur presbyteri, ab alio quam a pontifice docet confici posse“ (FRANCISCO DE VITORIA, [Anmerkung 52], p. 334). 50 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau vereinbaren sei53. Während Pedro De Soto seine sakramententheologi schen Reflexionen an der Praxis orientiert, ist Domingo De Soto († 1560) ein außergewöhnlicher Kenner der Tradition, vertraut mit den Schriften des Thomas von Aquin und auf die widerspruchsfreie Begründung seiner Thesen bedacht. Da es, so argumentiert er, nur ein Sakrament des sacerdotium gibt, empfangen die Bischöfe und Priester (nicht die Diakone!) dasselbe Sakrament; aber Erstere in vollkommener, Letztere in partizipativer Weise. Obwohl es kein achtes Sakrament gibt, ist die Bischofsweihe aus der Sicht Domingo de Sotos kein bloß jurisdiktioneller Akt — dies schon deshalb nicht, weil ein Bischof, der seines Amtes enthoben wird, dennoch Bischof bleibt54. Den an Thomas orientierten Dominikanern und Salmantizensern stehen Theologen der Franziskanerschule und die meisten Kanonisten entgegen. Der Scotus-Schüler Petrus Aureoli OFM († 1322) zum Beispiel lehrt in seinem Sentenzenkommentar, dass alle Unterschiede zwischen Bischof und Priester ausschließlich durch die Rechtsordnung der Kirche bedingt seien, dass also der Papst als Inhaber der vollen Rechtsgewalt einen Priester ohne vorherige Bischofsweihe nicht nur zum Firmspender, „Ex his igitur et vera ratio huius sacramenti, et dubia quae inter posteriores nostros scholasticos disputantur, sunt cognoscenda. Primo itaque illud certo constat, esse sacramentum ordinis in ecclesia, illud consummari et perfici in traditione potestatis illius ecclesiasticae. Cum vero disputatur, quomodo sit unum sacramentum ordinis, vel quae non plura, cum sint plures ordines magis de nomine quaestio est, quam de re aliqua. Unicum sacramentum ordinis dicimus, quanquam tradantur plures per illud ordines, et quidem […] diversarum specierum et rationum; tamen omnes ad unum (quod praecipuum et maximum est) sacerdotium scilicet, sive presbyterium, sive episcopatum referuntur de quo postea. Nunc satis est, quod attributione quadam omnia unum sacramentum dicuntur, quae ad unum illud praecipuum ordinantur. Haec est beati Thomae in hac materia sententia. Quando non desint qui ex eius verbis putent, id quod sit in aliis omnibus ordinibus, sacramentale quoddam esse; sicut quae fiunt in exorcismis ante baptismum, praeparationes sunt ad baptismum; ita omnia alia quae fiunt in omnibus ordinibus, praeparationes sunt ad sacerdotium. Et putant isti hoc probabiliter affirmari posse; atque ita unum tantum verissime esse sacramentum ordinis. Sed revera minus tuta est sententia, minus consentanea sensui ecclesiae; et ut specialius dicamus, certum est episcopatus, presbyterii, et diaconii ordines verissima esse sacramenta; quorum duo priora ex institutione Christi habentur, ut probatum est; et traduntur per ea distinctae potestates. Nam episcopi potestas superior est […] et ad multa in sacramentis Christi omnino necessaria. Igitur verissime per sacramentum illa traditur; sicut potestas confirmandi et ordinandi. Itaque episcopatus (de quo multi aut dubitant, aut diversitate nominis quaestionem constituunt) censendus est verus ordo […]“ (PEDRO DE SOTO, Tractatus de institutione sacerdotum: Prima pars - De sacramento ordinis. Dillingen, 1558, p. 346). 54 Vgl. DOMINGO DE SOTO, De Iustitia et Iure, l. 10, q. 1, a. 2. 53 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 367 368 MENKE, K.-H. sondern auch zur Spendung aller Ordinationen ermächtigen könne55. Der 1321 zum Erzbischof von Aix ernannte Franziskaner konnte sich für diese Position auch auf einen Kanonisten des Dominikanerordens, den 1276 zum Papst gewählten Petrus von Tarentaise (Papst Innozenz V.), berufen, weil dessen Rechtsdogmatik von der Überzeugung geprägt war, dass das Sakrament der Weihe allein die Konsekrationsgewalt über das corpus Christi verum überträgt und dass Papst, Bischof und Priester im Unterschied zum Diakon diese im Sakrament empfangene Gewalt gemeinsam haben, dass daher alle Unterschiede der Hierarchie durch die vom Papst ausgehende Ordnungsgewalt über das corpus Christi mysticum (die Kirche) bedingt sind. Der Papst kann, so folgert Petrus von Tarentaise, ohne zusätzliche Weihe einem Priester die Priesterweihe, einem Diakon die Diakonenweihe, einem Subdiakon die Subdiakonenweihe auftragen, niemals aber ohne vorherige Spendung des Ordo-Sakramentes die Konsekrationsvollmacht56. Ähnlich argumentieren in der Folgezeit die Kanonisten Antonius de Butrio († 1408), Nikolaus de Tudeschis († 1435) oder Johannes von Imola († 1436)57. Bis heute enthält der sogenannte Denzinger/Hünermann einige der Dokumente, die beweisen, dass bestimmte Päpste bestimmte Priester ohne vorherige Bischofsweihe zur Spendung aller Ordinationen mit Ausnahme der Bischofsweihe ermächtigt haben58. Nicht diese Fakten und auch nicht die besagten theologischen Gegensätze und Ungereimtheiten haben eine endgültige Klärung „Et ratio est, quia gradus episcopatus facit diversitatem non quoad ordinem, ut dictum est, sed quoad actum ordinis licet non quoad characterem“ (PETRUS AUREOLI, In IV, Sent, d. 24, a. 2, prop. 2). – Dazu: MÜLLER (Anmerkung 51), p. 50. 56 Vgl. PETRUS VON TARENTAISE, In IV Sent., d. 7, q. 3., a. 1. – Dazu: GILLMANN, F. Zur Lehre der Scholastik vom Spender der Firmung und des Weihesakramentes. Paderborn, 1920, p. 70–72. 57 Belege in: MÜLLER (Anmerkung 51), p. 52. 58 So Papst BONIFAZ IX. († 1404) durch die Bulle Sacrae religionis (1º Februar 1400) auf unbestimmte Zeit dem Augustinerabt von St. Osyth in Essex (DH 1145 f.); so Papst MARTIN V (†1431) durch die Bulle Gerentes ad vos (16 November 1427) für einen Zeitraum von fünf Jahren dem Zisterzienserabt von Altzelle in der Diözese Meißen (DH 1290); so Papst INNOCENZ VIII (†1492) durch die Bulle Exposcit tuae devotionis vom 9. April 1498 (DH 1435) auf unbestimmte Zeit das Privileg der Subdiakonen und Diakonenweihe an die Zisterzienseräbte von Cîteaux, La Ferté, Pontigny, Clairvaux und Morimond. – Dazu: BEYER, J. Nature et position du sacerdoce, in: NRTh n. 76, 1954, p. 356–373, p. 469–480. BERTRAMS, W. De quaestione circa originem potestatis iurisdictionis episcoporum in Concilio Tridentino non resoluta, in: PRMCL n. 59, 1970, p. 458-213. 55 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau des Verhältnisses von sakramentaler und rechtlicher Hierarchie, von Weihe- und Jurisdiktionsgewalt erzwungen. Dazu kam es erst im Gefolge der Reformation. Denn die Reformatoren haben mit ihrer Lehre von der Christusunmittelbarkeit aller Gläubigen jede sakramentale Repräsentation des ‚Voraus‘ Christi gegenüber der Kirche in Frage gestellt59. Deshalb musste das Konzil von Trient nicht nur erklären, warum die Einzigkeit des Mittlertums Christi einer Repräsentation in Gestalt des besonderen Priestertums bedarf; sondern auch, warum die Heilsuniversalität Christi der hierarchisch geordneten Kirche bedarf. Gegenüber der reformatorischen Leugnung des Unterschiedes zwischen dem besonderen Priestertum der Ordinierten und dem Taufpriestertum der übrigen Gläubigen war unter den Konzilsvätern von Trient unstrittig, dass es eine sakramentale Repräsentation der Differenz Christi zur Kirche, des Gegenübers des Hauptes zum Leib, des ‚Voraus‘ des Erlösers gegenüber den Erlösten gibt60. Unstrittig war auch, dass die Eucharistiefeier die sakramentale Vergegenwärtigung des Erlösungsopfers Christi ist und dass die Kirche sich stets neu aus der sakramentalen Vergegenwärtigung der Selbstverschenkung des Erlösers empfängt. Unklar aber war, ob das Sakrament des Ordo nur der sakramentalen Vergegenwärtigung der Erlösung beziehungsweise Rechtfertigung oder auch der Ordnung der Kirche dient. Unklar zum Beispiel war, ob die Stufung des Ordo (Bischof, Priester, Diakon) eine bloße Sache der rechtlichen Ordnung oder eine Sache der von Christus der Kirche eingestifteten Apostolizität ist. Auf dem Konzil von Trient standen sich zwei Parteien gegenüber — in der zweiten und in der dritten Sitzungsperiode so erbittert, dass der Erfurter Dogmatiker Josef Freitag, zurzeit der wohl beste Kenner der Materie, von einer Kriegsfront spricht, die das Konzil fast Dazu: AARTS, J. Die Lehre Martin Luthers über das Amt in der Kirche: Eine genetisch-systematische Untersuchung seiner Schriften von 1512 bis 1525. Helsinki, 1972; ders., Einige katholische Bemerkungen zu Luthers Theologie vom kirchlichen Amt, in: KuD n. 20, 1974, p. 158-165. 60 Vgl. PETER, B. Der Streit um das kirchliche Amt: Die theologischen Positionen der Gegner Martin Luthers. Mainz, 1997, besonders 91-190. 59 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 369 370 MENKE, K.-H. gespalten hätte61. Auf der einen Seite stand die kuriale beziehungsweise papale Partei, repräsentiert vor allem von den italienischen Konzilsvätern. Ihre Grundthese: Alle Jurisdiktionsgewalt in der Kirche liegt auf Grund göttlicher Anordnung (iure divino) beim Papst. Denn die Kirche ist, wenn auch in Ortskirchen gegliedert, eine einzige; also bedarf es einer höchsten, inappellablen Gewalt, die alles andere ordnet. Die Bischöfe haben so gesehen nicht mehr Weihegewalt empfangen als der einfache Priester; also betrachtet die papale Partei — mit Berufung auf Hieronymus und eine lange kanonistische Tradition — die Bischofsweihe nicht als Sakrament, sondern lediglich als liturgisch zelebrierte Übertragung der vom Papst verliehenen Jurisdiktionsgewalt in einer bestimmten Diözese. Auf der anderen Seite standen vor allem spanische und französische Konzilsväter. Sie betonten, dass der Bischof seine Leitungsgewalt nicht vom Papst empfängt, sondern durch die Bischofsweihe. So gesehen setzt die Leitungsgewalt des Bischofs der Leitungsgewalt des Papstes Grenzen. Sie ist ja nicht vom Papst, sondern im Sakrament der Bischofsweihe unmittelbar durch Christus übertragen. Dieses war exakt der Punkt, der enormes Konfliktpotenzial enthielt: Beschränkung der universalen Jurisdiktion des Papstes durch Ableitung der bischöflichen Jurisdiktionsgewalt aus der Bischofsweihe. Die Formulierungen der canones 6 und 762 auf die sich die bis zuletzt streitenden Parteien am 15 Juli 1563 geeinigt haben, sind mehr Ausdruck eines Kompromisses als Vgl. FREITAG, J. Sacramentum ordinis auf dem Konzil von Trient: Ausgeblendeter Dissens und erreichter Konsens. Innsbruck, 1991, p. 204-350. 62 „Kan. 6. Wer sagt, in der katholischen Kirche gebe es keine durch göttliche Anordnung [divina institutione institutam] eingesetzte Hierarchie, die aus Bischöfen, Priestern und Diakonen besteht: der sei mit dem Anathema belegt. – Kan. 7. Wer sagt, die Bischöfe stünden nicht höher als die Priester: oder sie besäßen keine Vollmacht, zu firmen und zu weihen oder die, die sie besitzen, sei ihnen mit den Priestern gemeinsam; oder von ihnen gespendete Weihen seien ohne Zustimmung oder Berufung des Volkes oder der weltlichen Macht ungültig; oder diejenigen, die weder von der kirchlichen und kanonischen Macht rechtmäßig geweiht oder beauftragt wurden, sondern anderswoher kommen, seien rechtmäßige Diener des Wortes und der Sakramente: der sei mit dem Anathema belegt“ (DH 1776 f.). 61 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau einer theologisch durchdachten Lösung. Die spanischen Konzilsväter63 wollten ausdrücklich erklärt wissen, dass nicht nur die Einheit, sondern auch die Differenzierung (Trias von Bischöfen, Priestern und Diakonen) des Ordo von Christus selbst gewollt sei. Aber sie mussten sich damit abfinden, dass die von ihnen favorisierte Formulierung „hierarchia a Christo Domino instituta“ durch die viel offener gehaltene Wendung „hierarchia divina ordinatione instituta“ ersetzt wurde. Die Italiener beteuerten gegenüber den Spaniern, sie würden gar nicht in Abrede stellen, dass die Bischöfe von Christus eingesetzt würden. Aber sie dachten dabei an eine mittelbare Einsetzung durch die von Christus autorisierte Jurisdiktionsgewalt des Papstes64. Die genaue Rekonstruktion der Genese des Trienter Weihedekretes durch Josef Freitag lässt erkennen, dass der Dualismus von potestas ordinis und potestas iurisdictionis durch die Beschlüsse des Trienter Konzils zwar nochnicht überwunden, wohl aber relativiert wird. Denn das sacerdotium wird nicht mehr „als bloße Konsekrationsgewalt aufgefasst, sondern als potestas spiritualis, die alle nötigen Vollmachten umgreift und sich von daher ausdifferenziert in ein bischöfliches und nichtbischöfliches Kardinal Gabriele Paleotti notiert in seinem Konzilstagebuch am 10 Juli 1563: „Orator regis Hispani simul cum multis praelatis Hispanis adeunt legatos magnoque studio petunt, ut in 6. Canone dicatur hierarchiam esse a Christo institutam aut esse ex Christi institutione. Aiunt enim illa verba divina ordinatione esse nimis generalia, cum et caetera in universo orbe a divina ordinatione procedant. Alias denuntiant se canonem illum non accepturos, praesertim cum adsit et octavus, qui ait episcopos assumptos a Romano pontifice etc., quasi episcopi nusquam a Christo, sed tantum a Romano pontifice proficiscantur, Deo ita ordinante“ (PALEOTTI, Gabrielis. Acta concilii Tridentini annis MDLXII et MDLXIII originalia, in: CT Band III, herausgegeben von MERKLE, S. Freiburg i. Br., 1931, 231–762, 689,7–13). 64 „[…] nec enim negant Itali episcopos esse a Christo institutos, sed aiunt hoc egere declaratione, quod videlicet sunt a Christo non immediate, sed mediante summo pontifice. Item quod sunt a Christo quoad ordinem, non quoad iurisdictionem“ (ebd. 691, 30-32). 63 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 371 372 MENKE, K.-H. Priestertum“65. In can. 7 wird gesagt, dass die Bischofsweihe mehr Weihegewalt überträgt als die Priesterweihe. Also haben die spanischen Konzilsväter zumindest dies erreichen können, dass nicht nur die Priesterweihe, sondern auch die Bischofsweihe als Sakrament bezeichnet wird66. Dies bedeutet: Die Überordnung des Bischofs über die Presbyter trägt nicht nur rechtlichen (iure ecclesiastico), sondern auch sakramentalen (iure divino) Charakter. Von einem Sieg der Spanier über die Italiener aber kann man nicht sprechen. Ganz im Gegenteil: Die zentrale Streitfrage, ob die Bischofsweihe nicht nur mehr Weihegewalt als die Priesterweihe, sondern auch die Jurisdiktionsgewalt über die jeweilige Diözese überträgt, wurde im Sinne der papal-kurialen Partei entschieden. Bilanzierend muss man festhalten: Es bleibt auch nach dem Trienter Konzil bei einer Parataxe von sakramentaler und rechtlicher Hierarchie. Obwohl die Bischofsweihe den Empfänger unwiderruflich zum Bischof erhebt, macht sie ihn nicht auch zum Leiter seiner Diözese; dies geschieht allein durch den Papst als den Inhaber aller Jurisdiktionsgewalt. Robert Kardinal Bellarmin († 1621), der mit Abstand bedeutendste Ekklesiologe der Gegenreformation, hält sich an die Vorgaben des Trienter Konzils, verbindet diese aber mit der für ihn charakteristischen FREITAG (Anmerkung 62), 256. – Die von Karl Josef Becker (BECKERM K. J. Der Unterschied von Bischof und Priester im Weihedekret des Konzils von Trient und nach der Kirchenkonstitution des IIº Vatikanischen Konzils: Rückschreitende oder fortschreitende Dogmenentwicklung?, in: RAHNER, K. [Hg.], Zum Problem der Unfehlbarkeit: Antworten auf die Anfrage von Hans Küng. Freiburg i. Br., 1971, p. 289–327; besonders 312–315) und Gerhard Fahrnberger (FAHRNBERGER, G. Bischofsamt und Priestertum in den Diskussionen des Konzils von Trient: Eine rechtstheologische Untersuchung. Wien, 1970, p. 116–122; sowie ders., Episkopat und Presbyterat in den Diskussionen des Konzils von Trient, in: Cath[M] n. 30 [1976] p. 119–152) vertretene Auffassung, das Trienter Weihedekret halte bei aller Betonung der Superiorität des Bischofs über den Priester an der These der beiden gemeinsamen Weihegewalt fest, darf durch die Untersuchung von Josef Freitag als widerlegt gelten. 66 Zwar gibt es nach dem Konzil von Trient noch einzelne Theologen wie Francisco Suárez SJ (1548–1617), JeanBaptiste Gonet OP (1616–1681) oder Charles-René Billuart (1685–1757), welche die Sakramentalität der Bischofsweihe in Frage stellen. Aber insgesamt setzt sich Trient durch.Jedenfalls kann Pietro Gasparri (1852– 1934) im 19. Jahrhundert unwidersprochen feststellen: „Plerique scholastici putant episcopatum non esse ordinem proprie dictum, distinctum a presbyteratu […]. Sed salva tot ac tantorum theologorum auctoritate, haec doctrina, obscura, confusioni idearum innixa, hodie ab omnibus derelicta et derelinquenda prorsus est ac firmiter tenendum episcopatum esse ordinem proprie dictum distinctum a presbyteratu“ (GASPARRI, P. Tractatus canonicus de sacra ordinatione: Band I. Paris, 1893, 10 f.). 65 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Lehre von der Kirche als päpstlicher Monarchie67. Er betrachtet den Papst nicht von den Bischöfen her, sondern umgekehrt die Bischöfe vom Papst her. Unter Absehung von jeder historisch-kritischen Befragung der kirchengeschichtlichen Fakten stellt er die These auf, Christus habe die Apostel im Abendmahlssaal zu Priestern geweiht, wohingegen Petrus nach der Auferstehung dieselben Apostel zu Bischöfen geweiht habe. Also sind die Apostelnachfolger beides zugleich: sakramentale Repräsentanten Christi und des Petrus (nachfolgers)68 gegenüber der Kirche. Obwohl Bellarmin im Sinne des Trienter Konzils betont, dass die Bischofsweihe sakramentales Handeln Christi ist69, unterstreicht er doch ebenso die Vermittlung aller Apostelnachfolge (successio apostolica) durch Petrus70. Wörtlich bemerkt er: Darin zumindest kommen alle überein, dass die Jurisdiktion der Bischöfe wenigstens insgesamt betrachtet göttlichen Rechtes ist. Denn Christus selbst hat die Kirche so geordnet, dass es in ihr Hirten und Lehrer usw. gibt. […] Wenn es nicht so wäre, könnte der Papst diese Ordnung ändern und z. B. festlegen, dass es keinen Bischof in der Kirche geben solle, was er aber offensichtlich nicht kann. Aber die Frage bleibt, ob gültig erwählte Bischöfe ihre Jurisdiktion von Gott erhalten, so wie der Papst, oder ob sie die ihrige vom Papst erhalten. Es gibt dazu drei Meinungen unter den Theologen. Die erste wird von denen vertreten, die wollen, dass Apostel und Bischöfe ihre Jurisdiktion unmittelbar von Gott empfangen haben bzw. empfangen. […] Andere wollen, dass die Apostel ihre Jurisdiktion Dazu: MENKE, K.-H. Das Unfehlbarkeitsverständnis der „gegen-reformatorischen Konzilstraktate“, in: Cath(M) n. 45, 1991, p. 102-118. 68 „Der Papst ist […] als princeps ecclesiae […] das sichtbare Gegenüber der Kirche“ (DIETRICH, T. Die Theologie der Kirche bei Robert Bellarmin [1542–1621]: Systematische Voraussetzungen des Kontroverstheologen. Paderborn, 1999, p. 431). 69 Ohne die Siebenzahl der Sakramente in Frage zu stellen, charakterisiert Bellarmin die Bischofsweihe wie ein eigenes Sakrament über die Priesterweihe hinaus. Ja, er betont die Selbstständigkeit der Bischofs gegenüber der Priesterweihe so stark, dass diese die von der Priesterweihe übertragenen Gewalten nicht inkludiert. Wer nicht schon Priester ist, kann die Bischofsweihe nicht empfangen. Dazu: DIETRICH, T. (Anmerkung 69), p. 435–441. 70 Bellarmin „deutet die successio apostolica als successio petrinica. In dieser Sichtweise ist nicht nur das tiefere Fundament für die Unfehlbarkeitskonzeption, sondern auch für die Superioritätsentscheidung zu sehen. Die Einengung der iudex-Funktion der Kirche auf das Petrusamt umgreift sowohl die Lehrentscheidungen wie die Leitungskompetenz. Der Papst ist der Garant der certitudo fidei wie der unitas Ecclesiae“ (DIETRICH, T. [Anmerkung 69], 429). 67 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 373 374 MENKE, K.-H. nicht von Christus, sondern von Petrus, und dass die Bischöfe die ihrige nicht von Christus, sondern vom Nachfolger Petri empfangen haben bzw. empfangen. […] Eine dritte These wird von denen vertreten, die wollen, dass die Apostel ihre ganze Autorität unmittelbar von Christus empfangen haben, wohingegen die Bischöfe die ihrige allein vom Papst empfangen71. Bellarmin glaubt, mit seiner These, Christus (potestas ordinis) und Petrus (potestas iurisdictionis) beziehungsweise Christus und Petrusnachfolgerwürden im Sinne eines zeitlichen Nacheinanders die Hierarchie konstituieren, eine vermittelnde Position zwischen „Dualisten“ und „Monisten“ einzunehmen. Doch man darf bezweifeln, dass sein Versuch gelungen ist. Thomas Dietrich kommt zu dem folgenden Ergebnis: Bellarmin versucht die Einheit beider Gewalten (potestas ordinis und potestas iurisdictionis) zu bewahren, aber er opfert die Bindung der Jurisdiktionsgewalt an die Weihegewalt auf dem Altar einer Ekklesiologie, die den „vicarius Christi“ zum Vermittler aller bischöflichen Gewalt erklärt72. Bellarmin bezeichnet die Bischofsweihe als ein Sakrament, das die Priesterweihe zwar voraussetzt, aber nicht inkludiert; damit aber drängt sich die Frage auf, ob er trotz gegenteiliger Beteuerungen noch an der Siebenzahl der Sakramente beziehungsweise an der Einheit des OrdoSakramentes festhält. Er spricht mit Blick auf Priester- und Bischofsweihe von zwei Wirklichkeiten, die durch das gemeinsame Ziel der Auferbauung „Et quidem omnes in eo conveniunt, iurisdictionem Episcoporum saltem in genere esse de iure divino. Nam Christus ipse ita ordinavit Ecclesiam, ut in ea sint Pastores, Doctores etc. […] Et praeterea, nisi ita esset, posset pontifex mutare hunc ordinem, et instituere, ne ullus sit in Ec clesia Episcopus, quod sine dubio non potest facere. At quaestio est, an episcopi canonice electi accipiant a Deo suam iurisdictionem, sicut eam accipit summus Pontifex: an vero a Pontifice. Sunt autem tres de hac re Theologorum sententiae. Prima eorum, qui volunt tam Apostolos, quam caeteros Episcopos immediate a Deo accepisse, et accipere iurisdictionem. […] Altera est eorum, qui volunt, Apostolos non a Christo, sed a Petro, et Episcopos non a Christo, sed a Petri successore accepisse, vel accipere iurisdictionem. […] Tertia est media eorum, qui volunt, Apostolos quidem accepisse a Christo immediate omnem suam auctoritatem, tamen Episcopos non a Christo, sed a summo Pontifice eam accipere“ (BELLARMIN, Robert. Disputationes de controversiis Christianae fidei vol. I, contr. 3, cap. 22). 72 Vgl. DIETRICH (Anmerkung 69), p. 446. 71 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau der Kirche so aufeinander hingeordnet sind, dass sie untrennbar bleiben73. Ob der im Vorfeld des Vatikanums I mit der Revision des Konzilsschemas „De ecclesia Christi“ befasste Jesuitentheologe Joseph Kleutgen (1811–1883) hier anknüpft, kann nur vermutet werden74. Jedenfalls stellt er innd Kommentaren Folgendes heraus75. Weil man das Vgl. ebd. 452 f. Der entsprechende Text des Konzilsschemas lautet: „Sed neque omnes, qui in opus ministerii assumuntur, pari inter se potestate praediti sunt. Siquidem apostolica traditione patrumque consensu firmatum est, solis sacerdotibus prae ceteris ministris potestatem esse, corporis et sanguinis Domini sacramentum conficiendi atque fideles peccatorum vinculis solvendi; inter ipsos rursum sacerdotes episcopos, quos Spiritus Sanctus posuit regere ecclesiam Dei, presbyteris tam ordine quam iurisdictione ex divina institutione superiores esse. Neque enim presbyteris, sed episcopis tantum competit, sacerdotes aliosque ministros ordinare, atque ecclesias sibi commissas propria et ordinaria potestate regere. Itaque et singuli in sua quisque ecclesia et congregati in synodis de doctrina et disciplina decernunt, leges ferunt, iudicium exercent. Neque fas est presbyteris sive aliis clericis suo in gradu et munere quidquam sine antistitis auctoritate agere: ut ecclesia super episcopos constituatur, et omnis actus ecclesiae per eosdem praepositos gubernetur. Verum etiam supremi muneris docendi et gubernandi universam ecclesiam episcopi expertes non sunt. Illud enim ligandi et solvendi pontificium, quod Petro soli datum est, collegio quoque apostolorum, quo tamen capiti coniuncto, tributum esse constat […]“ (Schema constitutionis dogmaticae secundae de ecclesia Christi secundum reverendissimorum patrum animadversiones reformatum, in: Mansi n. 53, 308–317; 310). 75 Joseph Kleutgen kommentiert den revidierten Text des Konzilsschemas (siehe Anmerkung 75), wie folgt: „De hierarchia iam egit concilium Tridentinum in sessione modo laudata; decretum autem hoc, quod Vaticano concilio proponitur, a Tridentino eo differt, quod non ordinis potestatem tantum, sed etiam et amplius iurisdictionis pertractat. Patres enim Tridentini de hierarchia locuti sunt, doctrinam de sacramento ordinis tradentes; hic autem de ea sermo instituitur, ut ecclesiae constitutio qualis sit, demonstretur. Porro caput tres partes complectitur: in prima docetur, presbyteros reliquis ministris, episcopos presbyteris superiores esse; in altera agitur de parte, quam episcopi in docenda et gubernanda ecclesia universa habent; in tertia de eorum subiectione Romano pontifici. Itaque cum in Tridentino decreto episcopi dicantur presbyteris superiores simpliciter, in hoc decreto additur: tam ordine quam iurisdictione, et plura de potestate episcoporum, proprias dioeceses gubernandi adiiciuntur. – Neque vero eo, quod episcopi e divina institutione presbyteris iurisdictione superiores esse dicuntur, controversia, a qua diiudicanda patres Tridentini post longam deliberationem abstinuerunt, dirimitur, controversia scilicet, utrum episcopi iurisdictionem ab ipso Deo in consecratione, an a Romano pontifice accipiant. Etiamsi enim posterius, quod longe communior sententia tenet, ponatur; tamen verum est, episcopos ex institutione divina presbyteris etiam iurisdictione praecedere. Nam ipse episcopatus procul dubio divinitus institutus est; institutus autem est, non solum, ut episcopi sanctificent, sed etiam ut regnant ecclesiam Dei. Etsi igitur iurisdictionem per Romani pontificis electionem vel confirmationem accipiant; haec tamen est muneri eorum propria et ordinaria. Et quamvis summus pontifex efficere possit, ut hic prae illo sit alicuius ecclesiae antistites; non tamen potest efficere, ne sint in ecclesia antistites, qui assignatas sibi dioeceses propria illa et ordinaria potestate regant. […] dubitari non potest, quin episcopi in docenda et gubernanda universa ecclesia partem aliquam habent“ (Iosephi Kleutgen relatio de schemate reformato, in: Mansi n. 53, 317–332; 320 f). 73 74 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 375 376 MENKE, K.-H. eine Sakrament des Ordo auf verschiedene Weise empfangen kann, gibt es trotz der Einzahl des Ordo-Sakramentes Bischöfe und Priester. Vom Diakon spricht Kleutgen bezeichnenderweise nicht; die Diakonenweihe hält er offenbar nicht für ein Sakrament; vermutlich, weil der Diakon das mit der Konsekrationsgewalt identifizierte sacerdotium entbehrt. Was Bischof und Presbyter betrifft, ist für ihn klar: Die Bischofsweihe überträgt die entsprechende Jurisdiktion beziehungsweise Leitungsgewalt (propria et ordinaria potestas), die jeder Bischof in Communio mit dem im Papst sichtbar geeinten Bischofskollegium ausübt. Der Priester empfängt dasselbe Sakrament des Ordo, um in Unterordnung unter seinen Bischof zu lehren, zu heiligen und zu leiten. Wenn man von der Ausklammerung des Diakonates aus dem sakramentalen Ordo absieht, hat Kleutgen in seinem Beitrag vorausgenommen, was erst das Zweite Vatikanische Konzil endgültig geklärt hat. Im Nachhinein muss man bedauern, dass der Deutsch-Französische Krieg eine Diskussion der Konzilsväter über Kleutgens Darlegungen und also auch die Verabschiedung eines entsprechenden ekklesiologischen Dokumentes verhindert hat. Die These, dass den Bischöfen auf Grund ihrer Weihe eine nicht vom Papst delegierte Jurisdiktion zukommt, hätte nicht nur jeden Dualismus von potestas ordinis und potestas iurisdictionis beseitigt, sondern auch jene Interpretationen des Unfehlbarkeitsdogmas von 1870 verhindert, welche die Kirche im Sinne Bellarmins als päpstliche Monarchie beschreiben. Kleutgen war schon um die Jahrhundertwende weithin vergessen. Im Gefolge des unseligen Antimodernismus entstand 1917 ein kirchliches Gesetzbuch, welches nur die Priesterweihe als Sakrament betrachtet und also den Dualismus zwischen potestas ordinis und potestas iurisdictionis fortschreibt. Hubert Müller merkt an: „Der c. 94976, der alle höheren und niederen Weihen vom Presbyterat bis zum Ostiariat der Reihe nach aufzählt, erwähnt nicht den Episkopat, betrachtet ihn also nicht einmal als 76 „Can. 949. In canonibus qui sequuntur, nomine ordinum maiorum vel sacrorum intelliguntur presbyteratus, diaconatus, subdiaconatus; minorum vero acolythatus, exorcistatus, lectoratus, ostiariatus“ (CIC 1917). Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Weihestufe. Er wird erst im folgenden c. 95077 zusammen mit der Tonsur (!) aufgeführt“78. Der CIC von 1917 war unvereinbar mit der vor allem durch Pius XII forcierten Corpus-Christi-mysticum-Ekklesiologie. Denn eine Kirche, die alsrechtlich geordnete Institution Leib Christi sein will, darf ihre juridischenicht von ihrer sakramentalen Ordnung trennen. Es war kein Zufall, dass derselbe Papst, der 1943 die Enzyklika „Mystici Corporis“ verfasst hat, den Problemüberhang des tridentinischen Ordo-Dekretes beseitigen wollte. Er war sich nämlich des Zusammenhangs bewusst, der zwischen zwei Problemen besteht: dem der Verhältnisbestimmung von potestas ordinis und potestas iurisdictionis und dem der Einheit des OrdoSakramentes trotz unterschiedlicher Weihen beziehungsweise Stufen. In dem am 30. November 1947 promulgierten Dokument Sacramentum ordinis (DH 3857–3861) erklärt der Papst, dass die Kirche keine Vollmacht hat, die ein für alle Mal festgeschriebene Siebenzahl der Sakramente zu ändern, und dass es deshalb auch nur ein Ordo-Sakrament geben könne. Für die Folgezeit von kaum zu überschätzender Bedeutung ist, dass Pius XII. von einer dreifachen Möglichkeit spricht, ein und dasselbe Sakrament zu empfangen. Wie schon bei den griechischen Vätern und ungebrochen in der Tradition der russischen und griechischen Orthodoxie, so wird nun auch in einem päpstlich approbierten Dokument die Diakonenweihe mit Bischofs- und Priesterweihe als eine Gestalt des einen Ordo-Sakramentes bezeichnet. Pius XII. lässt keinen Zweifel daran, dass die Bischofsweihe, sollte sie ohne vorherige Priester- und Diakonen-weihe gespendet werden, die ganze Fülle der drei Gestalten von Weihe und Jurisdiktionsgewalt impliziert. Anders gesagt: Das Sakrament des Ordo wird nicht mehr von der Konsekrationsgewalt des Priesters her erklärt, sondern von der Fülle seiner Höchstgestalt, von der Bischofsweihe her. Die Einheit des Ordo-Sakramentes — so lehrt der Papst in „Sacramentum ordinis“ — kommt in der allen drei Stufen gemeinsamen Handauflegung („Materie“ des Sakramentes) zum Ausdruck, das Unterscheidende („Form“ des Sakramentes) in den unterschiedlichen Bezeichnungen durch die Worte „Can. 950. In iure verba: ordinare, ordo, ordinatio, sacra ordinatio, comprehendunt, praeter consecrationem episcopalem, ordines enumeratos in can. 949 et ipsam primam tonsuram, nisi aliud ex natura rei vel ex contextu verborum eruatur“ (CIC 1917). 78 MÜLLER (Anmerkung 51), 57 f. 77 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 377 378 MENKE, K.-H. der Weihepräfation. Bei der Diakonenweihe betet der weihende Bischof: „Sende auf ihn, so bitten wir, Herr, den Heiligen Geist, damit er für die Aufgabe, deinen Dienst zu erfüllen, mit dem Geschenk deiner siebenförmigen Gnade gestärkt werde“ (DH 3860). Bei der Priesterweihe betet der Bischof: „Verleihe, so bitten wir, allmächtiger Vater, diesem deinem Diener die Würde des Priestertums; erneuere in seinem Herzen den Geist der Heiligkeit, damit er das von Dir, Gott, empfangene Amt des zweiten Ranges festhalte und durch das Bei]]spiel seines Lebenswandels die Zucht der Sitten fördere“ (DH 3860). Hier ist klar ausgesprochen, dass das Sakrament auch die Ordnung („zweiter Rang“) und also die entsprechende Jurisdiktionsgewalt überträgt. Was für die Priesterweihe gilt, gilt erst recht für die Bischofsweihe. In ihr lauten die entscheidenden Worte der Weihepräfation: „Vollende in deinem Priester die Fülle deines Dienstes und heilige den mit den Kostbarkeiten der ganzen Verherrlichung Ausgestatteten mit dem Tau himmlischen Salböls“ (DH 3860). Wenn man die skizzierte Entwicklung betrachtet, dann bezeichnen die entsprechenden Aussagen des Zweiten Vatikanischen Konzils den definitiven Abschluss eines langen Klärungsprozesses. Das eine Sakrament des Ordo ermöglicht die sakramentale Repräsentation Christi gegenüber der Kirche in dreifacher Gestalt, nämlich durch die Bischöfe gemeinsam mit den Priestern und Diakonen (LG 20; 28). Wer das Sakrament des Ordo empfängt, erhält nicht nur eine Gabe oder Begabung, sondern wird zugleich eingewiesen in eine Ordnung: der Bischof in ein im Petrusnachfolger sichtbar geeintes Kollegium (LG 22); der Priester in die sacerdotale (LG 28) und der Diakon in die kenotische Repräsentanz Christi (LG 29), und zwar im Gehorsam gegenüber dem zuständigen Bischof. Joseph Ratzinger hat die Bedeutung dieser Klärung wie folgt erfasst: Sie bedeutet die „Aufsprengung jener Mauer […], welche das Mittelalter von der alten Kirche und so den lateinischen Westen von den Kirchen des Ostens trennt“79. Das Konzil, so erklärt er, hat den u. a. von Petrus Lombardus, Albertus Magnus, Bonaventura, Thomas fortgeschriebenen 79 RATZINGER, J. Zur Lehre des Zweiten Vatikanischen Konzils: Formulierung – Vermittlung – Deutung. Freiburg i. Br., 2012, 467. Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Dualismus von potestas ordinis und potestas iurisdictionis endgültig verworfen. Besonders wichtig erscheint ihm jenes vordergründig unscheinbare Sätzchen (LG 22), „wonach jemand Glied des Bischofskollegiums wird durch die sakramentale Weihe“80. Er fährt fort: Kollegialität beruht nach dieser Aussage nicht auf einer vom Papst verliehenen Jurisdiktion, die dann dem Sakrament als einer bloß individuellen Gabe gegenübergestellt würde, sondern sie reicht ins Sakramentale hinein, das als solches eine innere Zuordnung zum Miteinander der Bischöfe in sich trägt81. Ausdrücklich sagt die Kirchenkonstitution des Zweiten Vatikanums über die Jurisdiktion der Bischöfe: Diese Vollmacht, die sie im Namen Christi persönlich ausüben, ist die eigene, ordentliche und unmittelbare, auch wenn ihr Vollzug letztlich von der höchsten Autorität der Kirche geregelt wird und im Hinblick auf den Nutzen der Kirche oder der Gläubigen mit bestimmten Grenzen umschrieben werden kann (LG 27). Von den Priestern wird gesagt, dass sie dasselbe Sakrament des Ordo empfangen — allerdings so, dass sie ihr sacerdotium nur in der Weise der Unterordnung unter ihren Bischof bzw. als dessen Repräsentanten realisieren können. Sie sind, obwohl sie die Bischofswürde nicht haben und in der Ausübung ihrer Vollmacht von den Bischöfen abhängen, dennoch mit ihnen in der priesterlichen Ehre verbunden und werden kraft des Sakraments der Weihe […] zum Verkündigen des Evangeliums, zum Leiten der Gläubigen und zur Feier des Gottesdienstes geweiht (LG 28). Sie repräsentieren Christus in der Weise der Repräsentation ihres Bischofs. Auch die Diakone empfangen analog dazu dasselbe (eine und einzige) Sakrament des Ordo (LG 29), um das ‚Prius‘ beziehungsweise ‚Voraus‘ Christi vor seiner Kirche zu repräsentieren – allerdings 80 81 Ebd. 467. Ebd. 468. Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 379 380 MENKE, K.-H. nicht auf dieselbe Weise wie Bischof und Priester, sondern durch die sakramentale Darstellung der kenotischen Dimension des kirchlichen Amtes. Die triadische Einheit des sakramentalen Ordo Wie oben skizziert, ging es jahrhundertelang — zumindest im Westen — immer nur um Priester- und Bischofsweihe. Von der Diakonenweihe war kaum oder gar nicht die Rede. Doch in der Heiligen Schrift werden die Diakone stets gemeinsam mit den Bischöfen genannt. Schon im 3. Jahrhundert hat sich die Trias von Bischof, Priester und Diakon flächendeckend etabliert. Erst die erwähnten Sentenzen des Hieronymus führten zur Reduktion des Ordo-Sakramentes auf die Priesterweihe (Übertragung der Konsekrationsgewalt). Im Hochmittelalter wirkten die Schriften, die fälschlicherweise dem Paulusschüler Dionysius Areopagita zugeschrieben wurden, wie ein Korrektiv. Es war aber ein langer Weg bis zur definitiven Erklärung der Dreigestaltigkeit des einen OrdoSakramentes und bis zur definitiven Bindung der Jurisdiktions an die Weihegewalt. Der Diakon erhält mit dem Empfang des Ordo-Sakramentes keinen Anteil am sacerdotium des Bischofs und des Priesters. Und doch wird auch er durch seine Weihe ermächtigt, das ‚Voraus‘ Christi vor und gegenüber der Kirche sakramental zu repräsentieren (LG 29)82 — nicht in der Weise, in der das Haupt den Leib beherrscht, sondern in der Weise 82 „Diacres, prêtres et évêque sont les uns pour les autres les vis-à-vis sacramentels du seul sacrament de l’ordre. Aucun ne peut être ce qu’il est sans les deux autres, et c’est ensemble qu’ils rendent présente la personne du Christ dans toute sa plénitude“ (BORRAS, A; POTTIER, B. La grâce du diaconat: Questions actuelles autour du diaconat latin. Bruxelles, 1998, P. 200). – Entsprechend auch: GONNEAUD, D. La sacramentalité du ministère diaconal, in: RTL n. 36, 2005, p. 3-20. Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau der dienenden Wegbereitung83. LG 29 erinnert daran, dass der Bischof der erste und vorrangige Diakon seiner Herde ist; dass Bischöfe und Priester in ihrem Lehren, Heiligen und Leiten nicht aufhören, Diakone zu sein. Aber der Diakon als solcher ist dazu geweiht, ausschließlich diese Dimension der Konstituierung von Kirche sakramental darzustellen84. Eine beträchtliche Anzahl von Theologinnen und Theologen hat die vom Vatikanum II geklärte Sakramentalität der Trias erneut in Frage gestellt85. Guido Bausenhart z. B. spricht vom Amt wie von einem Prinzip oder einer unbestimmten Größe, die von der Kirche immer wieder neu gestaltet und spezifiziert werden kann86. Er verweist auf die unbestreitbare Tatsache, dass der Begriff „Apostel“ zunächst kein eindeutig definierter Amtstitel ist; dass Lukas nur die Zwölf und dann auf Grund der Geschehnisse vor Damaskus auch Paulus ‚Apostel‘ nennt; Gegenüber vielfältigen Tendenzen, eine Art „Ersatzpriestertum“ des Diakons theologisch zu rechtfertigen, hat Papst Benedikt XVI. mit dem Motuproprio „Omnium in mentem“ (15 Dezember 2009) im CIC von 1983 die cc. 1008 f. verändert. Die Änderung richtet sich gegen die Einbeziehung der Diakone in das nur den Bischöfen und Priestern zukommende Lehren, Heiligen und Leiten in der Person Christi des Hauptes. Der dem c. 2009 neu beigefügte § 3 formuliert: „Jene, die in das Bischofsamt oder in das Priesteramt aufgenommen worden sind, erhalten die Aufgabe und die Fähigkeit, in der Person Christi des Hauptes zu handeln. Die Diakone dagegen werden dazu befähigt, dem Gottesvolk zu dienen in der Liturgie des Wortes und der Liebe“. 84 Leider ist es schon den Konzilsvätern nicht gelungen, eine Theologie des Diakonates zu entwickeln. Die aus der „Traditio“ des Hippolyt stammende Sentenz, dass den Bischöfen und Presbytern die Hände „ad sacerdotium“, den Diakonen aber „ad ministerium“ aufgelegt werden, wurde in LG 29 mit einer parataktischen Aufzählung einzelner „ministeria“ beziehungsweise Funktionen erläutert. Statt zu erklären, worin die spezifische Gestalt der Repräsentation des ‚Voraus‘ Christi vor seiner Kirche für die Diakone besteht, begnügt sich der Konzilstext mit einer additiven Nennung von Aufgaben, die Diakone schon einmal übernommen haben oder in Zukunft übernehmen könnten. Funktionalismus in der Praxis ist aber gerade das, was ein sakramentales Verstehen des Diakonates aushöhlt. Zur Reflexion dieser Problematik: BORRAS; POTTIER (Anmerkung 83), 73-157. SANDER, S. Gott begegnet im Anderen: Der Diakonat und die Einheit des sakramentalen Amtes. Freiburg i. Br., 2006. HARTMANN, R; REGER, F.; SANDER, S. (Hgg.), Ortsbestimmungen: Der Diakonat als kirchlicher Dienst. Frankfurt am Main, 2009. ARMBRUSTER, K.; MÜHL, M. (Hgg.), Bereit wozu? Geweiht für was? Zur Diskussion um den Ständigen Diakonat. Freiburg i. Br., 2009. 85 Vgl. BAUSENHART, G. Das Amt in der Kirche. Eine not-wendende Neubestimmung. Freiburg i. Br., 1999. MÜLLER, J. In der Kirche Priester sein. Das Priesterbild in der deutschsprachigen katholischen Dogmatik des 20 Jahrhunderts. Würzburg 2001. RAHNER, J. Creatura Evangelii: Zum Verhältnis von Rechtfertigung und Kirche. Freiburg i. Br., 2005. 86 Bausenhart (vgl. Anmerkung 86, 27–41), wendet sich gegen die Bezeichnung des kirchlichen Amtes als einer Einrichtung „göttlichen Rechtes“ im klassischen Sinn und plädiert für eine Ausweitung des Terminus „ius divinum“ auf Einrichtungen, welche die Gemeinde als für sich notwendig erkennt. 83 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 381 382 MENKE, K.-H. dass aber Paulus auch Mitarbeiter wie Apollos, Barnabas, Epaphras, Epaphrodites, Silvanus, Timotheus und Titus ‚Apostel‘ nennt. Er verweist auf die ursprüngliche Parallelität von presbyterialer und episkopaler Gemeindeverfassung. Und er relativiert den Monespiskopat als zeitbedingtes Mittel der Bekämpfung von Häresien. Aber die entscheidende Frage ist doch, ob die schließlich definitiv gewordene Dreistufung der postapostolischen Christusrepräsentation funktional oder sakramental bedingt ist. Ein funktionales Amtsverständnis betrachtet die „Ämter“ von Bischöfen, Priestern und Diakonen als das, was die Gemeinden schon im zweiten Jahrhundert als für sie notwendig erkannt haben, ohne dass dies immer so bleiben muss. Dieser Auffassung stellt die Kirchenkonstitution des Zweiten Vatikanischen Konzils das sakramentale Verständnis des Ordo entgegen. Denn die entsprechenden Texte (LG 18-29) lassen keinen Zweifel daran, dass die Trias des sakramentalen Amtes analog zur Kanonisierung bestimmter Schriften im Unterschied zu den Apokryphen und analog auch zur Festlegung von sieben Vollzügen der Kirche als Sakramente zu verstehen ist. Allerdings kann man die drei Stufen des Ordo ebenso wenig aus dem abstrakten „Wesen der Kirche“ deduzieren wie die Schriften des neutestamentlichen Kanons oder die sieben Sakramente. Das eben bedeutet ja der Primat der Inkarnation vor der Inspiration, dass alle Erkenntnis der Wahrheit Christi und der Kirche geschichtlich beziehungsweise inkarnatorisch vermittelt ist. Der Traum von einer Unmittelbarkeit des Einzelnen oder auch der Kirche insgesamt zum Willen Gottes ist das Kennzeichen aller Gnostizismen. Gegenüber einer supranaturalistischen Sicht der Geistwirksamkeit ist an die innere Einheit von Pneumatologie, Christologie und Ekklesiologie zu erinnern. Der erhöhte Christus wirkt im Heiligen Geist nicht an der geschichtlichen Ursächlichkeit vorbei (Schrift, Tradition, Hermeneutik der Auslegung der Offenbarung, Glaubenssinn des Volkes Gottes, Lehramt). Vielmehr bewirkt der Geist, dass die menschliche Artikulation der Offenbarung das bleibt, was sie sein soll, nämlich die Vergegenwärtigung des Wortes und Willens (1 Thess 2,13) des sich ein für alle Mal in Jesus geoffenbart haben den Gottes (Hebr 10,10)87. 87 MÜLLER, G. L. Priestertum und Diakonat. Der Empfänger des Weihesakramentes in schöpfungstheologischer und christologischer Perspektive. Einsiedeln/Freiburg i. Br., 2000, p. 61. Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Indem die Kirche von Christus her und auf Christus hin lebt, erkennt sie sich selbst, erkennt sie zum Beispiel ihre Apostolizität, erkennt sie den Unterschied zwischen Aposteln und Apostelnachfolgern, erkennt sie die Einheit des Ordo, aber auch die Notwendigkeit der Stufung von Bischöfen, Priestern und Diakonen88. In Analogie zu diesem Vorgang der Ausdifferenzierung des Ordo erklärt Karl Rahner den noch viel länger währenden Prozess der Erkenntnis der sieben Grundvollzüge der Kirche wie folgt: Die Kirche erlebt ihr eigenes Wesen […], indem sie es vollzieht. Indem sie es nun erfährt, begreift sie auch die Gestuftheit ihres Tuns hinsichtlich ihres eigenen Engagements in diesem Tun. Und so kann sie erkennen, dass bestimmte Vollzüge aus ihrem Wesen heraus grundlegende und absolute Vollzüge ihres eigenen Wesens und also das sind, was wir Sakramente nennen. Die Kirche könnte diese Erkenntnis nicht haben […], wenn dieses Wesen nur in einer abstrakten Idee und nicht in seinem realen Vollzug gegeben wäre, sie könnte also die Sakramente (und vor allem ihre Siebenzahl) nicht theoretisch allein aus dieser Idee her ableiten. Aber wenn sie ihr Wesen in dessen konkretem Vollzug besitzt und erkennt, kann sie daraus wohl begreifen, dass diese und jene bestimmten Vollzüge, die sie schon getan hat als unreflexen Wesensvollzug, der notwendig der Selbstreflexion vorausgeht, diesem Wesen wesentlich sind, ohne dass ihr dies nochmals eigens explizit mitgeteilt werden müsste89. Natürlich könnte man sich theoretisch weitere Grundvollzüge der Kirche ausdenken; und ebenso andere Stufen des Ordo; auch noch dreißig weitere Lebensjahre Jesu über die dreiunddreißig hinaus, die faktisch Gottes Selbstoffenbarung waren. Aber das gerade bedeutet ja Inkarnation, dass die faktische Geschichte Jesu und die faktische Kanonisierung bestimmter Zeugnisse über Jesus und die faktische Abschließung bestimmter dogmatischer Prozesse (Dreistufung des Ordo; Siebenzahl der Sakramente etc.) unbeliebig, nicht mehr revidierbar und also abgeschlossen sind. Dazu: SCHEFFCZYK, L. Tradition und Weihevorbehalt. Zur Bedeutung der Überlieferung in der Auseinandersetzung um das Weiheamt der Frau, in: Ders. (Hg.), Diakonat und Diakonissen. St. Ottilien, 2002, p. 107-148. 89 RAHNER, K. Kirche und Sakramente, in: Sämtliche Werke XVIII, Freiburg i. Br., 2003, 3-72; 42. 88 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 383 384 MENKE, K.-H. Karl Rahner unterscheidet zwischen irreversiblen Entscheidungen und offen bleibenden Prozessen des Erkennens und Handelns der Kirche90. Er weiß um die Frage, ob das eine sakramentale Amt nicht doch anders als durch die Trias von Bischof, Priester und Diakon gestaltet werden könne. Und er hält diese Frage für legitim, orientiert aber seine Antwort an der Intention der Konzilsväter, sakramentale und faktische Ordnung zu identifizieren, d. h. die Einheit des Ordo als ebenjene Trias vorzustellen, „die in der apostolischen Zeit von den Aposteln selbst vorgenommen wurde (selbst wenn sie nicht von Christus selbst ausdrücklich angeordnet wurde)“91. Theologinnen und Theologen, die Rahner in diesem Punkt widersprechen, orientieren sich an bestimmten kirchenpolitischen Interessen (Aufwertung der sogenannten Pastoralassistenten; Diakonat der Frau etc.). Sie betrachten das sakramentale Amt wie eine allgemeine Größe, die immer wieder neu geordnet beziehungsweise gestaltet werden kann. Der Preis, den sie dafür zahlen, ist die Rückkehr zum Dualismus zwischen sakramentaler und rechtlicher Ordnung, der vom Zweiten Vatikanum endgültig abgewiesen wurde. Man wendet sich vehement gegen die Vorstellung, der Papst könne das Amt in der Kirche wie einen Topf verwalten, aus dem er nach Belieben austeile. Doch was der Papst nicht darf, soll die sogenannte Basis dürfen. Nicht der Papst soll alles regeln und ordnen, sondern die Gemeinde. Bei Licht betrachtet ist solche Forderung schlicht reaktionär, weil sie die oben geschilderte Entwicklung hin zum sakramentalen Verständnis aller Leitungs- und Jurisdiktionsgewalt ignoriert und wieder eine Ordnungsmacht — diesmal „von unten“ statt „von oben“ — propagiert, die nicht sakramental rückgebunden ist an Christus und die Apostel. Aus den Praeparatoria und Antipraeparatoria des Zweiten Vatikanums lässt sich, wie Hubert Müller detailliert erwiesen hat92, eindeutig erheben, dass die Konzilsväter nicht nur die Einheit des Ordo, sondern auch dessen triadische Struktur als die sakramentale Repräsentation Vgl. RAHNER, K. Über den Begriff des „ius divinum“ im katholischen Verständnis, in: Sämtliche Werke X. Freiburg i. Br., 2003, p. 605-625. 91 RAHNER, K. Erwägungen über Inhalt und Aufbau eines Sammelwerkes über die zeitgemäße Erneuerung des Diakonats, in: Sämtliche Werke XVI. Freiburg i. Br., 2005, 357–408; 375. 92 Vgl. MÜLLER (Anmerkung 51), p. 261–367. 90 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau des ‚Voraus‘ Christi vor und gegenüber seiner Kirche bestimmen wollten. Gegenteilig urteilt Dorothea Reininger. Das Konzil, so meint sie, habe mit der Bestimmung des Presbyterates und des Diakonates vom Bischofsamt her nicht nur die mittelalterliche Fokussierung des Amtsverständnisses auf die eucharistische Konsekrationsvollmacht verabschiedet, sondern mit der Bestimmung aller sakramental verstandenen Ämter vom Bischofsamt her eine Einheit beschrieben, die für eine fortschreitende Vielfalt offensteht93. Es geht, so betont sie, um die Frage, ob das sakramentale Amt den Bedürfnissen immer neu angepasst werden kann oder nicht. Für die Beantwortung dieser Frage hängt aus ihrer Sicht alles „davon ab, wie das Weiheamt verstanden wird, […]: als untrennbare Einheit oder als Einheit in Vielfalt“94. Abgesehen davon, dass sie die in Christus ergangene Offenbarung für nicht abgeschlossen erklärt und deshalb auf die Revision der päpstlichen Entscheidung zum Frauenpriestertum hofft95, verweist Dorothea Reininger auf den Unterschied der Einheit der Bestandteile eines Herings einerseits und der Einheit der Kartoffeln in einem Kartoffelsack andererseits. Die Vielheit, die das sakramentale Amt in der Kirche verträgt, erscheint ihr so groß wie die der Kartoffeln in einem Kartoffelsack96. Eine ähnliche Position vertritt Regina Radlbeck-Ossmann. Sie bezeichnet die These von der Einheit des Ordo als Position des Mittelalters Vgl. REININGER, D. Diakonat der Frau. Gegenwärtige Realitäten und zukunftsweisende Visionen, in: Diak. n. 33, 2002, p. 277–286. 94 Ebd. 280. 95 „Das ist ja gerade der Grund, warum im katholischen Offenbarungsverständnis mit dem Ende biblischer Zeit die Offenbarung als noch nicht abgeschlossen gilt, sondern die Kirche darauf vertraut, dass ihr – trotz aller Unvollkommenheit und Sündhaftigkeit – in ihren konkreten Entscheidungen der Heilige Geist geschenkt ist. In der Gewissheit der Realpräsenz ihres Herrn im Heiligen Geist darf die Kirche solche Veränderungen ihrer Strukturen um ihres Auftrags willen und aus dringenden pastoralen Notwendigkeiten vornehmen“ (ebd.). 96 „Zur Vereinheitlichung dieser unterschiedlichen Bedeutung des Einheitsbegriffs mag folgender bildlicher Vergleich helfen: Auch die Einheit eines Herings ist eine andere als die Einheit, die ein Sack Kartoffeln bildet. Im ersten Fall wird man zu dem Schluss kommen, dass die Möglichkeit einer Zulassung von Frauen zur Diakonatsweihe bereits durch die negative Entscheidung bezüglich der Priesterweihe der Frau vorentschieden ist“ (ebd.). 93 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 385 386 MENKE, K.-H. und die These von der Vielfalt der Ämter als Position der Väter97. Sie geht davon aus, dass der Diakonat, den sie für Frauen geöffnet sehen will, im Mittelalter deshalb als Stufe des einen Ordo betrachtet worden sei, weil man ihn wie das Presbyterat einseitig auf die eucharistische Konsekration hingeordnet und also zu einer bloßen Durchgangsstufe für Priesteramtskandidaten degradiert habe. Das Gegenteil ist zutreffend: Gerade im Mittelalter wurde die Diakonenweihe nicht als Stufe oder Teil des sakramentalen Ordo beschrieben. Weil Radlbeck-Ossmann das Problem der Verhältnisbestimmung von potestas ordinis und potestas iurisdictionis von der Frage nach dem Verhältnis der Einheit zur Vielfalt des Ordo trennt, entgeht ihr die oben geschilderte Wirkungsgeschichte des Hieronymus. Wer sie zur Kenntnis nimmt, kann gar nicht leugnen, was bis zum Trienter Konzil durchgängig belegbar ist: nämlich die Ausklammerung von Bischofs- und Diakonenweihe aus dem ordosacramentalis. Radlbeck-Ossmann verkehrt den geschichtlichen Befund in sein Gegenteil. Ihre Interpretation von LG 18–29 ist von dem Interesse geleitet, die Schaffung eines sakramental verstandenen Frauendiakonates als mit den Konzilstexten vereinbar zu erweisen. Sie versteht die Einheit des Ordo nicht als triadische Einheit, sondern als die gemeinsame Abhängigkeit nachgeordneter (schon etablierter oder erst noch zu schaffender) Ämter von der Leitung des Bischofs. Der Priester, so erklärt sie, repräsentiert nicht Christus, sondern den Bischof98, und der Diakon empfängt in LG 29 sein Amt „nicht zum Priestertum, sondern zur Dienstleistung“. Damit sei evident, dass „das Konzil die mittelalterliche Konzeption von der „Die historische Prüfung zeigt, dass das Argument von der Einheit des Ordo spezifisch mittelalterliche Verhältnisse widerspiegelt und in seiner Aussagelogik von den ältesten Traditionen der Kirche abweicht. Blickt man auf die Tradition der alten Kirche, so zeigt sich, dass diese weder von primär einem Amt ausgeht noch mit der ‚Einheit des Amtes‘ argumentiert. In der Patristik rückt man im Gegenteil gerade die Vielfalt der Ämter in der Kirche immer wieder in den Vordergrund“ (RADLBECK-OSSMANN, R. Das Argument von der Einheit des Ordo: Fundament für die Ablehnung eines Diakonats der Frau?, in: WINKLER, D. W. [Hg.], Diakonat der Frau: Befunde aus biblischer, patristischer, ostkirchlicher liturgischer und systematisch-theologischer Perspektive, Wien/Berlin, 2010, 119–134, 125). 98 „Während der Bischof Jesus Christus selbst in der Kirche vergegenwärtigt (LG 21, 26), besteht die Aufgabe etwa der Priester lediglich darin, den Bischof (!) in ihrer örtlichen Gemeinde präsent zu halten (LG 28)“ (ebd. 128). 97 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Einheit des Ordo nicht teilt“99. Das Zweite Vatikanum nenne verschiedene Ordines; zumindest der Diakonat bilde einen eigenen Ordo. Also stehe der Zulassung von Frauen zur Diakonenweihe selbst dann nichts entgegen, wenn man ihnen weiterhin Bischofs- und Priesterweihe verweigere100. Radlbeck-Ossmann sieht die in LG aufgezählten Aufgaben eines Diakons in Wortverkündigung und Sakramentenspendung zentriert101. Sie spricht von der Notwendigkeit, die faktisch von Frauen realisierten Dienste durch eine Weihe in das sakramentale Amt einzubeziehen102. Abgesehen von den Folgen dieses Postulats für das gemeinsame Priestersein aller Getauften und Gefirmten übersieht die Hallenser Theologieprofessorin, dass die Konzilstexte den wesentlichen Unterschied des besonderen gegenüber dem gemeinsamen Priestertum in der sakramentalen Repräsentation des ‚Voraus‘ und des ‚Gegenübers‘ Christi zur Kircheverorten. Eben diese Christusrepräsentation begründet die Einheit des triadischen Ordo. Und wenn der Priester seine Christusrepräsentation nur in der Weise der Repräsentation des Bischofs wahrnehmen kann, dann ist er dennoch wahrer Christusrepräsentant. Denn an der dreifachen Vollmacht des Lehrens, des Heiligens und des Leitens haben die Presbyter solchen Anteil, dass wahres Weihepriestertum in ihnen verwirklicht ist und ihnen der Name des Priesters nicht bloß in übertragener Weise zukommt. Dies kraft der Ordination, eines im vollen Sinne sakramentalen Aktes, der sie dem Bilde Christi, des Urbildes neutestamentlichen Priestertums, ähnlich macht. Diese Gestaltung nach Ebd. 129. Vgl. ebd. 131-134. 101 In Bezug auf die Aufzählung der Aufgaben eines Diakons in LG 29 bemerkt Radlbeck-Ossmann: „Es ist geradezu markant, welch geringen Raum in dieser Aufgabenbeschreibung der Dienst am Nächsten einnimmt, den man klassisch als vorrangigen Dienst des Diakons betrachtet. Dieser kommt – zumindest in der deutschen Übersetzung – in lediglich acht Worten zum Ausdruck, die noch nicht einmal zu einem Hauptsatz verbunden, sondern partizipisch verkürzt dargeboten werden, während der auf den Dienst an Wort und Sakrament bezogene Einsatz über volle acht Zeilen hin beschrieben wird“ (ebd. 129 f.). 102 „Wenn Frauen diakonisch in der Kirche arbeiten, tun sie dies ehrenamtlich, im kirchlichen Laienberuf oder als Ordensfrau. Damit wird in ihrem Dienst zwar das bewundernswerte Engagement zahlreicher einzelner Christinnen sichtbar, nicht aber, dass sich in ihrem Dienst ein Grundvollzug der Kirche ereignet. Diese Bedeutung kann nur dann vermittelt werden, wenn der diakonische Dienst auch und gerade von amtlichen Vertretern der Kirche geleistet wird“ (ebd. 133). 99 100 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 387 388 MENKE, K.-H. dem Bilde Christi, des Hohenpriesters, ist grundgelegt in der sakramentalen Aussonderung, Bestellung und Ausrüstung für die Verkündigung des Wortes, zur Leitung der Gläubigen und zur Feier des Gottesdienstes — dies freilich „auf der Stufe des Dienstes“103, der dem einzelnen Presbyter von seinem Bischof aufgetragen ist104. Gegen die traditionelle Bestimmung des Priesters von seinen kultischen Funktionen her behält das Konzil in Bezug auf die Presbyter die selbe Reihenfolge in der Trias von Lehren, Heiligen und Leiten bei, die in der Beschreibung der Christusrepräsentation der Bischöfe genannt wird. Was den Diakonat betrifft: Darin besteht doch gerade die Klarstellung des Vatikanums II (in Anknüpfung an die Apostolische Konstitution „Sacramentum Ordinis“), dass der Diakonat keinen eigenen Ordo darstellt, sondern — untrennbar vom Bischof mit seinem Presbyterium — das ‚Voraus‘ Christi vor der Kirche auf eigene (nicht sacerdotale) Weise sakramental repräsentiert. Das Eigene des Diakons besteht nicht in der Bündelung von Funktionen, die auch jeder getaufte und gefirmte Christ wahrnehmen kann, sondern in der Darstellung der kenotischen Dimension der Autorität Christi gegenüber der Kirche. Diese darf auch bei Bischöfen und Priestern nicht fehlen, wird aber von den Diakonen eigens veranschaulicht. Regina Radlbeck-Ossmann beruft sich vor allem auf den emeritierten Tübinger Theologen Peter Hünermann105. Dieser allerdings stützt seine Argumente für eine Anpassung des triadischen Ordo an die Bedürfnisse der Zeit weniger auf LG 18–29 als auf die konziliare Beschreibung der Kirche als Grundsakrament. Daraus folgert er in seiner jüngsten Veröffentlichung (Dezember 2012): GRILLMEIER, A. Kommentar zum III. Kapitel, Artikel 28 der Dogmatischen Konstitution über die Kirche, in: LThK; Erg. Band I (Freiburg i. Br. 1966), 247–255, 251. 104 Dazu: SCHEFFCZYK, L. Die Christusrepräsentation als Wesensmoment des Priesteramtes, in: Ders., Schwerpunkte des Glaubens. Gesammelte Schriften zur Theologie. Einsiedeln, 1977, p. 367–386. 105 Radlbeck-Ossmann (Anmerkung 98, 12523), unterstreicht, dass sie sich den „Vorarbeiten von Peter Hünermann“ (HÜNERMANN, P. Theologische Argumente für die Diakonatsweihe von Frauen, in: Diakonat. Ein Amt für Frauen in der Kirche – Ein frauengerechtes Amt? [dort: Anmerkung 112], 98-128), anschließen wolle. 103 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Das ganze Volk Gottes ist zum Mitvollzug des Hirtendienstes Jesu Christi an den Menschen, in Diakonie und Caritas berufen. […] Weil der Ordo wesentlich Dienst am und für das Volk Gottes ist, und das Volk Gottes das Mysterium Jesu Christi gar nicht anders als unter den gegebenen zeitgeschichtlichen Umständen leben und bezeugen kann, muss der Dienst, den die Ordinierten zu leisten haben, entsprechend gestaltet werden […]106. Hünermann leitet aus der Sakramentalität der Kirche ab, dass diese ihre Struktur selbst gestalten kann. Er bezweifelt nicht, dass zur Sakramentalität der Kirche stets auch das kirchenleitende Amt gehört. Aber er ist zugleich der Meinung, dass dieses Amt nicht an die Trias von Bischof, Priester und Diakon gebunden sei. Der Hirtendienst Christi, so schreibt er, subsistiere zwar in der gegenwärtigen Amtsstruktur, sei aber nicht mit dieser identisch. Die Kirche als Grundsakrament habe also die Möglichkeit, den Ordo zu verändern und auch ein Diakonat einzuführen, zu dem beide Geschlechter Zugang haben107. Die Einheit des Ordo und die Frage nach einem Diakonat der Frau Schon Ende des zweiten Jahrhunderts ist die sakramentale Repräsentation des ‚Voraus‘ Christi vor der Kirche eine Trias. Kein Kirchenvater spricht von einer Frau als Trägerin dieses sakramentalen Amtes108. Wer versucht, die singuläre Bezeichnung einer Frau als „Diakon“ (Röm 16,1 f.) vom Gesamtbefund des NT und von der nachapostolischen Entwicklung abzukoppeln oder gar zur Grundlage eines sakramentalen Diakonates der Frau in apostolischer Zeit zu erklären, setzt sich dem Verdacht aus, den historisch-kritisch eruierbaren HÜNERMANN, P. Zum Streit über den Diakonat der Frau im gegenwärtigen Dialogprozess – Argumente und Argumentationen, in: ThQ n. 192, 2012, 342–375, 371. 107 Vgl. ebd. 371 f. 108 Radlbeck-Ossmann (Anmerkung 98, 121), fordert gerade deshalb eine aktualisierende Umkehrung zum Beispiel des Origenes-Diktums: „Was sollen die Heiden denken, wenn bei Christen sich die Männer niedersetzen, um sich von Frauen belehren zu lassen?“. 106 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 389 390 MENKE, K.-H. Befund bestimmten Interessen unterzuordnen109. Anni Hentschel hat nachgewiesen, dass im NT klar unterschieden wird zwischen den vielen Tätigkeiten, die mit dem Wortfeld διακονειν/διάκονοι bezeichnet werden, und dem Diakonenamt110. Eindeutig belegt ist, dass es seit dem 3. Jahrhundert „Diakonissen“ gab; aber ebenso unbestreitbar ist, dass diese nie als Trägerinnen des sakramentalen Amtes betrachtet worden sind. Im Gegenteil: Die entsprechenden Dokumente sind geradezu ängstlich darauf bedacht, diese Schlussfolgerung aus der gemeinsamen Bezeichnung von Frauen und Männern als διάκονοι zu vermeiden. Aimé Georges Martimort und Gerhard Ludwig Müller haben alle zugänglichen Dokumente gesichtet, in denen von Diakonissen die Rede ist. Sie kommen zu dem Ergebnis, dass der Unterschied zwischen dem nichtsakramentalen Diakonissenamt und dem sakramentalen Amt der Diakone in jedem Fall gewahrt oder gar deutlich bezeichnet wird111. Hans Jorissen — im Unterschied zu Martimort und Müller ein Befürworter der Zulassung von Frauen zum Sakrament des Ordo — bilanziert seine historischen Studien mit der Feststellung: Das Diakonissenamt stellt sich im historischen Rückblick als ein äußerst komplexes, geographisch und zeitlich sehr verschieden ausgeprägtes Phänomen dar. Es fehlt die Kontinuität der Überlieferung. Auch da, wo die Weihe von Diakoninnen unter Handauflegung und Epiklese analog zur Diakonenweihe gestaltet ist, wie in den Apostolischen Marlis Gielen (GIELEN, M. Frauen als Diakone in paulinischen Gemeinden, in: Winkler [Anmerkung 98], 11–40), folgert aus der einzigen Bibelstelle (Röm 16,1 f.), in der eine Frau διάκονο genannt wird, und aus der Vergleichbarkeit von Tätigkeiten biblisch bezeugter Frauen mit denen biblisch bezeugter „Amtsträger“ die ursprüngliche Einbeziehung von Frauen in die Gemeindeleitung sowie den soziologisch bzw. kirchenpolitisch bedingten Abfall von diesem Ursprung in nachapostolischer Zeit. 110 Vgl. HENTSCHEL, A. Diakonia im Neuen Testament. Studien zur Semantik unter besonderer Berücksichtigung der Rolle von Frauen. Tübingen, 2007, besonders 180–184.374–382. 111 Zu dem durchgängig klar bezeugten Unterschied zwischen dem sakramentalen Diakonat der Männer und dem nichtsakramentalen Dienst der Diakonissen: MARTIMORT, A. G. Les diaconesses. Essai historique, Rom 1982; MÜLLER, G. L. Der Empfänger des Weihesakramentes: Quellen zur Lehre und Praxis der Kirche, nur Männern das Weihesakrament zu spenden. Würzburg, 1999. 109 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Konstitutionen112 und vor allem im späteren byzantinischenRitus113, erlauben die historischen Befunde es nicht, von einer Gleichrangigkeit beider Ordinationen zu sprechen. Mit Recht folgert Martimort: „Wie groß auch die Feierlichkeit gewesensein mag, die den Ritus umgab, und die äußere Ähnlichkeit mit der Weihe zum Diakon, so ist die byzantinische Diakonisse doch kein Diakon; es ist ein völlig anderes Amt“114. Bedenkenswert ist vor allem der ausdrückliche Ausschluss der Diakonin von jeglichem liturgischen Altardienst, von der öffentlichenAusübung des Verkündigungsdienstes und von der feierlichen Taufspendung. „Die Gründe hierfür liegen (eindeutig) im Ausschluss der Frau vom Amtspriestertum.“ Männlicher und weiblicher Diakonat sind in der Alten Kirche nicht zwei gleichartige Zweige des einen diakonalen Amtes. Die Sakramentalität (im heutigen dogmatischen Verständnis) eines eigenständigen Diakonats ohne inneren, in der Diakonatsweihe begründeten Bezug zum bischöflich-priesterlichen Amt lässt sich mit guten Gründen nicht historisch stützen115. Jorissen folgert aus der vom Zweiten Vatikanum endgültig affirmierten Trias des sakramentalen Ordo, dass die Zulassung der Frau zum Diakonat deren Zulassung auch zur Bischofs- und Priesterweihe implizieren würde116. Folgerichtig konstatiert er, dass die Forderung der In den Apostolischen Konstitutionen, in denen die Weihe der Diakonin unter Handauflegung und Epiklese dokumentiert ist, heißt es zugleich: „Die Diakonin segnet nicht, sie tut nichts von dem, was Priester und Diakone tun, sie bewacht nur die Türen und leiht dem Priester ihren Dienst bei der Taufe der Frauen, aus Gründen der Schicklichkeit“ (Constitutiones Apostolicae VIII, 28,6 [SC 336,230]). 113 Dazu: KARRAS, V. A. Female Deacons in the Byzantine Church, in: ChH n. 73, 2004, p.272–316. MITSIOU, E. Weibliches Mönchtum und Diakonat in byzantinischer Zeit, in: WINKLER (Anmerkung 98), p. 59-70. 114 MARTIMORT (Anmerkung 112), 155. 115 JORISSEN, H. Theologische Bedenken gegen die Diakonatsweihe von Frauen, in: Diakonat. Ein Amt für Frauen in der Kirche – Ein frauengerechtes Amt?, herausgegeben von HÜNERMANN, P. [u. a.], Ostfildern, 1997, 86– 97; 94 f. 116 „Ein eigenständiger Diakonat postuliert (gegenüber der Einheit des Ordo/Weihesakramentes im traditionellen dogmatischen Verständnis) ein zweites, nicht als besondere Ausprägung des einen Ordo zu verstehendes Weihesakrament, dessen sakramentale Begründung aber völlig ungeklärt bleibt – es sei denn, man würde der Kirche eine diesbezügliche Kompetenz zuschreiben, was aber zu Schwierigkeiten mit den lehramtlichen Äußerungen führen würde, wonach die Kirche keine Vollmacht über die Substanz der Sakramente hat. Aus alledem ist zu folgern: Die Möglichkeit eines sakramentalen Frauendiakonats steht und fällt mit der Möglichkeit des Frauenpriestertums“ (JORISSEN [Anmerkung 116], 95). 112 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 391 392 MENKE, K.-H. Würzburger Synode117 nach Zulassung von Frauen zu einem sakramental verstandenen Diakonat von falschen Voraussetzungen ausgeht und wohl nur vor dem Erscheinen der römischen Dokumente „Inter insigniores“ (15.10.1976), Mulieris dignitatem (15.8.1988) und „Ordinatio sacerdotalis“ (22.5.1994) denkbar war118. Natürlich kann man darüber nachdenken, ob die Institutionalisierung der Dienste und Charismen von Frauen in Gestalt eines dem altoder ostkirchlichen Institut ähnlichen Amtes sinnvoll wäre. Aber dabei kann es nicht darum gehen, alles, was in der Kirche getan wird, zu verbeamten oder zu klerikalisieren. Man sollte zudem beachten, dass zumindest im Westen das Institut der Diakonissen durch karitativ tätige Frauenorden abgelöst wurde. Der berechtigten Forderung nach mehr Partizipation von Frauen in der Kirche würde durch Einweisung in ausschließlich dienende Funktionen kaum oder gar nicht entsprochen. Frauen, die als Diakonissen bezeichnet, aber den Diakonen nicht gleichgestellt wären, würden sich wohl eher diskriminiert als aufgewertet fühlen119. Die in süddeutschen Diözesen installierte Vorbereitung von Frauen auf ein (sakramental oder nichtsakramental?) verstandenes „Viele Frauen üben in vielen Kirchenprovinzen, nicht nur in Missionsgebieten, eine Fülle von Tätigkeiten aus, die an sich dem Diakonenamt zukommen. Der Ausschluss dieser Frauen von der Weihe bedeutet eine theologisch und pastoral nicht zu rechtfertigende Trennung von Funktion und sakramental-vermittelter Heilsvollmacht. Ein weiterer Grund liegt darin, daß die Stellung der Frau in Kirche und Gesellschaft es heute unverantwortlich erscheinen läßt, sie von theologisch möglichen und pastoral wünschenswerten amtlichen Funktionen in der Kirche auszuschließen. Schließlich läßt die Hineinnahme der Frau in den sakramentalen Diakonat in vielfacher Hinsicht eine Bereicherung erwarten, und zwar für das Amt insgesamt und für die in Gang befindliche Entfaltung des Diakonats im besonderen“ (Beschluss: Dienste und Ämter, 4.2.2, in: OGGSB I, Freiburg i. Br. 1976, 617). 118 Zur Irreversibilität dieser Dokumente: LÜDECKE, N. Also doch ein Dogma? Fragen zum Verbindlichkeitsanspruch der Lehre über die Unmöglichkeit der Priesterweihe für Frauen aus kanonistischer Perspektive, in: TThZ n. 105, 1996, p. 161–211. MÜLLER, G. L. Hat die Kirche die Vollmacht, Frauen das Weihesakrament zu spenden?, in: StZ n. 137, 2012, p. 374-384. – Zu den Konvenienzgründen, die man für die Position des Lehramtes anführen kann: MENKE, K.-H. Sakramentalität: Wesen und Wunde des Katholizismus. Regensburg, 2012, p. 75–87. 119 Dazu: VON STRITZKY, M. B. Der Dienst der Frau in der Alten Kirche, in: LJ n. 28, 1978, 136- 154; besonders 152–154. 117 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 Zweiten Vatikanischen Konzil und die triadische Einheit des Ordo und die Frage nach einem Diakonat der Frau Diakonenamt120 hat Erwartungen geweckt, die nur in Frustration münden können121. Dennoch: Hier soll in keiner Weise bestritten werden, dass der Anspruch von Frauen berechtigt ist, nicht nur in Wirtschaft, Wissenschaft und Politik, sondern auch in der Kirche mehr Verantwortung zu übernehmen. Aber dieses Anliegen sollte nicht mit der Forderung nach dem Zugang der Frau zum sakramentalen Ordo verknüpft werden. Natürlich ist die sakramentale Repräsentation des ‚Voraus‘ Christi gegenüber der Kirche immer auch mit der Übertragung von Autorität verbunden. Aber diese Macht wird missbraucht, wenn ihre Träger sich nicht zugleich in die Nachfolge des fußwaschenden, herabsteigenden, gekreuzigten Erlösers stellen lassen. Gisbert Greshake hat in seinem Buch „Priester sein in dieser Zeit“ eine Erneuerung der Kirche unter ebendieser Prämisse angemahnt. Dazu gehört neben einer Revision der Priesterausbildung ganz wesentlich auch die Geschlechtergerechtigkeit. Es mag utopisch klingen. Aber ein Papst könnte, ohne die sakramentale Ordnung zu verletzen, das Kirchenrecht dahingehend ändern, dass jeweils zur Hälfte mit Frauen und Männern besetzte Gremien den Bischof und auch den Papst wählen; dass unterhalb von Generalvikar und Offizial Verwaltungsämter paritätisch mit Frauen besetzt werden und dass Entscheidungen auf allen Ebenen den Rat von Frauen voraussetzen122. Erhalten: 04/08/2014 Received: 08/04/2014 Genehmigt: 27/11/2014 Approved: 11/27/2014 Ein als Notifikation betiteltes Schreiben der zuständigen römischen Kongregationen hat 2001 alle Initiativen verboten, „die in irgendeiner Weise darauf abzielen, Frauen auf die Diakonenweihe vorzubereiten“ (AkathKR 170 [2001] 481 f.). – Zu den Initiativen in den Bistümern Rottenburg-Stuttgart und Freiburg im Sinne einer ausdrücklichen Ermutigung, den eingeschlagenen Weg „konstruktiven Ungehorsams“ weiterzugehen: REININGER (Anmerkung 94), 284 f. 121 Regina Radlbeck-Ossmann (Anmerkung 98), 120, sieht die Zulassung von Frauen zu einem sakramental verstandenen Diakonat durch die römische Abweisung der besagten Initiativen nicht berührt. – Dazu: M. Hauke, Die Diskussion um den Diakonat der Frau. Eine kritische Bilanz, in: SCHEFFCZYK, L. (Hg.), Diakonat und Diakonissen, St. Ottilien, 2002, p. 11–65. DÜREN, S. Über den beharrlichen und zugleich sinnlosen Versuch, Frauen den Empfang der sakramentalen Diakonatsweihe zu ermöglichen, in: Ebd. p. 149–231. 122 Vgl. GRESHAKE, G. Priester sein in dieser Zeit. Freiburg i. Br., 2000, p. 158–167. 120 Rev. Pistis Prax., Teol. Pastor., Curitiba, v. 7, n. 2, p. 349-393, maio/ago. 2015 393
https://openalex.org/W4378716833	https://boris.unibe.ch/183014/1/s13063-023-07399-6.pdf	English	null	N-acetyl-L-leucine for Niemann-Pick type C: a multinational double-blind randomized placebo-controlled crossover study	Trials	2,023	cc-by	8,361	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Trials Trials Trials Fields et al. Trials (2023) 24:361 https://doi.org/10.1186/s13063-023-07399-6 Open Access N‑acetyl‑L‑leucine for Niemann‑Pick type C: a multinational double‑blind randomized placebo‑controlled crossover study T Fields1* , T M. Bremova2, I Billington1, GC Churchill3, W Evans4,5, C Fields1, A Galione3, R Kay6, T Mathieson4,6, K Martakis7, M Patterson8, F Platt3, M Factor1 and M Strupp9 T Fields1* , T M. Bremova2, I Billington1, GC Churchill3, W Evans4,5, C Fields1, A Galione3, R Kay6, T Mathieson4,6, K Martakis7, M Patterson8, F Platt3, M Factor1 and M Strupp9 Abstract Background Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal disease characterized by multiple symptoms such as progressive cerebellar ataxia and cognitive decline. The modified amino acid N-acetyl-leucine has been associated with positive symptomatic and neuroprotective, disease-modifying effects in various studies, including animal models of NPC, observational clinical case studies, and a multinational, rater- blinded phase IIb clinical trial. Here, we describe the development of a study protocol (Sponsor Code “IB1001-301”) for the chronic treatment of symptoms in adult and pediatric patients with NPC. Methods This multinational double-blind randomized placebo-controlled crossover phase III study will enroll patients with a genetically confirmed diagnosis of NPC patients aged 4 years and older across 16 trial sites. Patients are assessed during a baseline period and then randomized (1:1) to one of two treatment sequences: IB1001 followed by placebo or vice versa. Each sequence consists of a 12-week treatment period. The primary efficacy endpoint is based on the Scale for the Assessment and Rating of Ataxia, and secondary outcomes include cerebellar functional rating scales, clinical global impression, and quality of life assessments. Discussion Pre-clinical as well as observational and phase IIb clinical trials have previously demonstrated that IB1001 rapidly improved symptoms, functioning, and quality of life for pediatric and adult NPC patients and is safe and well tolerated. In this placebo-controlled cross-over trial, the risk/benefit profile of IB1001 for NPC will be evaluated. It will also give information about the applicability of IB1001 as a therapeutic paradigm for other rare and common neuro- logical disorders. Trial registrations The trial (IB1001-301) has been registered at www.clinicaltrials.gov (NCT05163288) and www.clini caltrialsregister.eu (EudraCT: 2021–005356-10). Registered on 20 December 2021. Keywords Niemann-Pick type C (NPC), N-acetyl-L-leucine, Pharmaceutical intervention, Symptomatic treatment, Randomized controlled trial, Cerebellar ataxia, Lysosomal storage disease Keywords Niemann-Pick type C (NPC), N-acetyl-L-leucine, Pharmaceutical intervention, Symptomatic treatment, Randomized controlled trial, Cerebellar ataxia, Lysosomal storage disease tfields@intrabio.com Full list of author information is available at the end of the article tfields@intrabio.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Fields et al. Trials (2023) 24:361 Fields et al. Trials (2023) 24:361 Page 2 of 9 potential clinical benefits compared to the racemic mix- ture. N-acetyl-L-leucine is a pro-drug of L-leucine and has a unique transport mechanism [10]. One mechanism of action of N-acetyl-L-leucine is the activation of cer- ebral glucose metabolism in the cerebellum, correlated with enhanced cerebellar activity [11, 12]. In an animal model of NPC, N-acetyl-DL-leucine and its enantiom- ers significantly reduced ataxia in Npc1−/− mice, when treated symptomatically (from 8 to 9 weeks of age) and pre-symptomatically (from 3 weeks of age) [6]. These studies specifically identified the L-enantiomer as the neuroprotective isomer, observed to significantly delay the onset of functional decline (gait abnormalities, motor dysfunction), the decline in general health and condi- tion, as well as slowing disease progression and prolong- ing survival (whereas the D-enantiomer did not). Similar effects of N-acetyl-L-leucine were found in an animal model of another lysosomal storage disease, the Sandhoff (hexb−/−) mouse [13]. It is important to note that the dos- age used in these in vivo studies (0.1 g per kg per day) approximates the dose used in previous observational clinical studies with the racemate, the phase IIb studies with IB1001, and the current IB1001-301 clinical trial. N‑acetyl‑L‑leucine N-acetyl-L-leucine (Sponsor Code “IB1001”) is the L-enantiomer of N-acetyl-DL-leucine, a modified, acety- lated derivative of a natural essential amino acid (Leu- cine) that has been available in France since 1957 as a racemate (equal amounts of both D- and L-enantiomers) under the trade name Tanganil™ (Pierre Fabre Labo- ratories, France) as a treatment for acute vertigo. Prior observational studies assessing the effect of N-acetyl-DL- leucine in patients with NPC suggest a beneficial symp- tomatic as well as neuroprotective disease-modifying effect of this agent. In a case series, short-term treatment with N-acetyl-DL-leucine was found to improve ataxia, cognition, and quality of life in 12 patients with NPC [4]. Subsequent long-term case series and pre-clinical stud- ies demonstrated the neuroprotective, disease-modifying effect of treatment in NPC [5, 6]. In all studies, the com- pound was well tolerated with no serious side effects. Background g Niemann-Pick disease type C (NPC) is a rare (1:120,000 live births), prematurely fatal, autosomal recessive, neu- rovisceral lysosomal disease that predominantly affects children. The disease typically begins in early childhood, is chronic, progressive, and severely reduces quality of life. Adolescent and adult-onset cases are being increas- ingly recognized [1]. The presentation of NPC is charac- terized by broad heterogeneity in systemic, psychiatric, and neurological symptoms, though in general, the ear- lier the age of onset, the more rapidly progressive the symptoms [2]. There is broad inter-individual phenotypic variability, including the age of onset and rate of progres- sion. This renders an assembly of well-matched cohorts of NPC patients for controlled trials difficult to achieve. Treatment of NPC is currently limited to reducing the rate of disease progression with the substrate reduction therapy drug miglustat (Zavesca™), which is approved in the European Union and several other countries, but not in the USA [3]. Finally, pharmacokinetic studies demonstrate that the D-enantiomer is not metabolized and could accumulate relative to the L-enantiomer during chronic administra- tion of the racemate, having the potential for long-term negative effects [14]. Therefore, in this clinical trial, the effects of N-acetyl-L-leucine will be evaluated. Trial rationaleh The primary objective of the IB1001-301 clinical trial is to demonstrate the symptomatic benefits of IB1001 treat- ment given: The primary objective of the IB1001-301 clinical trial is to demonstrate the symptomatic benefits of IB1001 treat- ment given: (i) There are no treatments for the symptoms of NPC, which are highly debilitating and significantly det- rimental to functioning, quality of life, and health span. (ii) NPC is a life-limiting condition with an extremely high unmet medical need, mandating greater urgency for trials to be conducted as efficiently as possible to maximize the chance they can be made available before the window of therapeutic oppor- tunity is lost [8]. f Recently, two multinational, phase IIb clinical trials with N-acetyl-L-leucine for NPC and the related lyso- somal storage disorder GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases) were completed [7–9]. Both tri- als were successful, demonstrating a statistically signifi- cant change on the primary and secondary endpoints and clinically meaningful improvement in symptoms, func- tioning, and quality of life for children and adults with NPC and GM2. In both studies, N-acetyl-L-leucine was also well tolerated with no serious side effects. Accordingly, although there is data supporting both the symptomatic and neuroprotective, disease-modifying effects of IB1001 for NPC, the IB1001-301 pivotal trial prioritizes the investigation of the symptomatic benefit to expedite the development and availability of this promis- ing drug candidate. Given the extreme heterogeneity of the disease, a crossover study design is utilized so that each patient serves as their own control and the clinical f Animal studies in the NPC mouse model have shown that the L-enantiomer, i.e., N-acetyl-L-leucine, has Fields et al. Trials (2023) 24:361 Page 3 of 9 information about the rationale, design, and personal implications of the study. meaningfulness of the effect can be assessed for each patient. meaningfulness of the effect can be assessed for each patient. Patient population and eligibility criteria Patients will be screened for eligibility according to the inclusion and exclusion criteria. To be eligible for the respective study, patients aged ≥ 4 years with a confirmed diagnosis of NPC must present with clinical symptoms, provide appropriate informed consent, and undertake a washout of any prohibited medications (if applicable). These include any variant of N-acetyl-DL-leucine (e.g., Tanganil™). For a detailed description of the inclusion and exclusion criteria, see Table 1. Methods/design Study oversighth The IB1001-301 clinical trial is a double-blind placebo- controlled crossover trial. Patients will be first assessed during a baseline period (with or without a study run- in) and randomized (1:1) to one of two treatment sequences: IB1001 followed by placebo, or vice-versa. Each treatment period will last approximately 12 weeks (84–91 days). Patients will be assessed twice during each period to allow an assessment of intra-patient variability. The IB1001-301 trial is conducted in accordance with the International Conference for Harmonisation (of Techni- cal Requirements for Pharmaceuticals for Human Use)— Good Clinical Practice Guideline, the General Data Protection Regulator, and the Declaration of Helsinki. The study protocol was designed in accordance with the SPIRIT 2013 statement and the study is conducted in accordance with the SPIRIT reporting guidelines [15]; the SPIRIT Figure for both the Parent Study and the Exten- sion Phase is included as Supplementary Tables 1 and 2, and the SPIRIT checklist has been completed. The study has been approved by the ethics committees of each par- ticipating center and the regulatory authorities in each respective country. The safety, integrity, and feasibility of the trial is monitored by an independent data safety mon- itoring board (DSMB) consisting of three independent, non-participating members (including two clinicians and a statistician). The function of the DSMB is to monitor the course of the studies and, as applicable, recommend to the sponsor of the trial for discontinuation, modifica- tion, or continuation of the study. The roles and responsi- bilities of the DSMB are defined in a DSMB charter. Randomization At visit 2, patients will be randomized (1:1) to their respective sequence. Patients will be centrally rand- omized using Medpace’s ClinTrak Interactive Response Technology (IRT) web-based system. Medpace is respon- sible for the generation of the allocation sequence, and the IRT system will automatically assign participants to their respective sequence. The study utilizes a permuted block design for randomization. The LIVE randomization list is generated by an independent statistician based on the approved randomization plan. As the study is double- blinded, patients, their families, the study team, and the sponsor will be blinded to the randomization scheme and the sequences to which the patient is assigned. The rand- omization list will not be available to any person involved in the conduct of the study or the evaluation of the trial until the trial database is locked. The bioanalytical Screening At the initial screening visit, patients will be classified as either “naïve” or “non-naïve” depending on their use of prohibited medications within the past 6 weeks (42 days). The schedule of events during the initial screening visit and throughout the baseline period (through visit 1) will vary depending on the patient’s classification as either “naïve” or “non-naïve.” Given the known unlicensed use of the racemate (Tanganil™), for all patients, a urine sam- ple will be taken at visit 1 to detect N-acetyl-D-leucine using a validated liquid chromatography mass spectrom- etry/mass spectrometry method. Provided the level of N-acetyl-D-leucine is below the permitted threshold, the initial screening visit will be confirmed as visit 1 (baseline 1). If a patient classified as “Naïve” unexpectedly tests positive for levels of N-acetyl-D-leucine above the per- mitted threshold, at the direction of their principal inves- tigator, a run-in wash-out period of 6 weeks (42 days) is requested before they are eligible to return for a repeat visit 1. Patients who fail two urine N-acetyl-D-leucine tests (e.g., visit 1 and repeat visit 1) are ineligible for the study. Individuals who meet all of the following criteria are eligible to participate in the study: Females of childbearing potential, defined as a premenopausal female capable of becoming pregnant, will be included if they are either sexually inactive (sexually abstinenta for 14 days prior to the first dose and confirm to continue through 28 days after the last dose) or using one of the following highly effective contraceptives (i.e., results in < 1% failure rate when used consistently and correctly) 14 days prior to the first dose continuing through 28 days after the dose: drug”) for at least 42 days prior to visit 1. At the discretion of the investigator, medical monitor, and sponsor, the washout period for specific IMPs may be longer based on the pharmacological activity and pharmacoki- netics of the drug y a) Intrauterine device (IUD); b) Surgical sterilization of the partner (vasectomy for 6 months minimum); c) Combined (estrogen or progestogen containing) hormonal contraception associated with the inhibition of ovulation (either oral, intravaginal, or transdermal); c) Combined (estrogen or progestogen containing) hormonal contraception associated with the inhibition ovulation (either oral, intravaginal, or transdermal); ovulation (either oral, intravaginal, or transdermal); d) Progestogen only hormonal contraception associated with the inhibition of ovulation (either oral, injectable, or implantable); d) Progestogen only hormonal contraception associated with the inhibition of ovulation (either oral, injecta or implantable); 3. Patients with a physical or psychiatric con- dition which, at the investigator’s discretion and in consultation with the medical moni- tor and sponsor (as applicable), may put the patient at risk, may confound the study results, or may interfere with the patient’s participation in the clinical study, i.e., reliably perform study assessments e) Intrauterine hormone releasing system (IUS); f) Bilateral tubal occlusion f) Bilateral tubal occlusion 4. Females of non-childbearing potential who have undergone one of the following sterilization procedures at least 6 months prior to the first dose: 4. Females of non-childbearing potential who have undergone one of the following sterilization procedures at least 6 months prior to the first dose: a) Hysteroscopic sterilization; b) Bilateral salpingectomy; 4. Known or persistent use, misuse, or dependency of medication, drugs, or alcohol 5. Current or planned pregnancy or women who are breastfeeding c) Hysterectomy; d) Bilateral oophorectomy; OR be postmenopausal with amenorrhea for at least 1 year prior to the first dose and follicle stimulating hor- mone (FSH) serum levels consistent with postmenopausal status. FSH analysis for postmenopausal women will be done at screening. Individuals who meet all of the following criteria are eligible to participate in the study: Individuals who meet any of the following criteria are not eligible to participate in the study: g g p p y 1. Written informed consent signed by the patient and/or their legal representative/parent/impartial witness 2. Non-US: Male or female aged ≥ 4 years with a confirmed diagnosis of NPC at the time of signing informed consent. Confirmed diagnosis includes one of the following: g g p p y 1. Written informed consent signed by the patient and/or their legal representative/parent/impartial witnessi y 1. Written informed consent signed by the patient and/or their legal representative/parent/impartial witness 2 Non-US: Male or female aged≥4 years with a confirmed diagnosis of NPC at the time of signing informed 2. Non-US: Male or female aged ≥ 4 years with a confirmed diagnosis of NPC at the time of signing informed consent. Confirmed diagnosis includes one of the following: 1. Patients who have any known hypersensi- tivity or history of hypersensitivity to: i g g a) Clinical features and positive biomarker screen and/or filipin test without genetic tests results (has not been performed) a. Acetyl-leucine (DL-, L-, D-) or derivatives b. Excipients the IB1001 sachet (namely isomalt, hypromellose, and strawberry flavor) y ( , , ) b. Excipients the IB1001 sachet (namely isomalt, hypromellose, and strawberry flavor) p g c) Clinical features and positive biomarker screen and/or filipin test but only one NPC mutation identified on genetic test c) Clinical features and positive biomarker screen and/or filipin test but only one NPC mutation identified on genetic test c. Excipients the placebo sachet (namely isomalt, hypromellose, strawberry flavor, citric acid, microcrystalline cellulose, lactose, denatonium benzoate) g d) Clinical features with positive biomarker screen and/or filipin test and positive genetic testi d) Clinical features with positive biomarker screen and/or filipin test and positive genetic test US: Male or female aged ≥ 4 years with a confirmed genetic diagnosis of NPC at the time of signing informed consent. Patients must have clinical features of NPC and a positive genetic test for mutations in both copies of NPC1 or in both copies of NPC2 2. Simultaneous participation in another clinical study or participation in any clinical study involving administration of an inves- tigational medicinal product (IMP; “Study 3. a Sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual intercourse during the entire period of risk associated with the study treatments. In this trial abstinence is only acceptable if in line with the patient’s preferred and usual lifestyle. Period abstinence (calendar, symptothermal, post-ovulation methods), withdrawal (coitus interruptus), spermicides only, and lactational amenorrhoea method (LAM) are not acceptable methods of contraception. As well, female condom and male condom should not be used together Recruitment and patient involvementh The principal investigator at each site will be responsible for the enrolment of patients. Patients will be screened at 16 centers across Australia, the Czech Republic, Germany, the Netherlands, Slovakia, Switzerland, the UK, and the USA. The list of study sites is available via www.clinicaltrials.gov (NCT05163288). Patients will be recruited via personal correspondence, routine care appointments, and referrals. In addition, there is collabo- ration and support from multinational patient organiza- tions representing these rare disease communities. All eligible patients who agree to participate in the study are provided with a full verbal explanation of the trial and the Patient Information Sheet. This includes detailed Page 4 of 9 Fields et al. Trials (2023) 24:361 Fields et al. Trials (2023) 24:361 Inclusion criteria Individuals who meet any of the following criteria are not eligible to participate in the study: 1. Patients who have any known hypersensi- tivity or history of hypersensitivity to: a. Acetyl-leucine (DL-, L-, D-) or derivatives b. Excipients the IB1001 sachet (namely isomalt, hypromellose, and strawberry flavor) c. Excipients the placebo sachet (namely isomalt, hypromellose, strawberry flavor, citric acid, microcrystalline cellulose, lactose, denatonium benzoate) 2. Simultaneous participation in another clinical study or participation in any clinical study involving administration of an inves- tigational medicinal product (IMP; “Study drug”) for at least 42 days prior to visit 1. At the discretion of the investigator, medical monitor, and sponsor, the washout period for specific IMPs may be longer based on the pharmacological activity and pharmacoki- netics of the drug 3. Patients with a physical or psychiatric con- dition which, at the investigator’s discretion and in consultation with the medical moni- tor and sponsor (as applicable), may put the patient at risk, may confound the study results, or may interfere with the patient’s participation in the clinical study, i.e., reliably perform study assessments 4. Known or persistent use, misuse, or dependency of medication, drugs, or alcohol 5. Current or planned pregnancy or women who are breastfeeding 6. Patients with severe vision or hearing impairment (that is not corrected by glasses or hearing aids) that, at the investigator’s discretion, interferes with their ability to perform study assessments 7. Patients who have been diagnosed with arthritis or other musculoskeletal disorders affecting joints, muscles, ligaments, and/or nerves that by themselves affects patient’s mobility and, at the investigator’s discretion, interferes with their ability to perform study assessments 8. Patients unwilling and/or not able to undergo a 42-day washout period from any of the following prohibited medication prior to visit 1 (baseline 1) and remain without prohibited medication through visit 6 a) N-acetyl-DL-leucine (e.g., Tanganil®); b) N-acetyl-L-leucine (prohibited if not provided as IMP in the IB1001-301 trial); c) Sulfasalazine; d) Rosuvastatin Individuals who meet all of the following criteria are eligible to participate in the study: Individuals who meet all of the following criteria are eligible to participate in the study: FSH levels should be in the postmenopausal range as determined by the central laboratory 5. Non-vasectomized male patient agrees to use a condom with spermicide or abstain from sexual intercourse during the study until 90 days beyond the last dose of study medication and the female partner agrees to comply with inclusion criteria 3 or 4. For a vasectomized male who has had his vasectomy 6 months or more prior to study start, it is required that they use a condom during sexual intercourse. A male who has been vasectomized less than 6 months prior to study start must follow the same restrictions as a non-vasectomized malei 6. Patients with severe vision or hearing impairment (that is not corrected by glasses or hearing aids) that, at the investigator’s discretion, interferes with their ability to perform study assessments 7. Patients who have been diagnosed with arthritis or other musculoskeletal disorders affecting joints, muscles, ligaments, and/or nerves that by themselves affects patient’s mobility and, at the investigator’s discretion, interferes with their ability to perform study assessments p y 6. If male, patient agrees not to donate sperm from the first dose until 90 days after their last dose y 6. If male, patient agrees not to donate sperm from the first dose until 90 days after their last dose 7. Patients must fall within: 6. If male, patient agrees not to donate sperm from the first dose until 90 days after their last dose 7 P ti t t f ll ithi 7. Patients must fall within: a) A SARA score of 7 ≤ X ≤ 34 points (out of 40) a) A SARA score of 7 ≤ X ≤ 34 points (out of 40) Table 1 Inclusion and exclusion criteria for patient selection in the Parent Study Exclusion criteria AND i. Within the 2–7 range (0–8 range) of the Gait subtest of the SARA scale OR OR ii. Be able to perform the 9-Hole Peg Test with Dominant Hand (9HPT-D) (SCAFI subtest) in 20 ≤ X ≤ 150 s 8. Weight ≥ 15 kg at screening 9. Patients are willing to disclose their existing medications/therapies for (the symptoms) of NPC, including those on the prohibited medication list. Non-prohibited medications/therapies (authorized medicines for NPC [e.g., p p p miglustat], speech therapy, and physiotherapy are permitted provided: a) The investigator does not believe the medication/therapy will interfere with the study protocol/results b) P i h b bl d /d i d f h f l 42 d b f i i 1 (b a) The investigator does not believe the medication/therapy will interfere with the study protocol/results b) Patients have been on a stable dose/duration and type of therapy for at least 42 days before visit 1 (baseline 1) c) Patients are willing to maintain a stable dose/do not change their therapy throughout the duration of the t d c) Patients are willing to maintain a stable dose/do not change their therapy throughout the duration of the study c) Patients are willing to maintain a stable dose/do not change their therapy throughout the duration of the study y 10. An understanding of the implications of study participation, provided in the written patient information and informed consent by patients or their legal representative/parent, and demonstrates a willingness to comply with instructions and attend required study visits (for children this criterion will also be assessed in parents or appointed guardians) a Sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual intercourse during the entire period of risk associated with the study treatments. In this trial abstinence is only acceptable if in line with the patient’s preferred and usual lifestyle. Period abstinence (calendar, symptothermal, post-ovulation methods), withdrawal (coitus interruptus), spermicides only, and lactational amenorrhoea method (LAM) are not acceptable methods of contraception. As well, female condom and male condom should not be used together Fields et al. Trials (2023) 24:361 Page 5 of 9 assessments together with pre-planned time points for clinic visits in the Extension Phase. laboratory staff are authorized to receive the randomi- zation list prior to the study conclusion to determine which samples should be analyzed according to standard operating procedures. AND As the trial is a crossover design, in which each patient will receive IB1001 and placebo, and serve as their own control, no stratification by age is performed. Study drugh The dosage form of N-acetyl-L-leucine or the match- ing placebo is granules for oral suspension in a sachet (manufactured by Patheon France S.A.S., Bourgoin Facil- ity, France) which are suspended in 40 mL water, orange juice, or almond milk. A marked measuring cup is pro- vided to each patient. Intervention periods Figure 1 displays the naïve and non-naïve study schemes for the Parent Study. Suppl. Tables 1 lists the schedule of enrolment and assessments together with pre-planned time points for clinic visits. Patients who have completed Visit 6 of the Parent Study have the opportunity to con- tinue treatment with N-acetyl-L-leucine (IB1001) in an Extension Phase if the principal investigator determines it is in their best interest. The Extension Phase consists of a 1-year (351–379 day) treatment period followed by a 6 weeks (42–56 days) washout period. Table 2 lists the inclusion criteria for the Extension Phase. Administration and study drug dosage During the treatment periods for both treatment sequences, the dosing of the study drug is as follows: patients aged ≥ 13 years or aged 4–12 years weigh- ing ≥ 35 kg will take 4 g/day (2 g in the morning, 1 g in the afternoon, and 1 g in the evening). Patients aged 4–12 years weighing 25 to < 35 kg will take 3 g/day (1 g in the morning, 1 g in the afternoon, and 1 g in the even- ing). Patients aged 4–12 years weighing 15 to < 25 kg will take 2 g/day (1 g in the morning and 1 g in the evening). Medication should be taken at least 30 min before or at Figure 2 displays the Extension Phase study schema. Suppl. Table 2 lists the schedule of enrolment and Fig. 1 Parent Study schema. a Naïve patients screening pathway: patients who have not used any prohibited medications within 42 days of screening are “naïve.” Their initial screening visit is treated as visit 1 (baseline 1). b Non-naïve patient screening pathway: patients who have used or are unable to confirm or deny if they have used, any prohibited medication within the past 42 days are “non-naïve.” Patient will be given the opportunity to undergo a minimum of 42 days washout before returning for a repeat visit 1 (baseline 1). From visit 2 onward, the visit schedule is the same for naïve and non-naïve patients Fig. 1 Parent Study schema. a Naïve patients screening pathway: patients who have not used any prohibited medications within 42 days of screening are “naïve.” Their initial screening visit is treated as visit 1 (baseline 1). b Non-naïve patient screening pathway: patients who have used or are unable to confirm or deny if they have used, any prohibited medication within the past 42 days are “non-naïve.” Patient will be given the opportunity to undergo a minimum of 42 days washout before returning for a repeat visit 1 (baseline 1). From visit 2 onward, the visit schedule is the same for naïve and non-naïve patients Fig. 1 Parent Study schema. a Naïve patients screening pathway: patients who have not used any prohibited medications within 42 days of screening are “naïve.” Their initial screening visit is treated as visit 1 (baseline 1). Administration and study drug dosage b Non-naïve patient screening pathway: patients who have used or are unable to confirm or deny if they have used, any prohibited medication within the past 42 days are “non-naïve.” Patient will be given the opportunity to undergo a minimum of 42 days washout before returning for a repeat visit 1 (baseline 1). From visit 2 onward, the visit schedule is the same for naïve and non-naïve patients Fields et al. Trials (2023) 24:361 Fields et al. Trials (2023) 24:361 Page 6 of 9 Table 2 Inclusion criteria for patient participation in the Extension Study Inclusion criteria 1. Completed visit 6 of the Parent Study 2. The principal investigator determines further treatment with IB1001 to be in patient’s best interest 3. Written informed consent signed by the patient and/or their legal representative/parent/impartial witness for participation in the Extension Phase 4. Patients are willing to continue to remain without the following prohibited medication from visit 6 throughout the duration the Extension Phase: e) N-acetyl-DL-leucine (e.g., Tanganil®); f) N-acetyl-L-leucine (prohibited if not provided as investigational medicinal product [IMP]); g) Sulfasalazine; h) Rosuvastatin Fig. 2 Extension Phase schema. Patients will be assessed approximately 4 times over a 64-week period: at the start of the Extension Phase, after 6 months of treatment, 1 year of treatment, and after a 42-day (+ 14 day) post-extension-phase treatment washout Fig. 2 Extension Phase schema. Patients will be assessed approximately 4 times over a 64-week period: at the start of the Extension Phase, after 6 months of treatment, 1 year of treatment, and after a 42-day (+ 14 day) post-extension-phase treatment washout Safety and efficacy parameters Primary efficacy endpointh least 2 h after a meal. Compliance will be assessed upon a review of the inventory of IB1001 sachets returned by patients. The original SARA scale is an eight-item clinical rating scale (range 0–40, where 0 is the best neurological sta- tus and 40 is the worst). It is a reliable and valid clinical scale with a high internal consistency that measures the severity of symptoms and ataxia and increases with dis- ease stage [16]. Study objectivesh A detailed description of the safety parameters is provided in Suppl. Material I. Accordingly, the mSARA was developed as a six-item clinical rating scale consisting of the original Gait, Speech Disturbance, Finger Chase, Nose-Finger test, Fast Alter- nating Hand Movement, and Heel-shin slide domains. It ranges from a score of 0 to 30, where 0 is the best neuro- logical status and 30 is the worst. The SARA scale will be the basis for the primary effi- cacy estimand. The mSARA scale will be considered as a supplementary estimand. Withdrawal from study medi- cation due to adverse events and the taking of prohib- ited medication will be considered as intercurrent events (ICEs) and a treatment policy strategy will be considered for these ICEs. Withdrawal or early termination from the study for unspecified reasons and lost to follow-up will not be considered as ICEs, and the subsequent data will be considered as “missing.” Should these events occur, a last observation carried forward approach will be adopted based on observations at visits 2, 3 for period 1 and visits 4, 5 for period 2. All statistical analyses will be detailed in a separate statistical analysis plan. For each of the primary and secondary endpoints, there will be evaluations within key subgroups: naïve versus non-naïve as determined at screening; age (pediatric versus adult at the time of enrollment); age/weight/dosing group; age of diagnosis: early-infantile (< 2 years), late-infantile (2 to < 6 years), juvenile (6 to < 15 years), adolescent/adult (≥ 15 years); disease severity based on SARA below/above the median SARA score at visit 1; gender (male versus female); region (US versus rest of world); patients taking miglustat ver- sus patients not taking miglustat; intra-patient variability between SARA score at visit 1 (baseline 1) versus visit 2 (baseline 2) (below/above median). These evaluations will be based on plotting treatment differences together with 90% confidence intervals within each subgroup. The model for analysis will be an analysis of covariance model with the difference in the SARA scores at the end of periods I and II as the dependent variable and SARA at baseline (visit 2) as the independent variable together with an indicator variable for sequence [17]. The esti- mated coefficient of the indicator for sequence will pro- vide the least squares estimate of the difference in the treatment means on division by 2. Study objectivesh The Parent Study and Extension Phase enable the investi- gation of both the symptomatic (12-week) and long-term (1-year) safety and efficacy of treatment with N-acetyl- L-leucine. The primary objective of the Parent Study is to evaluate the efficacy of N-acetyl-L-leucine versus pla- cebo based on the Scale for the Assessment and Rating of Ataxia (SARA) or modified SARA (mSARA). In the Extension Phase, the primary objective is to evaluate the effects of N-acetyl-L-leucine based on the modified (5-domain) Niemann-Pick disease type C Clinical Sever- ity Scale (NPC-CSS). The original unmodified SARA was selected as the primary endpoint based on advice from EU National Regulatory Authorities (including Germany, Portu- gal, Spain, and the Netherlands) and the UK Medi- cines and Healthcare product Regulatory Authority. In the USA, the primary endpoint is the modified SARA (mSARA). This modification was based on the explicit advice of the US Food and Drug Administration, which requested the instrument be modified to: For both study phases, the secondary objectives are: • Include the domains that are the most clinically meaningful towards understanding the functional aspects of ataxia/symptoms in NPC patients • To assess the clinical efficacy (symptomatic and long- term) of N-acetyl-L-Leucine on symptoms of ataxia, functioning, and quality of life for patients with NPC • Remove domains where there is no/little move- ment and which may impact the interpretation and power of the tool (i.e., improve the reliability and sensitivity of the instrument) • To evaluate the safety and tolerability of N-acetyl-L- leucine at 4 g/day in patients with NPC aged ≥ 4 years and older • Extension Phase only: characterize the pharmacoki- netics of N-acetyl-L-leucine in NPC patients Fields et al. Trials (2023) 24:361 Fields et al. Trials (2023) 24:361 Page 7 of 9 throughout the study. Sparse pharmacokinetic blood sampling will be conducted in the Parent Study (visit 1–visit 6). Blood samples for the quantification of N-acetyl-L-leucine in plasma will be obtained at visit 7 and visit 9. Urine samples will also be collected for measuring concentrations of N-acetyl-D-leucine at the time points designated on the schedule of events (Suppl. Tables 1 and 2). At visit 1, this urine sample serves as a key enrollment criterion testing for the use of the prohibited medication N-acetyl-DL-leucine. Vital signs, physical exams, height/weight, and elec- trocardiograms will also be collected at the time points designated on the schedule of events (Suppl. Tables 1 and 2). Study objectivesh Secondary endpoints will measure other symptoms and evaluate quality of life (Spinocerebellar Ataxia Functional Index (SCAFI) [18]; Modified Disability Rating Scale (mDRS) [19, 20]; Qual- ity of Life EQ-5D-5L for patients aged ≥ 18; EQ-5D-Y for patients aged < 18 years [21]; and Investigator, Caregiver, Patient Clinical Global Impression Scale (CGI)) [22]. Descriptive statistics will be provided for these measures at each visit, and select secondary endpoints will be eval- uated statistically based on a comparison of the period differences (period II–period I) between the two treat- ment sequences in an analysis of covariance (ANCOVA) model with terms for baseline and treatment sequence. i In a one-sided test at the p = 0.05 level, a total sam- ple size of 46 in a two-treatment, two-period placebo- controlled cross-over trial achieves approximately 80% power for treatment comparisons in relation to the SARA/mSARA total score assuming a true mean differ- ence of 1.0/0.85 (respectively) and a standard deviation for the total SARA/mSARA score between 7.5 and 8.5 and 6.375 and 7.225 (respectively) based on an analy- sis of covariance with the baseline SARA/mSARA score at the start of period I as the covariate. These results, based on extensive simulations, assume a correlation of 0.95 between each of the pairwise outcomes: baseline for period I, endpoint for period I, and endpoint for period II. The values for the standard deviation and the correlations used in the calculation are guided by the results of the IB1001-201 study. These levels of power are maintained when there is a positive or negative period effect of up to 0.5 and 0.425 units on the SARA/ mSARA scale, respectively. Availability of data and materials All investigators will be provided with full access to all the final data set in the study. Data sharing is not applicable to this article as no datasets were gener- ated or analyzed during the current studies (study protocols). Acknowledgements We thank each of the study sites and principal investigators participating in the IB1001-301 clinical trial for their participation. We thank the multinational Patient Organizations representing the NPC community for their essential involvement, including the following NPC Organizations: Australian Niemann- Pick Type C Foundation, Niemann-Pick Canada, Fundación Niemann-Pick de España (Spain), Associazione Italiana Niemann Pick (Italy), Volwassenen, Kinderen en Stofwisselingsziekten (VKS), International Niemann-Pick disease Alliance (Worldwide), National Niemann-Pick Disease Foundation (USA), Niemann-Pick Association of Fuenlabrada (Spain), Niemann-Pick Selbsthil- fegruppe (Germany), Niemann-Pick Suisse (Switzerland), Niemann-Pick UK (UK), the Firefly Foundation (USA), and the Ara Parseghian Medical Research Foundation (USA). Finally, we are very grateful to all the patients and their families who participated in these studies. Authors’ contributions MS conceived the initial studies. MS and TBE initiated the initial case series, and FMP, AG, GC, and MS initiated the non-clinical studies, together establish- ing the current study concept and rational. All authors made contributions to the conception and/or the trial design. TF and MS wrote and revised the study protocol. MF, CF, MP, TB, IB, KM, and RK made contributions to the study protocol and revised the final protocol for content. RK has the main responsibility for statistical analyses and made substantial contributions to the study protocol. TF contributed decisively to ethics approval to ethics and regulatory authorities. MF is the sponsor’s delegated person. MS is the spon- sor’s delegated clinician. TF is responsible for the implementation of the study. TF and MS wrote and revised the manuscript. RK developed the statistical concept and performed the sample size calculation. MF, CF, MP, IB, KM, TB, WE, TM, FMP, AG, and GC revised the final manuscript for content. All authors read and approved the final manuscript. Trial status At the time of manuscript submission, the protocols for IB1001-301 (US V1.2 07-Jan-2022; Global: V2.0, 08-Mar-2022; Swiss V2.1 14-Jul-2022) have been accepted/approved in each country where the study will be conducted, including the US Food and Drug Administrations, UK Medicines and Healthcare prod- ucts Regulatory Authority, German Federal Institute for Drugs and Medical Devices, Slovakia Štátny ústav pre kontrolu liečiv, Switzerland Swissmedic, Netherlands Central Committee on Research Involving Human Sub- jects, Czech Republic State Institute for Drug Control, and Australia Therapeutic Goods Administration as well as respective research ethics committees (REC)/ institutional review boards (IRB) (active approved pro- tocol version varies based on status of enrollment per site/per study). The first study participant was enrolled on 30 June 2022. Recruitment is ongoing and expected to complete December 2022. Authors’ information M. Strupp is joint chief editor of the Journal of Neurology and editor in chief of Frontiers of Neuro-otology and Section Editor of F1000. Funding This study was financially supported by IntraBio (https://intrabio.com). The authors (TF, IB, MF) are officers in IntraBio, CF is an employee of IntraBio, and the authors (MP, GC, MS, AG, RK, and FP) were paid for consultancy work for IntraBio. Authors, acting in their capacity as consultants for IntraBio, played roles in the study design, decision to publish, or preparation of the manu- script. IntraBio is not involved in the data collection and analysis. Abbreviations ANCOVA Analysis of covariance CGI Clinical Global Impression DSMB Data safety monitoring board EP Extension Phase GM2 GM2 gangliosidoses ICE Intercurrent events IRB Institutional review boards IRT Interactive Response Technology mDRS Modified Disability Rating Scale mSARA Modified Scale for the Assessment and Rating of Ataxia NPC Niemann-Pick type C NPC-CSS NPC clinical severity scale PI Principal investigator REC Research ethics committees SARA Scale for the Assessment and Rating of Ataxia SCAFI Spinocerebellar Ataxia Functional Index Discussion Given the lack of global symptomatic or disease-mod- ifying therapies for NPC and other lysosomal diseases, there is an urgent need for effective and well-tolerated drug treatments. This multi-center, multinational ran- domized double- blind placebo-controlled crossover phase III trial was designed through a collaboration between national regulatory agencies, leading clinical experts, patient organizations, and the industry spon- sor. This design addresses the unique ethical and prac- tical challenges of conducting clinical trials for these orphan, heterogeneous patient populations in order to better capture N-acetyl-L-leucine’s therapeutic effect and thus expedite the development and availability of this promising drug candidate [23]. The online version contains supplementary material available at https://doi. org/10.1186/s13063-023-07399-6. The online version contains supplementary material available at https://doi. org/10.1186/s13063-023-07399-6. Additional file 1: Supplementary Material I. Safety Parameters. Sup‑ plementary Material II. Data Collection. Additional file 1: Supplementary Material I. Safety Parameters. Sup‑ plementary Material II. Data Collection. Additional file 2: Supplementary Table 1. Parent Study schedule of enrolment, interventions, and assessments. Supplementary Table 2. Extension Phase schedule of enrolment, interventions, and assessments. Additional file 3. SPIRIT Checklist for Trials. Additional file 3. SPIRIT Checklist for Trials. Declarations Ethics approval and consent to participate Safety parameters Ad ( Adverse events (serious and non-serious), concomi- tant drug and non-drug therapies, safety laboratory blood samples (hemoglobin, erythrocytes, hematocrit, thrombocytes, leukocytes, sodium, potassium, urea, creatinine, serum bilirubin level, aspartate aminotrans- ferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, follicle-stimulating hormone for postmenopausal women only), and urine samples (leukocytes, nitrite, urobilinogen, protein, pH, occult blood (erythrocytes, leucocytes), specific gravity, ketones, bilirubin, glucose) will be collected routinely A description of data collection is provided in Suppl. Material II. Fields et al. Trials (2023) 24:361 Page 8 of 9 Author details 1 19. Iturriaga C, Pineda M, Fernández-Valero EM, Vanier MT, Coll MJ. Niemann- Pick C disease in Spain: clinical spectrum and development of a disability scale. J Neurol Sci. 2006;249:1–6. 1 IntraBio Ltd, Begbroke Science Park, Begroke Hill, Woodstock Road, Oxford OX5 1PF, UK. 2 Department of Neurology, Inselspital, University Hospital Bern, and University of Bern, Bern, Switzerland. 3 Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. 4 Niemann-Pick UK, Suite 2, Vermont House, Concord, Tyne and Wear, Washington NE37 2SQ, UK. 5 Primary Care Stratified Medicine (PRISM), Division of Primary Care, University of Nottingham, Nottingham, UK. 6 RK Statistics, Brook House, Mesne Lane, Bakewell DE45 1AL, UK. 7 Department of Pediatric Neurology, University Chil- dren’s Hospital (UKGM) and Medical Faculty, Justus Liebig University of Gies- sen, Giessen, Germany. 8 Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. 9 Department of Neurology and German Center for Vertigo and Balance Disorders, Ludwig Maximilians University, Munich, Germany. 20. Pineda M, Perez-Poyato MS, O’Callaghan M, et al. Clinical experience with miglustat therapy in pediatric patients with Niemann-Pick disease type C: a case series. Mol Genet Metab. 2010;99:358–66. 21. EuroQol Group. EQ-5D Instruments \| About EQ-5D. 2017. 2017. Accessed at: https://euroqol.org/eq-5d-instruments/eq-5d-5l-about. Accessed 15 Feb 2020. 22. Quinn TJ, Dawson J, Walters MR, Lees KR. Variability in modified Rankin scoring across a large cohort of international observers. Stroke. 2008;39:2975–9. 23. Kempf L, Goldsmith JC, Temple R. Challenges of developing and conduct- ing clinical trials in rare disorders. Am J Med Genet A. 2018;176:773–83. Received: 29 September 2022 Accepted: 22 May 2023 Competing interests IB2020/056096, and PCT/IB2021/050236 relating to treatment of lysosomal storage disorders, neurodegenerative diseases, and neurodegeneration with acetyl-leucine and its analogs. 18. Schmitz-Hübsch T, Giunti P, Stephenson DA, et al. SCA Functional Index: a useful compound performance measure for spinocerebellar ataxia. Neurology. 2008;71:486–92. Received: 29 September 2022 Accepted: 22 May 2023 Consent for publication Not applicable. 9. Martakis K, Claassen J, Gascon-Bayarri J, et al. N-acetyl-L-leucine improves symptoms and functioning in GM2 Gangliosidosis (Tay-Sachs & Sandhoff). medRxiv; 2021. p. 2021.09.24.21264020. Accessed at: https://www.medrxiv. org/content/10.1101/2021.09.24.21264020v1. Accessed 3 Aug 2022. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Competing interests 10. Churchill GC, Strupp M, Factor C, et al. Acetylation turns leucine into a drug by membrane transporter switching. Sci Rep. 2021;11:15812. MF is a co-founder, shareholder, and chairman of IntraBio. TF and IB are officers and shareholders of IntraBio. CF Is an employee and shareholder of IntraBio. FP is a cofounder, shareholder, and consultant to IntraBio and consultant to Actelion. MS is a shareholder to IntraBio and consultant for Abbott, Actelion, AurisMedical, Heel, IntraBio, Sensorion, and Vertify; he has received speaker’s honoraria from Abbott, Actelion, Auris Medical, Biogen, Eisai, Grünenthal, GSK, Henning Pharma, Interacoustics, Johnson & Johnson, MSD, Otometrics, Pierre- Fabre, TEVA, and UCB. AG and GC are cofounders, shareholder sand consult- ants to IntraBio. MP is a shareholder of IntraBio and has received consulting fees, honoraria, and research grants from Actelion Pharmaceuticals Ltd. and Biomarin. RK is a consultant to IntraBio. TBE received honoraria for lecturing from Actelion and Sanofi Genzyme and fees for the blinded rater services from IntraBio. y 11. Günther L, Beck R, Xiong G, et al. N-acetyl-L-leucine accelerates vestibular compensation after unilateral labyrinthectomy by action in the cerebel- lum and thalamus. PLoS one. 2015;10:e0120891. 12. Tighilet B, Leonard J, Bernard-Demanze L, Lacour M. Comparative analysis of pharmacological treatments with N-acetyl-DL-leucine (Tanganil) and its two isomers (N-acetyl-L-leucine and N-acetyl-D-leucine) on vestibular compensation: Behavioral investigation in the cat. Eur J Pharmacol. 2015;769:342–9. 13. Kaya E, Smith DA, Smith C, Boland B, Strupp M, Platt FM. Beneficial effects of acetyl-DL-leucine (ADLL) in a mouse model of Sandhoff disease. J Clin Med. 2020;9:1050. 14. Churchill GC, Strupp M, Galione A, Platt FM. Unexpected differences in the pharmacokinetics of N-acetyl-DL-leucine enantiomers after oral dos- ing and their clinical relevance. PLoS one. 2020;15:e0229585. IntraBio owns patents EP3359146, EP3416631, EP3482754, USPTO10905670, and USPTO11400067 (application 16/324,301) related to treatment of lyso- somal storage disorders and neurodegenerative diseases with acetyl-leucine and its analogs. 15. Chan A-W, Tetzlaff JM, Gøtzsche PC, et al. SPIRIT 2013 explanation and elaboration: guidance for protocols of clinical trials. BMJ. 2013;346:e7586. IntraBio has pending patent applications EP19174007.5, EP20215877.0, PCT/IB2017/054928, PCT/IB2017/054929, PCT/US2018/056420, PCT/ IB2018/054676, PCT/US2018/018420, PCT/IB2019/060525, PCT/ IB2019/051214, PCT/GB2017/051090, PCT/IB2020/051767, PCT/ 16. Schmitz-Hübsch T, du Montcel ST, Baliko L, et al. Scale for the assessment and rating of ataxia: development of a new clinical scale. Neurology. 2006;66:1717–20. 17. Mehrotra DV. A recommended analysis for 2 × 2 crossover trials with baseline measurements. Pharm Stat. 2014;13:376–87. 2. Vanier MT, Millat G. Niemann-Pick disease type C. Clin Genet. 2003;64:269–81. 4. Bremova T, Malinová V, Amraoui Y, et al. Acetyl-dl-leucine in Niemann-Pick type C: a case series. Neurology. 2015;85:1368–75. 1. Geberhiwot T, Moro A, Dardis A, et al. Consensus clinical manage- ment guidelines for Niemann-Pick disease type C. Orphanet J Rare Dis. 2018;13:50. 3. Patterson MC, Clayton P, Gissen P, et al. Recommendations for the detec- tion and diagnosis of Niemann-Pick disease type C: an update. Neurol Clin Pract. 2017;7:499–511. Ethics approval and consent to participate The study has been approved by the responsible central research ethics com- mittees/institutional review boards, including the Ethics Committee of Ludwig Fields et al. Trials (2023) 24:361 Page 9 of 9 Maximilian University of Munich (21–1269), National Institute of Child Diseases Bratislava Ethics Committee (EudraCT 2021–005356-10), Ethics Committee of General University Hospital in Prague (56/22 S-MEK), East Midlands – Derby Research Ethics Committee (1004498), Amsterdam UMC Locatie AMC Central Ethics Committee (NL79787.018.21), Mayo Clinic Institutional Review Board (22–001734), Emory University Institutional Review Board (STUDY00003227) Switzerland Business Administration System for Ethics Committees (2022–00638), and Victoria Human Research Ethics Committee (HREC/86167/ MH-2022). 5. Cortina-Borja M, Te Vruchte D, Mengel E, et al. Annual severity increment score as a tool for stratifying patients with Niemann-Pick disease type C and for recruitment to clinical trials. Orphanet J Rare Dis. 2018;13:143. 6. Kaya E, Smith DA, Smith C, et al. Acetyl-leucine slows disease progression in lysosomal storage disorders. Brain Commun. 2020;fcaa148, https://doi. org/10.1093/braincomms/fcaa148. 6. Kaya E, Smith DA, Smith C, et al. Acetyl-leucine slows disease progression in lysosomal storage disorders. Brain Commun. 2020;fcaa148, https://doi. org/10.1093/braincomms/fcaa148. 7. Bremova-Ertl T, Claassen J, Foltan T, et al. Efficacy and safety of N-acetyl-L- leucine in Niemann-Pick disease type C. J Neurol. 2022;269:1651–62. leucine in Niemann Pick disease type C. J Neurol. 2022;269:1651 62. 8. Fields T, Patterson M, Bremova-Ertl T, et al. A master protocol to investi- gate a novel therapy acetyl-L-leucine for three ultra-rare neurodegenera- tive diseases: Niemann-Pick type C, the GM2 gangliosidoses, and ataxia telangiectasia. Trials. 2021;22:84. Informed consent will be obtained for all study participants by the principal investigator/delegated investigator. References 1. Geberhiwot T, Moro A, Dardis A, et al. Consensus clinical manage- ment guidelines for Niemann-Pick disease type C. Orphanet J Rare Dis. 2018;13:50.
https://openalex.org/W4291792037	https://www.researchsquare.com/article/rs-1162391/latest.pdf	English	null	Glucose feeds the tricarboxylic acid cycle via excreted ethanol in fermenting yeast	Nature chemical biology	2,022	cc-by	11,124	Abstract 11 Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized 12 glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individ 13 cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show tha 14 similar uncoupling occurs in the yeast Saccharomyces cerevisiae. Even in fermenting yeast that are net 15 releasing ethanol, media 13C-ethanol rapid enters and is oxidized to acetaldehyde and acetyl-CoA. This is 16 evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-trac 17 reveals that ethanol is also a major source of both NADH and NADPH, and this role is augmented under 18 oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via 19 rapid reversible reactions is an ancient and conserved feature of eukaryotic metabolism. 20 Introduction 21 Fermentation occurs widely across Kingdoms, converting glucose into organic waste products1–4. In mamm 22 the main such product is lactate. Until recently, it was commonly assumed that the liver and kidney were 23 special in their capacity to clear circulating lactate, reconverting the waste (lactate) into fuel (glucose). New 24 evidence suggests, however, that most mammalian tissues take up circulating lactate and oxidize it via the 25 TCA cycle5. Indeed, it seems that most carbohydrate oxidation in mammals, rather than occurring by a tiss 26 taking up glucose and fully oxidizing it to carbon dioxide, instead involves carbon flowing through circulatin 27 lactate as a metabolic intermediate. Thus, glycolysis is uncoupled from the TCA cycle via cellular uptake 28 and/or excretion of lactate5. Biochemically, this occurs through the rapid reversible reactions linking 29 intracellular pyruvate, via lactate dehydrogenase and monocarboxylate transporters, to circulating lactate. 30 Baker's yeast (Saccharomyces cerevisiae) is a prototypical fermentative unicellular organism6. Its rapid 31 catabolism of glucose into ethanol + CO2 plays a central role in human society, contributing to such divers 32 fields as baking, beverages, and biofuels. S. cerevisiae is capable of growing aerobically on substrates 33 Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by 12 glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual 13 cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Keywords: Biochemically, this occurs through the rapid reversible reactions lin 29 Authors 3 Tianxia Xiao1, Artem Khan1,3, Yihui Shen2, Li Chen2,4, Joshua Rabinowitz1,2 () 1 Lewis Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Washington 5 Road, Princeton, NJ 08544, USA 6 1 Lewis Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Washington 5 Road, Princeton, NJ 08544, USA 6 2 Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, 08544, USA. 7 3 The David Rockefeller Graduate Program in Bioscience, The Rockefeller University, New York, NY, USA. 8 3 The David Rockefeller Graduate Program in Bioscience, The Rockefeller University, New York, NY, USA. 8 4 Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism & Integrative Biology, 9 Fudan University, Shanghai, 200433, China. 10 Abstract 11 Here we show that 14 similar uncoupling occurs in the yeast Saccharomyces cerevisiae. Even in fermenting yeast that are net 15 releasing ethanol, media 13C-ethanol rapid enters and is oxidized to acetaldehyde and acetyl-CoA. This is 16 evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-tracing 17 reveals that ethanol is also a major source of both NADH and NADPH, and this role is augmented under 18 oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via 19 rapid reversible reactions is an ancient and conserved feature of eukaryotic metabolism. 20 Keywords: Posted Date: December 28th, 2021 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 Nature Chemical Biology on August 15th, 2022. See the published version at https://doi.org/10.1038/s41589-022-01091-7. Glucose feeds the TCA cycle via environmental etha 1 fermenting yeast 2 Authors 3 Tianxia Xiao1, Artem Khan1,3, Yihui Shen2, Li Chen2,4, Joshua Rabinowitz1,2 () 4 1 Lewis Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University 5 Road, Princeton, NJ 08544, USA 6 2 Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, 08544, USA. 7 3 The David Rockefeller Graduate Program in Bioscience, The Rockefeller University, New York, NY 8 4 Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism & Integrativ 9 Fudan University, Shanghai, 200433, China. 10 Abstract 11 Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is 12 glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate 13 cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here w 14 similar uncoupling occurs in the yeast Saccharomyces cerevisiae. Even in fermenting yeast tha 15 releasing ethanol, media 13C-ethanol rapid enters and is oxidized to acetaldehyde and acetyl-C 16 evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl u 17 reveals that ethanol is also a major source of both NADH and NADPH, and this role is augment 18 oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived 19 rapid reversible reactions is an ancient and conserved feature of eukaryotic metabolism. 20 Introduction 21 Fermentation occurs widely across Kingdoms, converting glucose into organic waste products1 22 the main such product is lactate. Until recently, it was commonly assumed that the liver and kid 23 special in their capacity to clear circulating lactate, reconverting the waste (lactate) into fuel (glu 24 evidence suggests, however, that most mammalian tissues take up circulating lactate and oxidi 25 TCA cycle5. Indeed, it seems that most carbohydrate oxidation in mammals, rather than occurri 26 taking up glucose and fully oxidizing it to carbon dioxide, instead involves carbon flowing throug 27 lactate as a metabolic intermediate. Thus, glycolysis is uncoupled from the TCA cycle via cellul 28 and/or excretion of lactate5. Introduction 21 Fermentation occurs widely across Kingdoms, converting glucose into organic waste products1–4. In mammals, 22 the main such product is lactate. Until recently, it was commonly assumed that the liver and kidney were 23 special in their capacity to clear circulating lactate, reconverting the waste (lactate) into fuel (glucose). New 24 evidence suggests, however, that most mammalian tissues take up circulating lactate and oxidize it via the 25 TCA cycle5. Indeed, it seems that most carbohydrate oxidation in mammals, rather than occurring by a tissue 26 taking up glucose and fully oxidizing it to carbon dioxide, instead involves carbon flowing through circulating 27 lactate as a metabolic intermediate. Thus, glycolysis is uncoupled from the TCA cycle via cellular uptake 28 and/or excretion of lactate5. Biochemically, this occurs through the rapid reversible reactions linking 29 intracellular pyruvate, via lactate dehydrogenase and monocarboxylate transporters, to circulating lactate. 30 Fermentation occurs widely across Kingdoms, converting glucose into organic waste products1–4. In mammals, 22 the main such product is lactate. Until recently, it was commonly assumed that the liver and kidney were 23 special in their capacity to clear circulating lactate, reconverting the waste (lactate) into fuel (glucose). New 24 evidence suggests, however, that most mammalian tissues take up circulating lactate and oxidize it via the 25 TCA cycle5. Indeed, it seems that most carbohydrate oxidation in mammals, rather than occurring by a tissue 26 taking up glucose and fully oxidizing it to carbon dioxide, instead involves carbon flowing through circulating 27 lactate as a metabolic intermediate. Thus, glycolysis is uncoupled from the TCA cycle via cellular uptake 28 and/or excretion of lactate5. Biochemically, this occurs through the rapid reversible reactions linking 29 intracellular pyruvate, via lactate dehydrogenase and monocarboxylate transporters, to circulating lactate. 30 Baker's yeast (Saccharomyces cerevisiae) is a prototypical fermentative unicellular organism6. Its rapid 31 catabolism of glucose into ethanol + CO2 plays a central role in human society, contributing to such diverse 32 fields as baking, beverages, and biofuels. S. cerevisiae is capable of growing aerobically on substrates 33 Baker's yeast (Saccharomyces cerevisiae) is a prototypical fermentative unicellular organism6. Its rapid 31 catabolism of glucose into ethanol + CO2 plays a central role in human society, contributing to such diverse 32 fields as baking, beverages, and biofuels. S. cerevisiae is capable of growing aerobically on substrates 33 Baker's yeast (Saccharomyces cerevisiae) is a prototypical fermentative unicellular organism6. Fermenting yeast assimilate environmental ethanol 47 Ethanol can enter and exit cells via simple diffusion9. Thus, exogenous ethanol may enter yeast, even if they 48 are simultaneously excreting ethanol made internally from glucose. To differentiate two-carbon (2C) units from 49 environmental ethanol versus internal glucose catabolism, we grew yeasts in typical minimal media (yeast 50 nitrogen base, aerated, 30⁰C) with unlabeled glucose until mid-exponential phase. We then pelleted the cells 51 and resuspended them in yeast nitrogen base containing both glucose and ethanol, whose isotopic 52 composition is under experimental control. The glucose and ethanol concentrations in the resuspension media 53 were selected to approximate those naturally occurring during mid-exponential yeast growth in yeast nitrogen 54 base with glucose as the carbon source (recognizing that by mid-exponential phase yeast will have converted 55 a substantial amount of glucose into ethanol). Specifically, we provided glucose and ethanol at either 56 equimolar concentrations (42 mM each) or 1:1 mixture based on number of carbon atoms (28 mM glucose and 57 84 mM ethanol, “equicarbon”) (Figure 1A). 58 We used 13C NMR to measure rates of glucose uptake (fgluc_up) and conversion to ethanol via pyruvate 59 decarboxylase (fpdc) from the cultures with [U-13C]glucose and unlabeled ethanol (Figure 1B). The rates 60 measured by 13C NMR (fgluc_up and fpdc) are similar among strains of S. cerevisiae with different respiratory 61 capacity (FY4 and CEN.PK) and media substrate ratios (equimolar or equicarbon) (Supplementary figure 1A). 62 In parallel, net ethanol flux was measured by 1H NMR, revealing active fermentation (i.e. net ethanol excretion) 63 (Figure 1C). 64 We used 13C NMR to measure rates of glucose uptake (fgluc_up) and conversion to ethanol via pyruvate 59 decarboxylase (fpdc) from the cultures with [U-13C]glucose and unlabeled ethanol (Figure 1B). The rates 60 measured by 13C NMR (fgluc_up and fpdc) are similar among strains of S. cerevisiae with different respiratory 61 capacity (FY4 and CEN.PK) and media substrate ratios (equimolar or equicarbon) (Supplementary figure 1A). 62 In parallel, net ethanol flux was measured by 1H NMR, revealing active fermentation (i.e. net ethanol excretion) 63 (Figure 1C). 64 The yeast ethanol assimilation pathway involves oxidation of ethanol to acetate, which is converted into 65 cytosolic acetyl-CoA by acetyl-CoA synthetases10. Introduction 21 Its rapid 31 catabolism of glucose into ethanol + CO2 plays a central role in human society, contributing to such diverse 32 fields as baking, beverages, and biofuels. S. cerevisiae is capable of growing aerobically on substrates 33 including galactose, glycerol, and ethanol. But when provided with ample glucose, it will ferment even in the 34 presence of adequate oxygen7. When glucose runs out, after a delay to rewire metabolism, aerobic growth will 35 resume (the diauxic shift)8. As glucose fermentation in yeast parallels aerobic glycolysis in mammals, we were 36 curious whether it similarly involves reversible excretion and uptake of the "waste product" (ethanol), rather 37 than unidirectional waste elimination. We further wondered whether any such ethanol uptake during net 38 fermentative metabolism might contribute to yeast's metabolic robustness. 39 Understanding these questions is relevant both for basic science and for bioengineering (e.g. as ethanol 40 uptake might sometimes be disadvantageous). With these motivations in mind, we show that, even when 41 fermenting, yeast actively exchanges environmental ethanol for intracellular acetaldehyde at a sufficiently rapid 42 rate that intracellular acetyl units come substantially from environmental ethanol rather than directly from 43 glucose. Moreover, such exchange enables ethanol to be a major source of NADH and NADPH, especially 44 under oxidative stress conditions. 45 Fermenting yeast assimilate environmental ethanol 47 Su 91 are not known in mitochondria. Accordingly, we were curious whether environmental ethanol co 92 contribute to mitochondrial acetyl-CoA. To this end, using the same tracing strategy as above, w 93 whether environmental ethanol would label a metabolic product that is produced mitochondriall 94 CoA, N-acetylglutamate (NAG), an intermediate in the arginine biosynthesis pathway (Figure 3 95 either equimolar or equicarbon unlabeled glucose and [U-13C]ethanol, NAG was labeled both [M 96 while glutamate was only labeled [M+2] (Figure 3B, supplementary figure 3). To quantitate the 97 mitochondrial acetyl-CoA coming from environmental ethanol, we inferred mitochondrial acetyl- 98 from the observed mass isotope distribution of NAG and glutamate. The calculated [M+2] fracti 99 mitochondrial acetyl-CoA is around 60% (Figure 3C), similar to cytosolic acetyl-CoA. Thus, env 100 ethanol is a major source of both cytosolic and mitochondrial acetyl-CoA. 101 environmental ethanol (Figure 1E). This high [M+2] labeled fraction of acetyl-CoA is consistent across strains 69 and media compositions (Supplementary Figure 1B). 70 environmental ethanol (Figure 1E). This high [M+2] labeled fraction of acetyl-CoA is consistent across strains 69 and media compositions (Supplementary Figure 1B). 70 We built a 13C metabolic flux model to estimate the reversibility of the ethanol assimilation pathway (Figure 1F). 71 The model was constrained by the measured glucose uptake rate, the net ethanol excretion rate, PDC flux (the 72 flux representing gross glucose conversion to ethanol), and acetyl-CoA labeling from [U-13C]ethanol. The 73 model confirmed low PDH and high PDC flux, as typical for fermenting yeast (Figure 1G). Notably, it revealed a 74 fast exchange flux between ethanol and acetaldehyde, with ethanol a major source of acetaldehyde even 75 though net flux is in the direction of ethanol excretion (Figure 1G). This rapid exchange flux explains the 76 substantial acetyl-CoA labeling from environmental ethanol (Figure 1D). 77 We built a 13C metabolic flux model to estimate the reversibility of the ethanol assimilation pathway (Figure 1F). 71 The model was constrained by the measured glucose uptake rate, the net ethanol excretion rate, PDC flux (the 72 flux representing gross glucose conversion to ethanol), and acetyl-CoA labeling from [U-13C]ethanol. The 73 model confirmed low PDH and high PDC flux, as typical for fermenting yeast (Figure 1G). Environmental ethanol contributes to fatty acid synthesis 78 Acetyl-CoA exists as discrete cytosolic and mitochondrial pools. Fatty acid synthesis utilizes cytosolic acetyl- 79 CoA (Figure 2A), thus fatty acid labeling selectively represents cytosolic acetyl-CoA labeling. In yeast fed either 80 the equimolar or equicarbon mixture of unlabeled glucose and [U-13C]ethanol, a majority of the carbon in newly 81 synthesized fatty acids (i.e. those containing at least some label) was from environmental ethanol (Figure 2B, 82 Supplementary Figure 2). 83 To quantify fraction of cytosolic acetyl-CoA coming from environmental ethanol, we fit the observed fatty acid 84 mass isotope distribution to a binomial, reflecting the fact that each 2C unit incorporated into fat is selected 85 stochastically, with the assumption that only labeled fatty acids are newly synthesized. Such fitting revealed 86 around 60% environmental ethanol contribution (Figure 2C). Thus, rather than being derived mainly internally 87 by glycolysis and subsequent pyruvate catabolism, when environmental ethanol is present, cytosolic acetyl- 88 CoA in Baker’s yeast also comes from ethanol. 89 Fermenting yeast assimilate environmental ethanol 47 Notably, it revealed a 74 fast exchange flux between ethanol and acetaldehyde, with ethanol a major source of acetaldehyde even 75 though net flux is in the direction of ethanol excretion (Figure 1G). This rapid exchange flux explains the 76 substantial acetyl-CoA labeling from environmental ethanol (Figure 1D). 77 Fermenting yeast assimilate environmental ethanol 47 In attempt to directly trace potential ethanol uptake and 66 utilization, we directly measured cellular acetyl-CoA labeling distributions by LC-MS in yeasts grown with 67 unlabeled glucose and labeled ethanol (Figure 1D), finding substantial labeling (more than 50%) from 68 The yeast ethanol assimilation pathway involves oxidation of ethanol to acetate, which is converted into 65 cytosolic acetyl-CoA by acetyl-CoA synthetases10. In attempt to directly trace potential ethanol uptake and 66 utilization, we directly measured cellular acetyl-CoA labeling distributions by LC-MS in yeasts grown with 67 unlabeled glucose and labeled ethanol (Figure 1D), finding substantial labeling (more than 50%) from 68 environmental ethanol (Figure 1E). This high [M+2] labeled fraction of acetyl-CoA is consistent 69 and media compositions (Supplementary Figure 1B). 70 We built a 13C metabolic flux model to estimate the reversibility of the ethanol assimilation pathw 71 The model was constrained by the measured glucose uptake rate, the net ethanol excretion rat 72 flux representing gross glucose conversion to ethanol), and acetyl-CoA labeling from [U-13C]et 73 model confirmed low PDH and high PDC flux, as typical for fermenting yeast (Figure 1G). Nota 74 fast exchange flux between ethanol and acetaldehyde, with ethanol a major source of acetalde 75 though net flux is in the direction of ethanol excretion (Figure 1G). This rapid exchange flux exp 76 substantial acetyl-CoA labeling from environmental ethanol (Figure 1D). 77 Environmental ethanol contributes to fatty acid synthesis 78 Acetyl-CoA exists as discrete cytosolic and mitochondrial pools. Fatty acid synthesis utilizes cy 79 CoA (Figure 2A), thus fatty acid labeling selectively represents cytosolic acetyl-CoA labeling. In 80 the equimolar or equicarbon mixture of unlabeled glucose and [U-13C]ethanol, a majority of the 81 synthesized fatty acids (i.e. those containing at least some label) was from environmental ethan 82 Supplementary Figure 2). 83 To quantify fraction of cytosolic acetyl-CoA coming from environmental ethanol, we fit the obse 84 mass isotope distribution to a binomial, reflecting the fact that each 2C unit incorporated into fa 85 stochastically, with the assumption that only labeled fatty acids are newly synthesized. Such fit 86 around 60% environmental ethanol contribution (Figure 2C). Thus, rather than being derived m 87 by glycolysis and subsequent pyruvate catabolism, when environmental ethanol is present, cyt 88 CoA in Baker’s yeast also comes from ethanol. 89 Environmental ethanol supplies mitochondrial acetyl-CoA 90 Formation of cytosolic acetyl-CoA from acetate is catalyzed by acetyl-CoA synthetases11,12. Environmental ethanol supplies mitochondrial acetyl-CoA 90 Formation of cytosolic acetyl-CoA from acetate is catalyzed by acetyl-CoA synthetases11,12. Such synthetases 91 are not known in mitochondria. Accordingly, we were curious whether environmental ethanol could also 92 contribute to mitochondrial acetyl-CoA. To this end, using the same tracing strategy as above, we examined 93 whether environmental ethanol would label a metabolic product that is produced mitochondrially from acetyl- 94 CoA, N-acetylglutamate (NAG), an intermediate in the arginine biosynthesis pathway (Figure 3A). In yeast fed 95 either equimolar or equicarbon unlabeled glucose and [U-13C]ethanol, NAG was labeled both [M+2] and [M+4] 96 while glutamate was only labeled [M+2] (Figure 3B, supplementary figure 3). To quantitate the fraction of 97 mitochondrial acetyl-CoA coming from environmental ethanol, we inferred mitochondrial acetyl-CoA labeling 98 from the observed mass isotope distribution of NAG and glutamate. The calculated [M+2] fraction of 99 mitochondrial acetyl-CoA is around 60% (Figure 3C), similar to cytosolic acetyl-CoA. Thus, environmental 100 ethanol is a major source of both cytosolic and mitochondrial acetyl-CoA. 101 The enzyme succinyl-CoA acetate CoA transferase (Ach1) has been proposed as a potential means of 102 generating mitochondrial acetyl-CoA from acetate, but it is physiological role has remained unproven 13. Ach1 103 The enzyme succinyl-CoA acetate CoA transferase (Ach1) has been proposed as a potential means of 102 generating mitochondrial acetyl-CoA from acetate, but it is physiological role has remained unproven 13. Ach1 103 deletion completely abolished [M+4] NAG (Figure 3B, supplementary figure 3), with the inferred mitochondrial acetyl-CoA labeling zero in this deletion strain. Notably, the deletion strain nevertheless has similar whole cell [M+2] acetyl-CoA labeling from ethanol (supplementary figure 4), implying that only a small fraction of total cellular acetyl-CoA is mitochondrial, with Ach1 the key mitochondrial acetate assimilation enzyme. Mitochondrial acetyl-CoA contributes to the TCA cycle via citrate synthase (Figure 4A). Consistent with this, 108 from the 13C2-ethanol tracing, we observed [M+2] (iso)citrate, aconitate, α-ketoglutarate, and succinate (Figure 109 4B, supplementary figure 5). Fumarate, malate, and aspartate (whose carbon skeleton comes from 110 oxaloacetate) remained, however, largely unlabeled (Figure 4C, supplementary figure 5). The extensive 111 labeling of succinate with limited labeling of fumarate or oxaloacetate pinpoints succinate dehydrogenase 112 (complex II in the electron transport chain) as being functionally blocked during yeast fermentative growth 14. 113 Instead of being made by TCA turning, oxaloacetate and malate are generated by pyruvate carboxylase, using 114 pyruvate made from glucose. Environmental ethanol supplies mitochondrial acetyl-CoA 90 Nevertheless, acetate from environmental ethanol is assimilated into the TCA 115 cycle and drives conversion of these four-carbon TCA intermediates into citrate, α-ketoglutarate, and α- 116 ketoglutarate’s amino acid products. 117 Acetaldehyde oxidation feeds NADPH 166 Discussion 167 A fundamental metabolic question is, “Which pathways are coupled versus independent?” Here we 168 merely dilutes deuterium tracer signal from the actual hydride donors like [1-2H]glucose or [1-2H]acetaldehyde 140 (supplementary figure 6G,H,I, 9). Correcting for such exchange (and for extent of substrate labeling), we 141 observed that the oxPPP and Ald6 together account for most cytosolic NADPH, with the contribution of ethanol 142 via Ald6 roughly double that of glucose via the oxidative pentose phosphate pathway (Figure 5D). 143 Consistent with ethanol oxidation and oxPPP being alternative cytosolic NADPH production pathways, in 144 ∆ald6, oxPPP contribution to NADPH production (based on fatty acid labeling patterns) is nearly twice as high 145 as in wild type (Figure 5C, supplementary figure 7). In ∆zwf1, Ald6 contribution to NADPH production (based 146 on fatty acid labeling patterns) similarly doubles (Figure 5B, supplementary figure 8). Thus, ethanol is an 147 important source of both acetyl and hydride units in Baker’s yeast. 148 Acetaldehyde oxidation feeds NADPH Oxidation of ethanol to acetaldehyde generates NADH. Further oxidation of acetaldehyde into acetate via 119 aldehyde dehydrogenase generates NADPH. When the canonical main NAPDH-production pathway, the 120 oxidative pentose phosphate pathway, is deleted, the acetaldehyde dehydrogenase Ald6 is essential for yeast 121 growth15. We were curious about the extent to which acetaldehyde derived from environmental ethanol 122 contributes to NADH or NADPH. To explore this, we transferred fermenting yeast into glucose:ethanol as 123 above, with either the glucose or the ethanol deuterium labeled. Specifically, we compared NADH and NADPH 124 labeling from [1-2H]glucose (the labeled hydrogen is transferred to NADPH via G6PD, encoded by gene zwf1) 125 and [1,1-2H2]ethanol (the labeled hydrogen is transferred to NADH by ADH and to NADPH via Ald) (Figure 126 5A,D). Direct measurement of 2H-labeling in NADH and NADPH is technically challenging due to limited 127 abundance and stability, but nevertheless we observed clear isotope shifts upon exposure to the 2H-labeled 128 substrates, confirming contribution from glucose’s position 1 hydrogen to NADPH and from ethanol’s position 1 129 hydrogens to both NADH and NADPH (Supplementary Figure 5B,C,E,F). 130 To obtain more precise and compartment-specific information, we used fatty acid labeling to read out cytosolic NADPH labeling. Fatty acid synthesis incorporates 2 NADPH hydrides per acetyl group (Figure 5A)16. Strikingly, we observed greater deuterium labeling of fatty acids from [1,1-2H2]ethanol than from [1-2H]glucose (Supplementary figure 7,8). This reflects a major contribution of ethanol-derived acetaldehyde to cytosolic NADPH via Ald6 (Figure 5B). To obtain more precise and compartment-specific information, we used fatty acid labeling to read out cytosolic 131 NADPH labeling. Fatty acid synthesis incorporates 2 NADPH hydrides per acetyl group (Figure 5A)16. 132 Strikingly, we observed greater deuterium labeling of fatty acids from [1,1-2H2]ethanol than from [1-2H]glucose 133 (Supplementary figure 7,8). This reflects a major contribution of ethanol-derived acetaldehyde to cytosolic 134 NADPH via Ald6 (Figure 5B). 135 To convert the observed labeling into quantitative contributions to NADPH, we need to account for deuterium 136 loss from NADPH via hydrogen-deuterium exchange with water mediated by flavin enzymes16,17. Experiments 137 culturing cells in D2O revealed that about half of cytosolic NADPH hydrogen nuclei come from water via 138 hydrogen-deuterium exchange. Such exchange does not account for any NADPH’s high energy electrons, but 139 merely dilutes deuterium tracer signal from the actual hydride donors like [1-2H]glucose or [1-2H]ace 140 (supplementary figure 6G,H,I, 9). Acetaldehyde oxidation feeds NADPH Correcting for such exchange (and for extent of substrate labeling 141 observed that the oxPPP and Ald6 together account for most cytosolic NADPH, with the contribution 142 via Ald6 roughly double that of glucose via the oxidative pentose phosphate pathway (Figure 5D). 143 Consistent with ethanol oxidation and oxPPP being alternative cytosolic NADPH production pathway 144 ∆ald6, oxPPP contribution to NADPH production (based on fatty acid labeling patterns) is nearly twic 145 as in wild type (Figure 5C, supplementary figure 7). In ∆zwf1, Ald6 contribution to NADPH productio 146 on fatty acid labeling patterns) similarly doubles (Figure 5B, supplementary figure 8). Thus, ethanol 147 important source of both acetyl and hydride units in Baker’s yeast. 148 Ethanol becomes a yet greater NADH and NADPH source in response to peroxide stress 149 We were curious whether cells might shift between glucose or ethanol as an NAD(P)H source in res 150 environmental conditions. To explore this possibility, we grew yeast in glucose:ethanol with one sub 151 labeled as above, spiked in H2O2 to a final concentration of 20 mM, and rapidly sampled metabolites 152 labeling18,19 (Figure 6A). Upon adding H2O2, the NADH concentration and NADH/NAD+ ratio fell mar 153 (Figure 6B, C). Such a drop was expected, given that oxidative stress is known oxidize the GADPH’ 154 site cysteine and thereby block glycolytic flux and NADH production. Consistent with GAPDH being 155 addition to an increase of fructose-1,6-bisphosphate (FBP) (Supplementary Figure 10), we observed 156 NADH labeling from ethanol, which became the dominant NADH hydride source (Figure 6D). Thus, 157 catabolism is a crucial source for NADH when glycolysis is blocked by oxidative stress. 158 A classical rationale for glycolytic blockade by oxidative stress is to divert flux into the oxidative pent 159 phosphate pathway to help maintain NADPH homeostasis. Notably, the same concentration of hydr 160 peroxide that markedly suppressed NADH had no overt effect on NADPH pool size or the NADPH: N 161 (Figure 6E, F). However, rather than increasing the oxidative pentose phosphate pathway contributi 162 NADPH as measured by [1-2H]glucose, this contribution was decreased, with ethanol’s contribution 163 markedly increased (Figure 6G). Thus, in contrast to the common assumption that the main NADPH 164 route during oxidative stress is the oxidative pentose phosphate pathway, we observe an acute shift 165 greater NADPH contribution from ethanol oxidation under H2O2 stress15,20. Ethanol becomes a yet greater NADH and NADPH source in response to peroxide stress 149 174 While the net release of ethanol by fermenting yeast has been long appreciated, we are unaware of prior 175 demonstration that fermenting yeast simultaneously engage in extensive ethanol uptake. Through experiments 176 with 13C-ethanol, we show that, under typical mid-exponential fermentative growth conditions, environmental 177 ethanol, rather than mitochondrial pyruvate catabolism, supplies a majority of both cytosolic and mitochondrial 178 acetyl-CoA. In mitochondria, we prove that the ethanol assimilation pathway involves the CoA-transferase 179 Ach123–25. The assimilated ethanol was originally produced from glucose. But the pathway from glycolysis to 180 the TCA cycle (and other acetyl-CoA products like fatty acids and amino acids) flows through pyruvate 181 decarboxylase and environmental ethanol. 182 This metabolic design simplifies regulation of the fate of pyruvate, circumventing the challenge of partitioning 183 pyruvate optimally between ethanol and acetyl-CoA. Ethanol excretion is the default, and required two-carbon 184 units are re-assimilated as needed from environmental ethanol. This design further assures adequate 185 availability of both carbon and high-energy electrons even if glycolysis is impaired. Without any remodeling of 186 their internal metabolic machinery, yeast have access to both carbon and high-energy electrons from 187 environmental ethanol. Such access is particularly evident during acute redox stress, which impairs glycolysis 188 through inhibitory oxidation of the central glycolytic enzyme GAPDH. Under this circumstance, ethanol 189 becomes the predominant source of both NADH and NADPH, the latter being critical for survival of oxidative 190 stressors. Thus, uncoupling of glycolysis from the TCA cycle via ethanol provides yeast with metabolic 191 flexibility, decreases regulatory complexity, and enhances robustness. 192 Ethanol becomes a yet greater NADH and NADPH source in response to peroxide stress 149 We were curious whether cells might shift between glucose or ethanol as an NAD(P)H source in response to 150 environmental conditions. To explore this possibility, we grew yeast in glucose:ethanol with one substrate 2H- 151 labeled as above, spiked in H2O2 to a final concentration of 20 mM, and rapidly sampled metabolites and their 152 labeling18,19 (Figure 6A). Upon adding H2O2, the NADH concentration and NADH/NAD+ ratio fell markedly 153 (Figure 6B, C). Such a drop was expected, given that oxidative stress is known oxidize the GADPH’s active 154 site cysteine and thereby block glycolytic flux and NADH production. Consistent with GAPDH being shut off, in 155 addition to an increase of fructose-1,6-bisphosphate (FBP) (Supplementary Figure 10), we observed increased 156 NADH labeling from ethanol, which became the dominant NADH hydride source (Figure 6D). Thus, ethanol 157 catabolism is a crucial source for NADH when glycolysis is blocked by oxidative stress. 158 A classical rationale for glycolytic blockade by oxidative stress is to divert flux into the oxidative pentose 159 phosphate pathway to help maintain NADPH homeostasis. Notably, the same concentration of hydrogen 160 peroxide that markedly suppressed NADH had no overt effect on NADPH pool size or the NADPH: NADP+ ratio 161 (Figure 6E, F). However, rather than increasing the oxidative pentose phosphate pathway contribution to 162 NADPH as measured by [1-2H]glucose, this contribution was decreased, with ethanol’s contribution to NADPH 163 markedly increased (Figure 6G). Thus, in contrast to the common assumption that the main NADPH production 164 route during oxidative stress is the oxidative pentose phosphate pathway, we observe an acute shift towards a 165 greater NADPH contribution from ethanol oxidation under H2O2 stress15,20. 166 A fundamental metabolic question is, “Which pathways are coupled versus independent?” Here we present 168 evidence that uncoupling of glycolysis from the TCA cycle is an evolutionarily conserved design principle in 169 eukaryotic metabolism. Specifically, we show that fermenting Baker’s yeast simultaneously release and uptake 170 ethanol, much as many mammalian cells simultaneously produce and consume circulating lactate. Both lactate 171 and ethanol are redox-balanced with glucose21. Thus, their release allows glycolysis to run without need for the 172 TCA cycle or oxidative phosphorylation. Release of these electron-rich products anticorrelates with internal 173 NADH consumption by the electron transport chain22. Methods 193 Materials. Yeast were grown in yeast nitrogen base without amino acids (Sigma Aldrich, Y0626) with carbon 194 source added separately. BD DifcoTM YPD Broth (BD 242820) was used as the media for reviving frozen cells 195 or growing cells to be frozen. Glycerol (Sigma Aldrich, G5516) was added at 1:1 volume ratio to YPD yeast 196 cultures in cryovials (Nalgene®, 5000-1020, or Corning, 430289). Glucose (Sigma Aldrich, D9434), [U- 197 13C6]glucose (Cambridge Isotope Laboratories, CLM-1396), [13C2]ethanol (Sigma Aldrich, 427039), [1,1- 198 2H2]ethanol (Sigma Aldrich, 347434), ethanol (Decon Labs Inc., DSP-MD.43), and 1-2H-glucose (Omicron 199 Biochemistry, GLC-032) are used as carbon sources. Tap water filtered by Milli-Q® Reference Water 200 Purification System (Millipore Sigma, C79625) and D2O (Cambridge Isotope Laboratories, DLM-4) were used 201 as the water source for yeast cultures. MilliporeSigma™ Stericup™ Quick Release-HV Sterile Vacuum Bottle 202 Top Filtration Systems with 0.22 um PES filters were used to sterilize all media for yeast cultures. 0.45 μm 203 nylon filters (GVS North America, 123776) were used to filtering yeasts for metabolite extraction. 3- 204 (Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TMSP) (Sigma Aldrich, 269913) was used as 1H NMR 205 internal standard and sodium formate-13C (Sigma Aldrich, 279412) was used 13C NMR internal standard. 206 Experimental cultures were grown in 14 mL polypropylene round bottom tubes (FALCON, 352059). HPLC vials 207 (Thermo Scientific, 200-046, 501-313) were used for water-soluble metabolite samples and glass vials (Fisher 208 Scientific, 03-338A) were used for lipid hydrolysis and glass HPLC vials (Chemglass, CV-1152-1232, CV-3845- 209 13009) were used for fatty acid and lipid samples. 210 Yeast strains. S. cerevisiae strain FY4, derived from S288c, was taken from in-house frozen stocks. S. 211 cerevisiae strain CEN.PK was obtained from José Avalos. S. cerevisiae prototrophic mutant strains were 212 obtained from David Botstein, which were also derived from S288c through a diploid intermediate strain26,27. 213 Yeast strains. S. cerevisiae strain FY4, derived from S288c, was taken from in-house frozen stocks. S. 211 cerevisiae strain CEN.PK was obtained from José Avalos. S. cerevisiae prototrophic mutant strains were 212 obtained from David Botstein, which were also derived from S288c through a diploid intermediate strain26,27. 213 Yeast batch cell culture growth. S. cerevisiae colony was inoculated into an overnight culture, containing 6.7 214 g/L of Yeast Nitrogen Base (YNB) without amino acids and 20 g/L of glucose. Methods 193 After 24 h of growth at 30 °C, the 215 overnight culture was diluted 1:100 into appropriate experimental media, containing 6.7 g/L YNB without amino 216 acids and a carbon source/isotope tracer as specified for each experiment. For the prototrophic ∆zwf1 strain, 217 20 mg/L methionine was added to the media to accelerate growth. 218 1H qNMR measurement. Yeast were first grown to mid-exponential phase (OD=0.5), split into two equal 219 portions, and centrifuged to pellets. In the equimolar case, each pellet was redissolved at equal concentration 220 into media containing either (i) YNB + 42 mM unlabeled glucose and 42 mM [U-13C]ethanol or (ii) YNB+ 42 mM 221 [U-13C]glucose and 42 mM unlabeled ethanol. In the equicarbon case, each portion was switched into the 222 media containing either (i) YNB +28 mM unlabeled glucose and 84 mM [U-12C]ethanol or (ii) YNB+ 28 mM [U- 223 13C]glucose and 84 mM unlabeled ethanol. OD was taken before and after the incubation period for flux 224 calculation. Before switching and 1 hour after the media switch, 0.5 mL of the culture media was collected by 225 sampling and centrifugation. 450 µL of the supernatant was mixed with 50 µL of the 50 mM deuterated TMSP 226 standard solution in D2O and loaded onto NMR machine. 1H NMR δ: 0 ppm (s, 9H) for the TMS standard, 3.2 227 ppm (dd, 1H) for β-glucose, and 1.17 ppm (t, 3H) for ethanol. Water suppression is achieved by O1P=4.66 228 ppm, spw1=0.002. To achieve quantitative NMR, D1 is set to 5 s and 90˚ pulse (p1)to 11.69 μs. 229 13C qNMR measurement. The sample collection procedure and preparation were identical to the 1H NMR excretion profile, except that 13C sodium formate (0.1 M) was utilized as a standard instead of deuterated TMS. 13C NMR δ: 168 ppm (s, 1C) for the sodium formate standard, 161 ppm (d, 1C) for glucose, and 57 ppm (d, 1C) for ethanol. Samples of media that the cells are switched into are measured as baseline. We observed that unlabeled ethanol present in the media also gives a detectable 13C NMR signal (s, 1C at 57 ppm) due to natural 13C abundance. This natural abundance signal was not corrected for because it is < 5% signal of [U- 13C]ethanol. Methods 193 Relaxation time (D1) is set to a generous length of 40 s to ensure all spins reset properly and thus achieve quantification of 13C nuclei. Metabolite extraction. Metabolite extraction was performed according as previously28,29. 3 mL of the cell culture 238 at mid-exponential stage of growth (OD ~ 0.6) was vacuum filtered (0.45 µm, Millipore). The filter was 239 immersed in 1.6 mL of a cold (-20°C) extraction buffer (40% methanol: 40% acetonitrile: 20% water: 0.5% 240 formic acid by volume). Cells were washed out of the filter, and 88 µL of NH4(HCO3) (15 % w/v, Sigma-Aldrich) 241 solution per 1 mL extraction buffer was added to neutralize formic acid. The resulting solutions were 242 transferred to Eppendorf tubes and centrifuged for 10 min at 16,000 rotations per minute (rpm) in a cold room 243 (4 °C). The supernatant was collected and stored at -80 °C before loading onto a liquid chromatography-mass 244 spectrometer (LC-MS). 245 Water-soluble metabolite LC-MS Analysis. Prepared samples were loaded onto a quadrupole-orbitrap mass 246 spectrometer (Q Exactive Plus, Thermo Fisher Scientific) coupled to hydrophilic interaction chromatography 247 (HILIC) for analysis. Measurements of acetyl-CoA labeling were achieved by reversed-phase ion-pairing liquid 248 chromotography30 coupled to a standalone orbitrap (Exactive, Thermo Fischer Scientific). 249 Fatty acid extraction and LC-MS analysis. Fatty extraction was performed according to Zhang et al16. Cells 250 were pelleted in a 1.5 mL Eppendorf tube, and 1 mL of 0.3 M KOH in 90:10 methanol: water solution was 251 added. The resulting mixture with cells was transferred to a 4 mL glass vial. Saponification was performed by 252 placing the samples into a water bath at 80°C for one hour. Once the samples cooled down, 100 µL formic acid 253 (0.5 %) was added, followed by 1 mL hexane to extract the fatty acids. The extract was transferred into a glass 254 HPLC vial and dried under nitrogen flow. Afterward, it was diluted in 0.1 mL 50:50 acetonitrile: methanol 255 solution. The 0.1 mL solution was then added to a clean glass insert and placed inside an HPLC vial and the 256 cap sealed. All the samples were loaded onto the Exactive LC-MS employing a reversed-phase LC column 257 (C8) coupled with negative-mode ESI high-resolution MS. Methods 193 NADPH or acetyl-CoA labeled fractions were 258 inferred from observed fatty acid labeling patterns using a binomial model With unlabeled fat, which in part 259 reflects environmental contamination, omitted from the calculation. 260 Isotope tracing experiments. Cells were grown in YNB + 10g/L glucose up to OD=0.5. Then, the cells were 261 quickly centrifuged, the supernatant discarded, and switched to equimolar or equicarbon media with either 262 glucose or ethanol 13C- or 2H-labeled, or with both carbon sources unlabeled in 50% 2H2O. The cells were 263 allowed to grow in the labeled media for 1 hour before harvesting. A potential concern these experiments is 264 dilution of labeled ethanol tracer by unlabeled ethanol made from glucose. As no more than 2 mM unlabeled 265 ethanol is excreted into the media during the one-hour incubation, environmental ethanol remains more than 266 95% fully labeled during the experiments. OD was taken before and after the incubation period to assure the 267 cells were in exponential growth phase during the experiment. 268 Substrate correction for deuterium tracing with [1,1-2H2]ethanol. PDC flux dilutes the deuterium labeled fraction 269 of acetaldehyde in S. cerevisiae when fed with [1,1-2H2]ethanol. Contribution to NADPH by Ald6 is represented 270 by the labeled NADPH active hydride fraction from [1,1-2H2]ethanol divided by acetaldehyde labeling. The 271 acetaldehyde labeling is approximated by the fraction of acetyl-CoA labeled by ethanol in 13C isotope tracing 272 experiments. Due to the deuterium kinetic isotope effect, and PDH flux being small, if anything this will 273 overestimate 2H-labeling of acetaldehyde, and thus tend to underestimate Ald6 contribution to NADPH. Was 274 Water exchange activity calculation with 2H2O experiments. The fraction of NADPH in exchange with water is 75 represented by the fraction of NADPH active hydride labeled from 2H2O divided by the 2H2O enrichment 76 percentage (50%). 77 H2O2 stress experiments. The procedure was adapted from Christodoulou et al. with some modifications19. 278 Cells were incubated in the experimental culture media, containing either [1,1-2H2]ethanol or [1-2H]glucose 279 tracer, during the exponential growth stage. Metabolite extraction was performed with 1 mL of the culture for a 280 baseline measurement (“-1 s” time point). Afterward, 6 uL of 30 % hydrogen peroxide (Sigma Aldrich) was 281 added to the 3 mL culture to reach a final concentration of 20 mM H2O2 and samples were extracted after 15, 282 30, and 60 s. Methods 193 283 baseline measurement (“-1 s” time point). Afterward, 6 uL of 30 % hydrogen peroxide (Sigma Aldrich) was 281 added to the 3 mL culture to reach a final concentration of 20 mM H2O2 and samples were extracted after 15, 282 30, and 60 s. 283 Mitochondrial acetyl-CoA labeling calculation from [U-13C2] ethanol tracer. Acetyl-glutamate and α- 284 ketoglutarate labeling patterns were measured from the LC-MS analysis of cultures grown with [U-13C2]ethanol. 285 The matrix equation was set up as below, and solved by the least square method for the acetyl-CoA labeling. 286 [ M + 0 0 0 M + 1 M + 0 0 M + 2 M + 1 M + 0 0 M + 2 M + 1 0 0 M + 2] ( M + 0 M + 1 M + 2 ) = ( M + 0 M + 1 M + 2 M + 3 M + 4) 287 a-ketoglutarate acetyl-CoA acetyl-glutamate 288 a-ketoglutarate acetyl-CoA acetyl-glutamate 88 288 Data analysis and visualization. El-MAVEN v11.1 (Elucidata) software was used to process the LC-MS data31. 289 Metabolite identities were verified by both mass/charge (m/z) ratio and retention time match to authenticated 290 standards. For 2H- and 13C-isotope-labeled data analysis, natural isotope abundance correction was made 291 according to the binomial distribution model32. 13C-MFA was computed with INCA33. NADPH active hydride 292 labeling and acetyl-CoA labeling from fatty acids were calculated as previously16. MestReNova x64 software 293 was used for the NMR data processing. Statistical analysis are performed with GraphPad Prism, including two- 294 tailed t-test (with false discovery rate correction by the two-stage step up Benjamini, Krieger, and Yekutieli 295 method to confirm that any reported significant results involving statistical comparisons of multiple isotopic 296 forms of the same metabolite remain significant after correction for the multiple comparisons); ordinary one- 297 way ANOVA (when row matching is statistically significant, RM one-way ANOVA instead); and linear trend 298 (between column mean and by left-to-right column order, with p-value from F test). 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Services, results and/or products in support of the research project we 380 generated by the Rutgers Cancer Institute of New Jersey Metabolomics Shared Resource, supported, in part 381 with funding from NCI-CCSG P30CA072720-5923.This work was supported by DOE grant DE SC0018420 382 (CABBI). 383 Author contributions 384 T.X. and J.D.R. designed the study. T.X. performed NMR studies. T.X. and A.K. performed isotopic tracing 385 studies. T.X., Y.H., L.C. (Taylor & Francis Ltd, 2004). doi:10.1201/9780203503867 iScience 19, 1133–1144 (2019). 342 20. Minard, K. I. & Mcalister-henn, L. Sources of NADPH in Yeast Vary with Carbon Source . J. Biol. Chem. 343 280, 39890–39896 (2005). 344 21. van Hoek, M. J. A. & Merks, R. M. H. Redox balance is key to explaining full vs. partial switching to low- yield metabolism. BMC Syst. Biol. 6, 22 (2012). 22. Vemuri, G. N., Eiteman, M. A., McEwen, J. E., Olsson, L. & Nielsen, J. 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T.X. and A.K. analyzed the data. 386 J.D.R. and T.X. wrote the manuscript with the help from all authors. 387 Competing interests 388 None. 389 Materials & Correspondence 390 Correspondence and requests for materials should be addressed to J.D.R. 391 392 Additional Information 393 Supplementary Information is available for this paper. 394 34. MATLAB. 9.7.0.1190202 (R2019b). (The MathWorks Inc., 2018). 375 Acknowledgements 376 We thank S. Silverman and D. Botstein for access to the yeast knockout collection, T.TeSlaa, R. Ryseck, and 377 A.J. Cowan for feedback on the manuscript, and I. Pelczer and J. Eng for assistance with NMR and mass 378 spectrometry, C.M. Call for help with preliminary experiments, and M. Seyedsayamdost and members of the 379 Rabinowitz lab for helpful discussions. Services, results and/or products in support of the research project were 380 generated by the Rutgers Cancer Institute of New Jersey Metabolomics Shared Resource, supported, in part, 381 with funding from NCI-CCSG P30CA072720-5923.This work was supported by DOE grant DE SC0018420 382 (CABBI). 383 Author contributions 384 T.X. and J.D.R. designed the study. T.X. performed NMR studies. T.X. and A.K. performed isotopic tracing 385 studies. T.X., Y.H., L.C. and A.K. developed the computational models. T.X. and A.K. analyzed the data. 386 J.D.R. and T.X. wrote the manuscript with the help from all authors. 387 Competing interests 388 T.X. and J.D.R. designed the study. T.X. performed NMR studies. T.X. and A.K. performed isotopic tracing 385 studies. T.X., Y.H., L.C. and A.K. developed the computational models. T.X. and A.K. analyzed the data. 386 J.D.R. and T.X. wrote the manuscript with the help from all authors. 387 Figure 1. Environmental ethanol is a major source of acetyl units in fermenting S. cerevisiae. A. Schematic of the experimental design, in which fermenting yeast are transferred into fresh media containing both glucose and ethanol, whose isotopic composition is under experimenter control (Created with Biorender). B. Approach to measuring uptake/excretion fluxes. S. cerevisiae is switched into 13C-glucose + unlabeled ethanol media, and net glucose consumption (fgluc_in) and total production of ethanol from glucose (f13C_etoh_out) are measured by 13C-NMR. The net ethanol flux (of combined labeled and unlabeled Figure 1. Environmental ethanol is a major source of acetyl units in fermenting S. cerevisiae. A. 15, 1–8 (2015). Schematic of the experimental design, in which fermenting yeast are transferred into fresh media t i i b th l d th l h i t i iti i d i t t l Figure 1. Environmental ethanol is a major source of acetyl units in fermenting S. cerevisiae. A. Schematic of the experimental design, in which fermenting yeast are transferred into fresh media containing both glucose and ethanol, whose isotopic composition is under experimenter control (Created with Biorender). ( ) B. Approach to measuring uptake/excretion fluxes. S. cerevisiae is switched into 13C-glucose + unlabeled ethanol media, and net glucose consumption (fgluc_in) and total production of ethanol from glucose (f13C_etoh_out) are measured by 13C-NMR. The net ethanol flux (of combined labeled and unlabeled ethanol) is measured by 1H-NMR in a parallel experiment based on concentration change of the total ethanol pool (fetoh_out_net). B. Approach to measuring uptake/excretion fluxes. S. cerevisiae is switched into 13C-glucose + unlabeled ethanol media, and net glucose consumption (fgluc_in) and total production of ethanol from glucose (f13C_etoh_out) are measured by 13C-NMR. The net ethanol flux (of combined labeled and unlabeled ethanol) is measured by 1H-NMR in a parallel experiment based on concentration change of the total ethanol pool (fetoh_out_net). C. Uptake/excretion fluxes for S. cerevisiae FY4 in minimal media (YNB) with equimolar [U-13C]glucose: unlabeled ethanol (mean, SE, n=3 (biological replicates)). ( ( g p )) D. When S. cerevisiae is switched into unlabeled glucose + 13C-ethanol media, ethanol oxidation results in [M+2] acetyl-CoA, which is detectable by LC-MS. E. The whole-cell [M+2] acetyl-CoA fraction (mixture of cytosolic and mitochondrial origins) for S. cerevisiae FY4 in minimal media (YNB) with equimolar glucose: [13C2]ethanol (mean, n=3 (biological replicates)). p )) . Metabolic fluxes relevant to pyruvate and ethanol metabolism in yeast. G. 13C-metabolic flux analysis based on model in F and data in C and E. PDC flux is substantial while PDH flux is below detection, signifying acetaldehyde is the direct contributor for acetyl-CoA. High exchange flux at ADH implicates environmental ethanol as a major contributor to cellular acetaldehyde and acetyl- CoA (mean, UB/LB, n=3 (biological replicates)). Figure 2. Environmental ethanol feeds fatty acid synthesis in fermenting S. cerevisiae. A. When fed with labeled ethanol, the resulting 13C-labeled acetyl-CoA is incorporated into newly synthesized fatty acids. 15, 1–8 (2015). As both labeled and unlabeled cytosolic acetyl-CoA are randomly incorporated into growing fatty acid chains, the resulting fatty acid mass isotope distribution follows a binomial probability distribution. B Fatty acid (palmitate) labeling pattern from equimolar glucose: 13C-ethanol co-feeding experiment as in Figure 2. Environmental ethanol feeds fatty acid synthesis in fermenting S. cerevisiae. Figure 2. Environmental ethanol feeds fatty acid synthesis in fermenting S. cerevisiae. A. When fed with labeled ethanol, the resulting 13C-labeled acetyl-CoA is incorporated into newly synthesized fatty acids. As both labeled and unlabeled cytosolic acetyl-CoA are randomly incorporated into growing fatty acid chains, the resulting fatty acid mass isotope distribution follows a binomial probability distribution. B. Fatty acid (palmitate) labeling pattern from equimolar glucose: 13C-ethanol co-feeding experiment as in Figure 1D (mean, SE, n=3 (biological replicates)). Briefly, newly synthesized fatty acids are getting labeled by [M+2] acetyl-CoA, which is a result of uptaking 13C-ethanol from growth media by S. cerevisiae. C. Cytosolic acetyl-CoA labeling fitted from fatty acid labeling from equimolar glucose: 13C-ethanol co- feeding experiment as in Figure 1D (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0)). Figure 2. Environmental ethanol feeds fatty acid synthesis in fermenting S. Figure 2. Environmental ethanol feeds fatty acid synthesis in fermenting S. cerevisiae. A. When fed with labeled ethanol, the resulting 13C-labeled acetyl-CoA is incorporated into newly synthesized fatty acids. As both labeled and unlabeled cytosolic acetyl-CoA are randomly incorporated into growing fatty acid chains, the resulting fatty acid mass isotope distribution follows a binomial probability distribution. B. Fatty acid (palmitate) labeling pattern from equimolar glucose: 13C-ethanol co-feeding experiment as in Figure 1D (mean, SE, n=3 (biological replicates)). Briefly, newly synthesized fatty acids are getting labeled by [M+2] acetyl-CoA, which is a result of uptaking 13C-ethanol from growth media by S. cerevisiae. C. Cytosolic acetyl-CoA labeling fitted from fatty acid labeling from equimolar glucose: 13C-ethanol co- feeding experiment as in Figure 1D (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0)). A. When fed with labeled ethanol, the resulting 13C-labeled acetyl-CoA is incorporated into newly synthesized fatty acids. As both labeled and unlabeled cytosolic acetyl-CoA are randomly incorporated into growing fatty acid chains, the resulting fatty acid mass isotope distribution follows a binomial probability distribution. B. Fatty acid (palmitate) labeling pattern from equimolar glucose: 13C-ethanol co-feeding experiment as in Figure 1D (mean, SE, n=3 (biological replicates)). 15, 1–8 (2015). Briefly, newly synthesized fatty acids are getting labeled by [M+2] acetyl-CoA, which is a result of uptaking 13C-ethanol from growth media by S. cerevisiae. C Cytosolic acetyl-CoA labeling fitted from fatty acid labeling from equimolar glucose: 13C-ethanol co- A. When fed with labeled ethanol, the resulting 13C-labeled acetyl-CoA is incorporated into newly synthesized fatty acids. As both labeled and unlabeled cytosolic acetyl-CoA are randomly incorporated into growing fatty acid chains, the resulting fatty acid mass isotope distribution follows a binomial probability distribution A. When fed with labeled ethanol, the resulting 13C-labeled acetyl-CoA is incorporated into newly synthesized fatty acids. As both labeled and unlabeled cytosolic acetyl-CoA are randomly incorporated into growing fatty acid chains, the resulting fatty acid mass isotope distribution follows a binomial probability distribution. y y y y y p into growing fatty acid chains, the resulting fatty acid mass isotope distribution follows a binomial probability distribution. B. Fatty acid (palmitate) labeling pattern from equimolar glucose: 13C-ethanol co-feeding experiment as in Figure 1D (mean, SE, n=3 (biological replicates)). Briefly, newly synthesized fatty acids are getting labeled by [M+2] acetyl-CoA, which is a result of uptaking 13C-ethanol from growth media by S. cerevisiae. p y B. Fatty acid (palmitate) labeling pattern from equimolar glucose: 13C-ethanol co-feeding experiment as in Figure 1D (mean, SE, n=3 (biological replicates)). Briefly, newly synthesized fatty acids are getting labeled by [M+2] acetyl-CoA, which is a result of uptaking 13C-ethanol from growth media by S. cerevisiae. p y B. Fatty acid (palmitate) labeling pattern from equimolar glucose: 13C-ethanol co-feeding experiment as in Figure 1D (mean, SE, n=3 (biological replicates)). Briefly, newly synthesized fatty acids are getting labeled by [M+2] acetyl-CoA, which is a result of uptaking 13C-ethanol from growth media by S. cerevisiae. C. Cytosolic acetyl-CoA labeling fitted from fatty acid labeling from equimolar glucose: 13C-ethanol co- feeding experiment as in Figure 1D (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0)). C. Cytosolic acetyl-CoA labeling fitted from fatty acid labeling from equimolar glucose: 13C-ethanol co- feeding experiment as in Figure 1D (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0)). Figure 3. Environmental ethanol contributes to amino acid synthesis through mitochondrial acetyl- CoA. A. Synthesis of the arginine precursor N-acetylglutamate (NAG) in S. cerevisae takes place in mitochondria (Created with Biorender). 15, 1–8 (2015). A linear algebra deconvolution of the labeling fractions of glutamate and NAG can compute the mitochondrial acetyl-CoA labeling. B. Glutamate (Glu) and NAG labeling from the glucose: 13C-ethanol co-feeding experiment as in Figure 1D, including also data for ∆ach1 yeast (thereby identifying Ach1 as an enzyme essential for mitochondrial assimilation of ethanol-derived carbon into acetyl-CoA) (mean, SE, n=3 (biological replicates); , p <.001 by t-test). Briefly, given the observed highest isotopic label of Glu is [M+2], if Figure 3. Environmental ethanol contributes to amino acid synthesis through mitochondrial acetyl- CoA. A. Synthesis of the arginine precursor N-acetylglutamate (NAG) in S. cerevisae takes place in mitochondria (Created with Biorender). A linear algebra deconvolution of the labeling fractions of glutamate and NAG can compute the mitochondrial acetyl-CoA labeling. A. Synthesis of the arginine precursor N-acetylglutamate (NAG) in S. cerevisae takes place in mitochondria (Created with Biorender). A linear algebra deconvolution of the labeling fractions of glutamate and NAG can compute the mitochondrial acetyl-CoA labeling. g p y g B. Glutamate (Glu) and NAG labeling from the glucose: 13C-ethanol co-feeding experiment as in Figure 1D, including also data for ∆ach1 yeast (thereby identifying Ach1 as an enzyme essential for mitochondrial assimilation of ethanol-derived carbon into acetyl-CoA) (mean, SE, n=3 (biological replicates); , p <.001 by t-test). Briefly, given the observed highest isotopic label of Glu is [M+2], if mitochondrial acetyl-CoA are labeled [M+2] by 13C-ethanol uptaken by S. cerevisiae from growth media, newly synthesized NAG will have [M+4] isotopic label. B. Glutamate (Glu) and NAG labeling from the glucose: 13C-ethanol co-feeding experiment as in Figure 1D, including also data for ∆ach1 yeast (thereby identifying Ach1 as an enzyme essential for mitochondrial assimilation of ethanol-derived carbon into acetyl-CoA) (mean, SE, n=3 (biological replicates); , p <.001 by t-test). Briefly, given the observed highest isotopic label of Glu is [M+2], if mitochondrial acetyl-CoA are labeled [M+2] by 13C-ethanol uptaken by S. cerevisiae from growth media, newly synthesized NAG will have [M+4] isotopic label. e y sy t es ed G a e [ ] sotop c abe C. Mitochondrial acetyl-CoA [M+2] fraction fitted from glutamate and NAG labeling in B (mean, SE, n=3 (biological replicates)). y y [ ] p C. Mitochondrial acetyl-CoA [M+2] fraction fitted from glutamate and NAG labeling in B (mean, SE, n=3 (biological replicates)). 4 Figure 4. Carbons from ethanol feed into TCA intermediates. 5 A. 15, 1–8 (2015). Schematic of TCA cycle highlighting observed TCA labeling from glucose: 13C-ethanol co-feeding as in 6 Figure 1D, where [M+2] acetyl-CoA is a result of uptaking 13C-ethanol from growth media by S. 7 cerevisiae (Created with Biorender). 13C is in green while 12C is in white. 8 B. Labeling patterns of TCA intermediates on “right hand side” of TCA cycle (i.e. between citrate and 9 succinate) (mean, SE, n=3 (biological replicates)). 0 C. Labeling patterns of pyruvate and TCA intermediates on “left hand side” of TCA cycle (with aspartate as 1 a surrogate for oxaloacetate) (mean SE n=3 (biological replicates)) These metabolites are not labeled 2 Figure 4. Carbons from ethanol feed into TCA intermediates. Figure 4. Carbons from ethanol feed into TCA intermediates. Figure 4. Carbons from ethanol feed into TCA intermediates. A. Schematic of TCA cycle highlighting observed TCA labeling from glucose: 13C-ethanol co-feeding as in Figure 1D, where [M+2] acetyl-CoA is a result of uptaking 13C-ethanol from growth media by S. cerevisiae (Created with Biorender). 13C is in green while 12C is in white. B. Labeling patterns of TCA intermediates on “right hand side” of TCA cycle (i.e. between citrate and succinate) (mean, SE, n=3 (biological replicates)). ) ( , , ( g p )) C. Labeling patterns of pyruvate and TCA intermediates on “left hand side” of TCA cycle (with aspartate as a surrogate for oxaloacetate) (mean, SE, n=3 (biological replicates)). These metabolites are not labeled and thus originate exclusively from glucose, suggesting blockade of complex II and production of malate/fumarate through oxaloacetate reduction. C. Labeling patterns of pyruvate and TCA intermediates on “left hand side” of TCA cycle (with aspartate as a surrogate for oxaloacetate) (mean, SE, n=3 (biological replicates)). These metabolites are not labeled and thus originate exclusively from glucose, suggesting blockade of complex II and production of malate/fumarate through oxaloacetate reduction. Figure 5. Ald6 is a major source of the cytosolic NADPH. A. Schematic highlighting that, per acetyl-CoA, two NADPH active hydrides and one proton from H2O are incorporated into newly synthesized fatty acids. B. Cytosolic NADPH labeling (derived from fatty acid labeling, and corrected for substrate labeling fraction but not H-D exchange or kinetic isotope effect) from [1,1-2H2]ethanol in wild-type S. cerevisiae and ∆zwf1 strain (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0); , p<.001 by t-test). C. Cytosolic NADPH labeling as in B from [1-2H]glucose in wild-type S. cerevisiae and ∆ald6 strain (mean, Figure 5. Ald6 is a major source of the cytosolic NADPH. A. Schematic highlighting that, per acetyl-CoA, two NADPH active hydrides and one proton from H2O are incorporated into newly synthesized fatty acids. incorporated into newly synthesized fatty acids. B. Cytosolic NADPH labeling (derived from fatty acid labeling, and corrected for substrate labeling fraction but not H-D exchange or kinetic isotope effect) from [1,1-2H2]ethanol in wild-type S. cerevisiae and ∆zwf1 strain (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0); , p<.001 by t-test). C. Cytosolic NADPH labeling as in B from [1-2H]glucose in wild-type S. cerevisiae and ∆ald6 strain (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0); , p<.001 by t-test). D. Summary of data from B, C, and D2O tracing into fat shows sources of cytosolic NADPH redox active p y y y B. Cytosolic NADPH labeling (derived from fatty acid labeling, and corrected for substrate labeling fraction but not H-D exchange or kinetic isotope effect) from [1,1-2H2]ethanol in wild-type S. cerevisiae and ∆zwf1 strain (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0); , p<.001 by t-test). B. Cytosolic NADPH labeling (derived from fatty acid labeling, and corrected for substrate labeling fraction but not H-D exchange or kinetic isotope effect) from [1,1-2H2]ethanol in wild-type S. cerevisiae and ∆zwf1 strain (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0); , p y ) C. Cytosolic NADPH labeling as in B from [1-2H]glucose in wild-type S. cerevisiae and ∆ald6 strain (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0); *, p<.001 by t-test). D. Summary of data from B, C, and D2O tracing into fat shows sources of cytosolic NADPH redox active hydrogen nucleus in wild-type S. cerevisiae FY4 (mean, SE, n=6 (3 biological replicates with results from both C16:0 and C18:0)). The redox active hydrogen nucleus, but not the associated high energy electrons, is in a rapid H-D exchange with water, which explains the fractional contribution not accounted for by the pentose phosphate pathway and Ald6. Figure 6. Instead of oxPPP, ethanol oxidation provides major fraction of active hydrides in the presence of hydrogen peroxide. Figure 6. Instead of oxPPP, ethanol oxidation provides major fraction of active hydrides in the presence of hydrogen peroxide. Figure 6. Instead of oxPPP, ethanol oxidation provides major fraction of active hydrides in the presence of hydrogen peroxide. A. Figure 5. Ald6 is a major source of the cytosolic NADPH. Schematic of experimental workflow of administration of H2O2 oxidative stress to yeast (S.cerevisae FY4) cultures pre-incubated with equicarbon glucose:ethanol (Created with Biorender). Temporal changes within the first 60 s after H2O2 shock are captured by rapid quenching of metabolism at time points of 15, 30, and 60 s. p B. NADH pool size (mean, SE, n=6 (biological replicates), negative linear trend, p=.0016, ordinary one- way ANOVA). C. NADH: NAD+ ratio (mean, SE, n=6 (biological replicates), negative linear trend, p=.0006, ordinary one- way ANOVA). D. NADH active hydride labeling from [1,1-2H2]ethanol (orange) and [1-2H]glucose (grey) (mean, SE, n=3 (biological replicates), p=.011(), two-tailed paired t-test). Increase in the NADH active hydride from labeled ethanol is statistically significant (positive linear trend, p<0.001, ordinary one-way ANOVA). E. NADPH pool size (mean, SE, n=6 (biological replicates)). . NADPH pool size (mean, SE, n=6 (biological replicates)). NADPH: NADP+ ratio (mean SEM n=6 (biological replicates)) p ( ( g p )) F. NADPH: NADP+ ratio (mean, SEM, n=6 (biological replicates)). G. NADPH active hydride labeling from [1,1-2H2]ethanol (orange) and [1-2H]glucose (grey) (mean, SE, n=3 (biological replicates), p=.02, two-tailed paired t-test). Increase in the NADPH active hydride labeled by ethanol is statistically significant (positive linear trend, p<.001, ordinary one-way ANOVA). Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. NCHEMBA211214119Supplementary.pdf JRabinowitzEPC§atnew.pdf JRabinowitzEPC§atnew.pdf
https://openalex.org/W2895015116	https://europepmc.org/articles/pmc6748391?pdf=render	English	null	Modeling Urban Hydrology and Green Infrastructure Using the AGWA Urban Tool and the KINEROS2 Model	Frontiers in built environment	2,018	cc-by	13,250	EPA Public Access EPA Author Manuscript EPA EPA Author Manuscript 1School of Natural Resources and the Environment, University of Arizona, Tucson, AZ, United States 2Agricultural Research Service, Southwest Watershed Research Center, USDA, Tucson, AZ, United States 3Office of Research and Development, US Environmental Protection Agency, Las Vegas, NV, United States EPA Author Manuscript EPA Author Man *Correspondence: Yoganand Korgaonkar, yoganandk@email.arizona.edu. AUTHOR CONTRIBUTIONS DPG, DCG, IB, and WK were the original developers of AGWA. DCG and CU developed numerous features of KINEROS2. DPG, DCG, CU, and WK designed the La Terraza study. DPG and DCG were YK’s supervisors. YK and IB designed and developed the AGWA Urban tool. All authors helped write the paper. 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. Keywords y urban hydrology; green infrastructure; semi-arid; hydrologic model; stormwater; AGWA; KINEROS2; GIS EPA Author Manuscript EPA Autho EPA Author Manuscript Abstract Urban hydrology and green infrastructure (GI) can be modeled using the Automated Geospatial Watershed Assessment (AGWA) Urban tool and the Kinematic Runoff and Erosion (KINEROS2) model. The KINEROS2 model provides an urban modeling element with nine overland flow components that can be used to represent various land cover types commonly found in the built environment while treating runoff-runon and infiltration processes in a physically based manner. The AGWA Urban tool utilizes a Geographic Information System (GIS) framework to prepare parameters required for KINEROS2, executes the model, and imports results for visualization in the GIS. The AGWA Urban tool was validated on a residential subdivision in Arizona, USA, using 47 rainfall events (June 2005 to September 2006) to compare observed runoff volumes and peak flow rates with simulated results. Comparison of simulated and observed runoff volumes resulted in a slope of 1.00 for the regression equation with an R2 value of 0.80. Comparison of observed EPA Author Manuscript EPA Author Manuscript 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 (http://creativecommons.org/licenses/by/4.0/). 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. Wouter Buytaert, Imperial College London, United Kingdom y Juan Pablo Rodríguez Sánchez, University of Los Andes, Colombia David Glenn Chandler, Syracuse University, United States Specialty section: Juan Pablo Rodríguez Sánchez, University of Los Andes, Colombia David Glenn Chandler, Syracuse University, United States Specialty section: p y This article was submitted to Hydrosphere, a section of the journal Frontiers in Built Environment p y This article was submitted to Hydrosphere, a section of the journal Frontiers in Built Environment DATA AVAILABILITY Datasets are available on request. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. DATA AVAILABILITY Datasets are available on request. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. Abstract Page 2 Korgaonkar et al. and simulated peak flows had a slope of 1.12 with an R2 value of 0.83. A roof runoff analysis was simulated for 787 events, from January 2006 through December 2015, to analyze the water availability from roof runoff capture. Simulation results indicated a 15% capture of the average monthly rainfall volume on the watershed. Additionally, rainwater captured from roofs has the potential to provide for up to 70% of the domestic annual per capita water use in this region. Five different scenarios (S1 - base, S2 - with retention basins, S3 - with permeable driveways, S4 - with rainwater harvesting cisterns, and S5 - all GI practices from S2, S3, and S4) were simulated over the same period to compare the effectiveness of GI implementation at the parcel level on runoff and peak flows at the watershed outlet. Simulation results indicate a higher runoff volume reduction for S2 (53.41 m3 average capacity, average 30% reduction) as compared to S3 (average 14% reduction), or S4 (3.78 m3 capacity, average 6% reduction). Analysis of peak flows reveal larger peak flow reduction for S2. S3 showed more reduction of smaller peak flows as compared to S4. EPA Author Manuscript EPA Author EPA Author Manuscript Front Built Environ. Author manuscript; available in PMC 2019 September 17. INTRODUCTION Geographic Information Systems (GIS) provide capabilities and techniques to efficiently address these tasks, and visualize, analyze, and interpret spatial patterns (DeVantier and Feldman, 1993; Stuart and Stocks, 1993; Sui and Maggio, 1999; Vieux, 2001; Goodchild et al., 2005). The increasing availability of spatial data coupled with GIS capabilities, affords greater opportunity to simplify the process of using this data in hydrological models. Numerous hydrological models have been coupled with GIS to facilitate parameter extraction from spatial data and visualization of modeling results (Srinivasan and Arnold, 1994; Bhaduri et al., 2000; Miller et al., 2007; Chen et al., 2009; Lee et al., 2012). EPA Author Manuscript The US Department of Agriculture’s (USDA) Agricultural Research Service (ARS) developed the Kinematic Runoff and Erosion (KINEROS2) model. KINEROS2 is a spatially distributed, physically based, event driven model that simulates runoff and erosion for small watersheds (Smith et al., 1995; Goodrich et al., 2012). Overland flow is simulated using kinematic wave equations over rectangular elements with linear or curvilinear hillslopes, and concentrated flow in trapezoidal channels. Infiltration is simulated using a modified Smith- Parlange infiltration model (Parlange et al., 1982). KINEROS2 contains a rectangular urban element that consists of up to nine overland flow areas that contribute to one-half of a paved, crowned street. These nine overland flow areas include: (1) directly connected pervious (DCP) area, (2) directly connected impervious (DCI) area, (3) indirectly connected impervious (ICI) area, (4) indirectly connected pervious (ICP) area, (5) connecting pervious (CP) area, (6) connecting impervious (CI) area, (7) non-contributing (NC) area, (8) an infiltrating retention basin (RB) area, and (9) street half on to which the aforementioned overland flow areas contribute runoff. All nine flow areas are not required for each urban element for simulation. A single housing parcel in the urban area can have various surfaces that can affect hydrology differently. These surfaces include impervious roofs and driveways, pervious yards, and water sinks that can pond during rainfall events. With the help of the nine flow areas, the urban element can represent various flow-on/flow-off processes from these surfaces. These flow areas allow for model representation at a finer scale allowing better understanding of hydrological interactions and effects at a housing parcel level. The urban element in KINEROS2 can be used to represent a single housing parcel or a number of parcels in an urban development (Kennedy et al., 2013). INTRODUCTION Urbanization causes an increase in impervious surfaces (e.g., roofs, driveways, parking lots, and roads) by replacing vegetation and pervious natural areas. As a result, the area available for the infiltration of rainwater is substantially reduced. Additionally, soil compaction due to subdivision construction can result in reduced pore-water storage capacity thereby reducing infiltration rates (Gregory et al., 2006; Pitt et al., 2008; Woltemade, 2010; Yang and Zhang, 2011; Kennedy et al., 2013). Surface runoff volumes and peak discharges can increase significantly because of reduced infiltration and increases in connected impervious areas (Shuster et al., 2005). Increased surface runoff can also result in higher downstream loads of urban pollutants, such as lead, zinc, iron, suspended solids, fecal coliform bacteria, nitrogen, phosphorus, and hydrocarbons (Duda et al., 1982; Characklis and Wiesner, 1997; Norman et al., 2008; Bedan and Clausen, 2009). EPA Author Manuscript Green Infrastructure (GI, also referred to as Low Impact Development, or LID) has gained prominence in the management of stormwater to mitigate the effects of urbanization on watershed hydrology. There has been a shift in stormwater management techniques from traditional practices, such as curb-and-gutter systems, large detention basins, and hardened channels, to source control measures that use a variety of cost effective on-site design techniques to store, infiltrate, evaporate, and detain runoff. These practices include rain gardens, bioretention cells or basins, permeable pavements, green roofs, swales, infiltration trenches, roof runoff harvesting, and impervious disconnection (Dietz, 2007) that have varying degree of performance and effectiveness (Hopton et al., 2015). Analysis and mitigation of urban water quantity and quality issues, as well as the role of GI, requires a better understanding of the physical hydrological processes within urban areas. Various urban hydrological models have been successful in representing and simulating Page 3 Korgaonkar et al. urban hydrological processes as well as GI practices (Zoppou, 2001; Elliott and Trowsdale, 2007; Jayasooriya and Ng, 2014). All of these models have inherent assumptions, with a goal to represent hydrological processes accurately, while keeping the model simple and easy-to-use. Effective urban hydrological models are typically spatially distributed as they attempt to represent the heterogeneous nature of urban landscapes. By their nature, spatially distributed hydrologic models require copious spatial data on urban landscapes and hydrologically important watershed characteristics (e.g., land cover, land use, GI, soils, and topography). The process of acquiring this data and extracting model parameters can be tedious and lengthy. Front Built Environ. Author manuscript; available in PMC 2019 September 17. INTRODUCTION The ICI can be used to represent roofs, DCI for driveways, CP for front yards and DCP for all other yards, NC for swimming pools or walled areas, and RB for retention basins or rain gardens on the parcels. A typical urban watershed can be represented as a series of urban elements with the assumption that runoff Page 4 Korgaonkar et al. flows from each element into a street or alley half and follows the path along the street or alley to the watershed outlet. KINEROS2 requires parcel parameters in the form of dimensions, slope and fractional area of the urban overland flow areas; and street parameters in the form of width, grade, and cross slope. KINEROS2 also requires land cover parameters, which include values for hydraulic roughness for streets, impervious and pervious surfaces, interception depths for impervious and pervious surfaces, and canopy cover fractions. Additionally, KINEROS2 requires soils parameters in the form of saturated hydraulic conductivity (Ks), coefficient of variance of Ks to represent small-scale infiltration variability (Smith and Goodrich, 2000), mean capillary drive (potential of capillary suction in unsaturated soils), porosity, pore size distribution index, and volumetric rock fraction. Precipitation data in the form of time- intensity or time-accumulated depth pairs drives the KINEROS2 model. An urban simulation using KINEROS2 yields output in the form of runoff, infiltration, and storage for each individual element. Additionally, KINEROS2 can also output peak flow hydrographs at each element and at the outlet. The KINEROS2 urban element assumes negligible sediment production and therefore does not output sediment yield in this version. EPA Author Manuscript EPA Autho EPA Author Manuscript KINEROS2 was used to model runoff from a small residential development in Sierra Vista, Arizona in studies conducted by Kennedy (2007) and Kennedy et al. (2013). Kennedy et al. (2013) looked at three different discretization levels by lumping the 66 residential lots into 1, 5, and 23 KINEROS2 urban elements, and concluded that the accuracy of the model increased with the level of detailed representation of the development. Kennedy (2007) also performed a sensitivity analysis that indicated saturated hydraulic conductivity to be the primary estimation parameter to simulate runoff behavior. The study also highlighted the minimal uncertainty of parameter estimates in the urban watersheds, concluding that model uncertainty arose from model structural errors and input data error. Yatheendradas et al. Front Built Environ. Author manuscript; available in PMC 2019 September 17. INTRODUCTION Several studies to assess post-fire hydrological responses in watersheds have been conducted using AGWA (Canfield et al., 2005; Goodrich et al., 2005; Sidman et al., 2016). Other applications of AGWA include rangeland management (Goodrich et al., 2011; Weltzet al., 2011), flood hazard studies (Norman et al., 2010; Nedkov and Burkhard, 2012) and watershed assessment (Abdulla and Eshtawi, 2007; Yang and Li, 2011). EPA Author Manuscript EPA Author Manuscript This study presents a GIS approach to simulating urban hydrology and GI using the KINEROS2 model, and its integration with the AGWA tool. This study has three objectives: (1) Validate the capabilities of the AGWA Urban tool and KINEROS2 to model urban hydrology and green infrastructure; (2) Analyze the roof runoff availability for water use in a semiarid climate; and, (3) Analyze the effectiveness of GI practices on flood mitigation by reducing runoff volumes and peak flows at the watershed outlet. Study Area The La Terraza subdivision in Sierra Vista, Arizona (Figure 1) was selected as the study area based on Kennedy et al. (2013) study, and the availability of input datasets and high quality observations of rainfall and runoff. Sierra Vista is located in Cochise County in southeastern Arizona, at an elevation of approximately 1,300 m, with an annual average precipitation of 360 mm and annual mean temperature of 17.4°C based on records over the period 1981– 2000. Sierra Vista is located west of the San Pedro River and is contained in the Upper San Pedro HUC 15050202 watershed. The La Terraza subdivision is a residential development spanning 14 hectares located in the western part of the city. This study focused on an urban watershed consisting of 66 housing lots with an average parcel size of 1,780 m2, average house area of 380 m2, average driveway area of 108 m2, and 7.3m wide asphalt streets within the La Terraza subdivision. An undeveloped upland grassland watershed to the west contributes runoff to the urban watershed via a concrete channel. Runoff within the urban watershed flows over the streets, with the exception of a corrugated pipe that is located underneath La Terraza Drive that drains a 1.3 ha area in the northern part of the urban watershed to the outlet. Runoff exits at the outlet via another concrete channel to the southeast of the urban watershed. EPA Author Manuscript INTRODUCTION (2008) performed a comprehensive sensitivity analysis on the KINEROS2 model for flash flood forecasting, and concluded that the predominant source of uncertainty in modeled runoff response is due to depth/volume bias in rainfall estimates, followed by the saturated hydraulic conductivity, soil volumetric rock fraction and soil hydraulic roughness. The capabilities of KINEROS2 to model urban hydrology in detail forms the basis of the present study by representing each parcel in the watershed using the urban element. The Automated Geospatial Watershed Assessment (AGWA) tool was jointly developed by the USDA-ARS, the University of Arizona, and the EPA/ORD (Miller et al., 2007). AGWA is a GIS-based tool that uses existing spatial datasets in the form of digital elevation models (DEM), land cover maps, soil maps and weather data to prepare parameters for hydrological models. Currently, AGWA supports the Soil and Water Assessment Tool (SWAT; Arnold et al., 1998), the KINEROS2 model, and the Rangeland and Hillslope Erosion model (RHEM; Nearing et al., 2011). AGWA computes and supplies the parameters to these models, runs the models, and imports the results back in the GIS for visualization and analysis. AGWA is designed to provide qualitative estimates of runoff and erosion relative to landscape change. It, like virtually all watershed models, cannot provide quantitative estimates without careful calibration using high quality rainfall-runoff observations. Page 5 Korgaonkar et al. AGWA has been used to assess land use impacts on water resources in a number of studies (Hernandez et al., 2000; Miller et al., 2002; Baldyga et al., 2004; Kepner et al., 2004, 2009, 2012; Hamad et al., 2012; Nazarnejad et al., 2012; Baker and Miller, 2013; Burns et al., 2013; Barlow et al., 2014). Several studies to assess post-fire hydrological responses in watersheds have been conducted using AGWA (Canfield et al., 2005; Goodrich et al., 2005; Sidman et al., 2016). Other applications of AGWA include rangeland management (Goodrich et al., 2011; Weltzet al., 2011), flood hazard studies (Norman et al., 2010; Nedkov and Burkhard, 2012) and watershed assessment (Abdulla and Eshtawi, 2007; Yang and Li, 2011). AGWA has been used to assess land use impacts on water resources in a number of studies (Hernandez et al., 2000; Miller et al., 2002; Baldyga et al., 2004; Kepner et al., 2004, 2009, 2012; Hamad et al., 2012; Nazarnejad et al., 2012; Baker and Miller, 2013; Burns et al., 2013; Barlow et al., 2014). AGWA Urban Tool The AGWA Urban tool was designed and developed to harness the capabilities of the KINEROS2 model to represent urban areas in detail, and to create tools to use spatial datasets in parameter preparation for the model. The AGWA Urban tool was developed using the .NET Framework using the C# and VB.NET programming languages in the Microsoft Visual Studio Integrated Development Environment. ESRI provides an ArcObjects software development kit for the .NET Framework to build Windows applications with GIS functionalities. Using .NET and ArcObjects, Windows-based forms Front Built Environ. Author manuscript; available in PMC 2019 September 17. Korgaonkar et al. Page 6 Page 6 were developed that interface with existing GIS functionalities in ESRI ArcMap. The AGWA Urban tool is an ArcMap add-in that provides tools to prepare parameter files for the KINEROS2 model, runs the model, and imports the results into ArcMap for spatial visualization and analysis. Input parameters are generated from parcel, street, land cover, soils, and precipitation datasets. Additionally, inputs in the form of overland flow paths, and GI designs and locations can be manually provided. Each parcel is represented as a KINEROS2 urban element. The AGWA Urban tool executes the KINEROS2 model based on these input parameters. Runoff and infiltration results are visualized as maps and peak flow results are displayed as hydrographs. EPA Author Manuscript EPA Auth EPA Author Manuscript Front Built Environ. Author manuscript; available in PMC 2019 September 17. Steps Involved in an AGWA Urban Simulation Setup urban geodatabase—The AGWA Urban tool prepares the simulation workspace with a geodatabase to store spatial and tabular data prepared and used by an urban simulation. This step requires spatial data for the parcels and the streets. Required inputs are parcel and street maps that contain location data and dimensions (length, width, house size, driveway size, and slope) for each of the parcels and streets. The subsequent steps create files and relational tables that are stored in the simulation workspace and geodatabase, respectively. Flow routing—Overland stormwater flow paths are required as inputs in the flow routing step. These flow paths can be input as map layers, or can be created using the drawing tools provided by ArcMap. The AGWA urban tool links the flow paths to the parcels, performs checks to ensure continuity in the paths, and creates a table representing the flow route for the urban watershed. Parameterization—Parcel, street, land cover, and soils parameters are defined in the parameterization step. Parcels and street parameters can be defined based on existing data for each parcel (provided via the parcel map), or created homogeneously for all parcels. Similarly, land cover parameters can be created for all parcels. The AGWA Urban tool requires the nationally available Soil Survey Geographic Database (SSURGO) to extract soil parameters based on the input soils map. All parameters created in this step are stored in a parameterization table that maybe modified to reflect variations in parameters for each parcel. Note that SSURGO soils data are typically derived for a non-developed environment. Soil importation and compaction for subdivision and foundation pad construction may result in soil parameters substantially different from those represented in the SSURGO soils database (Kennedy et al., 2013). In this case, local, post-construction soil hydraulic property measurements are recommended. GI design and placement—Currently the AGWA urban tool provides options to design three different kinds of GI practices: (1) retention basins, (2) permeable driveways, and (3) rainwater harvesting cisterns. For retention basins, the size and depth of the basin are required, along with the hydraulic conductivity of the basin material. Hydraulic conductivity values for the driveways are required in order to model the driveways as permeable. To use the rainwater harvesting functionality, the volume of the capturing cistern is required. After these designs are created, they can then be applied to the parcels individually or collectively. Front Built Environ. Steps Involved in an AGWA Urban Simulation Author manuscript; available in PMC 2019 September 17. Korgaonkar et al. Page 7 Precipitation—The AGWA Urban tool accepts design storm precipitation data through the National Oceanic Atmospheric Administration (NOAA) Atlas 14-point precipitation frequency estimates databases, or user-defined depths and hyetographs, and converts them into a time-depth format required by KINEROS2. KINEROS2 also requires an initial soil saturation value that can be by rain gage, by element, or for the entire watershed. The AGWA Urban tool creates a precipitation text file based on the user inputs in the simulation workspace. EPA Author Manuscript EPA Autho EPA Author Manuscript KINEROS2 input files—KINEROS2 requires ASCII text files as inputs for model execution. The AGWA Urban tool converts the spatial and tabular data from the preceding steps into text files and saves them in the simulation workspace. A parameter file stores the input parameter data for each parcel and the sequence of execution of the parcels. A KINEROS2 control file directs the model with the parameter filename, precipitation filename, output filename, duration, and time steps for the simulation. A GI volumes file is created with initial volumes of the cisterns, and retention basins, for each parcel. The volumes in this file are updated after the simulation. A batch file is created that can execute the KINEROS2 model on the command line. Multiple simulations based on different combinations of flow routing, parameterization, and precipitation data can be created in this step. The AGWA Urban tool also provides an optional batch mode option to prepare and run KINEROS2 for multiple precipitation events. Model execution—KINEROS2 is a FORTRAN based model that runs on the Microsoft command line. The AGWA Urban tool executes the KINEROS2 model using the created input files. A command prompt displays the progress of the simulation and its status as success or if it encountered any errors. KINEROS2 creates an output text file, which summarizes water balances for each urban element and the entire watershed. Hydrograph files are also created for each parcel and the entire urban watershed. Importing and visualizing results—The AGWA Urban tool imports text file results from KINEROS2 simulations into ArcMap and links them to the spatial data, thereby allowing the user to visualize the data using the GIS interface. The user can visualize runoff and infiltration volumes for each individual parcel, as well as accumulated runoff along the streets as stormwater flows toward the outlet. Steps Involved in an AGWA Urban Simulation Additionally, the user can also compare runoff, infiltration, and accumulated runoff between simulations using absolute and percent change options. Hydrographs can be viewed at the outlet of the watershed as well as for each individual parcel. EPA Author Manuscript Front Built Environ. Author manuscript; available in PMC 2019 September 17. AGWA Urban Tool Validation The AGWA Urban tool was validated using forty-seven observed rainfall and runoff events from July 2005 through September 2006 for the La Terraza urban watershed. During this analysis, there were no GI practices installed on any of the parcels. Hence, GI was not included in the validation analysis. Rainfall data was extracted from four recording rain gauges (USDA Southwest Watershed Research Center, SWRC, gauges 401, 402, 403, and 404 - data available at https://www.tucson.ars.ag.gov/dap/; Figure 1) with areal average rainfall event totals ranging from 2 to 35 mm (events less than 2 mm were not used). Runoff, Front Built Environ. Author manuscript; available in PMC 2019 September 17. Page 8 Korgaonkar et al. Page 8 both in and out of the La Terraza urban watershed, was measured by v-notch weirs (U.S. Geological Survey, USGS, weirs 9470820 and 9470825, respectively; Figure 1). Because peak and runoff values are unreliable when the outlet “v” notch section of the weir is overtopped, events that met this criterion were excluded to provide a high-quality data set of 47 events. EPA Author Manuscript The AGWA Urban tool was used to setup the input parameters for the model. Parcel and street files were obtained from the Cochise County’s Information Technology department. Parcel dimensions and street widths were extracted from these files. Base map imagery, available in ArcMap, was used to determine the house and driveway areas on each parcel manually. Every parcel was assumed to have a front yard area and a noncontributing area equal to 10% of the total lot area. Values for slope, and land cover parameters were obtained from Kennedy et al. (2013). Soil parameters were obtained using the SSURGO database. The SSURGO dataset identified three different soil map units within the La Terraza urban watershed. Each parcel was assigned a specific set of soils parameters based on the soil map unit it intersected. Flow paths were drawn to represent the actual overland stormwater route converging toward three parcels (IDs 28, 39, 64) toward the southern part of the urban watershed (Figure 1). The adder element in KINEROS2 was utilized to combine these three parcels to represent the outlet. Stormwater was assumed to flow off the lots into the streets and along the streets to the outlet. This setup of modeling elements with associated parameters is hereafter referred to as the validation configuration. Roof Runoff Analysis Roof Runoff Analysis Each parcel in the above validation configuration was fitted with a virtual 37.85 m3 (10,000 gallon) cistern to estimate potential harvesting capacity, with the assumption that the cistern was emptied before every event. The cistern capacity was chosen in order to ensure that all roof-generated runoff was captured by the cistern, thereby simulating maximum roof runoff capture. Rainfall data for this analysis was extracted from SWRC Gauge 403 for the period ranging from January 2006 to December 2015, comprising 787 rainfall events. The analysis was run with a 1-min time step for each of the rainfall events in the 10-year period. The AGWA urban tool was used to design and place the cisterns on each of the parcels. The batch mode functionality was utilized to write the input files and execute the model for each of the events. Post-simulation cistern volumes for each parcel were extracted and compiled for roof runoff analysis. EPA Author Manuscript Front Built Environ. Author manuscript; available in PMC 2019 September 17. AGWA Urban Tool Validation Runoff from the upland grassland watershed was provided to the model using the injection functionality available in KINEROS2. The parameter text file created by the AGWA Urban tool was modified to incorporate an injection element. The AGWA urban tool created input files for each of the events, and executed the model 47 times. Every event had a measured initial soil saturation observation associated with each rain gage. Each simulation was run for a duration of 720 min with a time step of 30 s. Output runoff volumes and peak flows were then extracted for analysis. Case Study Scenarios Case Study Scenarios Five scenarios (Table 1) were designed to assess the impacts of various GI practices in the urban watershed. Scenario 1 (S1) is considered as the base scenario, where the validated Front Built Environ. Author manuscript; available in PMC 2019 September 17. Page 9 Korgaonkar et al. Page 9 configuration, described in section AGWA Urban Tool Validation, was simulated over the analysis period of 10 years without any GI practices. In scenario 2 (S2), a retention basin (RB) was installed in each parcel in the validation configuration. The retention basin was designed with a surface area equal to 10% of the total parcel area, a basin depth of 0.3 m, and a hydraulic conductivity of 8.3 mm/h. The retention basin was assumed empty at the start of each rainfall event. Note that runoff from driveways is captured by the retention basins to simulate retention of all available runoff from the parcel. Scenario 3 (S3) converted all driveways in the validation configuration to permeable driveways (PD) with a hydraulic conductivity of 8.3 mm/h. In scenario 4 (S4), each of the parcels in the validation configuration was installed with a 3.78 m3 (1,000 gallon) cistern to simulate rainwater harvesting (RH) off the roofs. The cistern was assumed empty at the start of each rainfall event. For scenario 5 (S5), GI designs from S2, S3, and S4 were all installed on each parcel in the validation configuration. The same 787 events from the roof runoff analysis were used to simulate these scenarios. Initial soil saturation was assumed as 0.2 for all events. The AGWA Urban tool was used to design and place the different GI practices on each lot, to create the input files, and execute the KINEROS2 model using the batch mode functionality. Runoff and peak flow results at the outlet of the urban watershed were extracted and compiled for analysis. EPA Author Manuscript EPA A EPA Author Manuscript EPA Author Manuscript EPA Autho EPA Author Manuscript Front Built Environ. Author manuscript; available in PMC 2019 September 17. The AGWA Urban Tool The AGWA Urban tool provides an easy-to-use framework to setup and execute the KINEROS2 model. Parcel ID 9 (Figure 1) and its representation using the urban element with and without GI practices, based on fractional areas of roofs, driveways, and yards is displayed in Figure 2. The roof area is represented by the indirectly connected impervious area, driveway by the directly connected impervious area, front yard by the connecting pervious area, and non-draining yards by the non-contributing areas. All remaining areas are represented by directly connected pervious areas using the KINEROS2 urban element. For GI practices, a retention basin can be represented by the retention basin area in KINEROS2 with an associated retention volume and hydraulic conductivity, a permeable driveway can be represented using the directly connected impervious flow area by specifying a hydraulic conductivity, and rainwater harvesting can be represented by specifying a cistern size to capture runoff from the indirectly connected impervious area. The user specified flow route in the AGWA Urban tool is converted to a KINEROS2 representation as shown in Figure 1. Although the flow route is drawn on the parcels, KINEROS2 assumes that each parcel contributes overland flow to the street half, and the subsequent runoff follows the specified flow route along the street. The AGWA Urban tool is capable of creating runoff, infiltration, and accumulated runoff maps (Figure 3). These maps can show absolute or percent difference in volumes between simulations for each individual parcel, demonstrated with percent increase in infiltration (Figure 3, left) from the S1 scenario (no GI) to the S5 scenario (all GI practices). Runoff volumes from a single scenario may also be displayed for each individual parcel, demonstrated with runoff from the S1 scenario (Figure 3, center). The accumulated runoff Front Built Environ. Author manuscript; available in PMC 2019 September 17. Korgaonkar et al. Page 10 Page 10 map (Figure 3, right) for scenario S1 (no GI) shows the cumulative runoff volume generated by the parcels and the streets combined as stormwater follows down the flow route toward the urban watershed outlet. Hydrographs can also be viewed, or exported from the AGWA Urban tool, for each parcel or at the urban watershed outlet (Figure 4). EPA Author Manuscript EPA EPA Author Manuscript Roof Runoff Analysis Roof Runoff Analysis Simulation results show an annual roof runoff capture of 7,078 m3 from all 66 parcels averaged over a period of 10 years. The average annual rainfall volume on all 66 parcels for the same period equals 47,280 m3. This means that the roofs are able to capture 15% of the total rainfall volume per year, for all the parcels combined, averaged over a 10-year period. The total roof area for all 66 parcels equals 25,085 m2, resulting in 28 cm of average annual depth of rainfall captured per unit area of the roofs for the La Terraza watershed. Each house in the La Terraza urban watershed has the potential to capture an average of approximately 107 m3 of rainfall annually. Cochise County has a domestic per capita water usage of 0.41 m3/day that amounts to approximately 150 m3/year (Maupin et al., 2014). Thus, roof rainwater harvesting has a capacity to provide for almost 70% of the domestic annual per capita water use, assuming constant water use every month. EPA Author Manuscript Figure 6 shows monthly average volumes of roof runoff captured as compared to the monthly per capita water usage. Approximately 44 m3 of average roof runoff can be available as surplus water after household use in the monsoon months of June, July, August and September, every year. AGWA Urban Tool Validation Runoff volumes and peak flows simulated by the AGWA Urban tool and KINEROS2 model are compared with observed values in Figure 5. Regression equation slopes of 1.00 for runoff volumes and 1.12 for peak flows are indicative of good model performance. Root Mean Square Error (RMSE) was calculated for both runoff volumes and peak flows using Equation (1), where n is the number of events, and Si and Oi represent the simulated and observed results for each event i. RMSE = ∑i = 1 n Si −Oi 2 n (1) Comparison of the runoff volumes results in an RMSE of 127.13 m3 (difference values ranging from −78.12 to 420.07), and peak flows results in an RMSE value of 0.06 m3/s (difference values ranging from −0.10 to 0.15) indicating a good fit of the model. Comparison of the runoff volumes results in an RMSE of 127.13 m3 (difference values ranging from −78.12 to 420.07), and peak flows results in an RMSE value of 0.06 m3/s (difference values ranging from −0.10 to 0.15) indicating a good fit of the model. Front Built Environ. Author manuscript; available in PMC 2019 September 17. Case Study Scenarios Table 2 summarize the average monthly runoff volumes at the watershed outlet for the five scenarios described in Table 1. Percent change in monthly runoff volumes were calculated using Equation (2), where S1 is the base scenario without GI practices, and n = 2, 3, 4, and 5 represent the four scenarios with GI practices, respectively. Korgaonkar et al. Korgaonkar et al. Page 11 Percent Change = Sn −S1 S1 × 100 (2) Percent Change = Sn −S1 S1 × 100 (2) EPA Author Manuscript EPA Autho EPA Author Manuscript For the simulated GI designs, retention basins in S2, and a combination of all GI practices in S5 have the highest reduction in average monthly runoff volumes at the watershed outlet, followed by permeable pavements in S3, and rainwater harvesting in S4, respectively. Scenarios S2 and S5 have identical results except for the month of July, when runoff volume for S5 is lower than S2. Percent reductions are greatest during the monsoon months as compared to the rest of the year for scenarios S2 S4 and S5 In contrast S3 has lower For the simulated GI designs, retention basins in S2, and a combination of all GI practices in S5 have the highest reduction in average monthly runoff volumes at the watershed outlet, followed by permeable pavements in S3, and rainwater harvesting in S4, respectively. Scenarios S2 and S5 have identical results except for the month of July, when runoff volume for S5 is lower than S2. Percent reductions are greatest during the monsoon months as compared to the rest of the year for scenarios S2, S4, and S5. In contrast, S3 has lower percent reductions during the monsoon months. In summary, scenarios S2, S3, S4, and S5 show average volume reductions of 30, 14, 6, and 30%, respectively when compared to S1. Further investigation of the near identical results of scenarios S2 and S5 revealed that the retention basins on each parcel had the capacity to retain and infiltrate all of the runoff generated from the respective parcel for most events. As a result, all runoff at the watershed outlet can be accounted for by rainfall excess generated by streets only. One rainfall event on July 11, 2014, however, simulated lower runoff volumes at the watershed outlet for S5 as compared to S2. Front Built Environ. Author manuscript; available in PMC 2019 September 17. The AGWA Urban Tool The AGWA Urban tool provides a user-friendly method to setup and execute the KINEROS2 model. Parcel data is commonly available from the local city or county office. With the availability of high-resolution imagery, each parcel can be digitized to extract roof, driveways and yard areas. Stormwater flow routes for an urban watershed can be digitized based on slope calculated from terrestrial airborne surveys, using Light Detection and Ranging (LiDAR), Structure from Motion (SfM), or another alternative. For the purpose of this study, the SSURGO dataset was utilized to extract soil parameters. However, in order to consider changes in soil properties due to compaction and landscape modifications, field measurements can be converted into spatial data for use in the GIS. Precipitation data is usually measured using a number of rain gauges depending on the size of the watershed. Additionally, estimated ground accumulated rainfall is available through precipitation products derived from Next Generation Weather Radar (NEXRAD) system from the NOAA’s National Centers for Environmental Information (NCEI) for a number of locations in the USA. The availability of these spatial datasets and the ability to import them into the GIS, helps simplify and speed up the process of setting up the KINEROS2 model using the AGWA Urban tool. The use of individual parcels to define the smallest modeling urban element in KINEROS2 enables a fine-scale simulation of urban hydrology. The nine urban overland areas can simulate a number of combinations of flow-off and flow-on areas at a detailed scale. It is important to note that the KINEROS2 representation of a parcel is always assumed rectangular. As a result, for non-rectangular shaped parcels, roofs, driveways, and yards, a distortion of physical reality must be expected in the representation. A combination of the detailed parcel representation and the ability to simulate sub-hour time steps for rainfall events enables the KINEROS2 model to represent the physical processes of infiltration and runoff for each parcel, and their responses to rainfall variability within an event. This gives a better understanding of the role of each individual parcel and its GI practices, as well as its contribution to the overall hydrology of the urban watershed. The infiltration and runoff maps from Figure 3 can help identify parcels with lower infiltration, and that generate higher runoff. DISCUSSION EPA Author Manuscript EPA Auth EPA Author Manuscript Case Study Scenarios This rainfall event had a total depth of 83 mm over a 190-min duration amounting to a volume of 11,600 m3 over the entire watershed. The mean for all 787 events was 600 m3 (with a standard deviation of 1,006 m3) indicating that the July 11, 2014 event was indeed an outlier. This event compares to a 3-h, 200-year recurrence interval event from NOAA Atlas 14’s point precipitation frequency estimates for the Sierra Vista station. The July 11, 2014 event was the only event within the 10-year period capable of generating runoff from the parcels with the designed GI practices. The combination of retention basins, permeable driveways and the harvesting cistern in S5 was capable of capturing and reducing the parcel generated runoff, explaining the lower runoff volume for S5 as compared to S2 for the month of July. This event was a good indicator of the ability of GI to mitigate flood risks for higher recurrence interval events. Figure 4 shows a comparison of peak flow hydrographs for all five scenarios for this event at the watershed outlet. Scenario S5 has the highest peak attenuation followed by S2. The second peak in the hydrograph (around 37min into the event) for S2 and S5 is due to the retention basins exceeding their retention capacity and overflowing onto the streets. The smaller permeable driveway areas in S3 contribute very little to infiltration, and result in peak flows similar to the base scenario S1. Similarly, the 3.78m3 cistern on every parcel in S4, only manages to capture a small portion of the rainfall from the roofs of the houses. Percent change in peak flows at the watershed outlet for each of 787 rainfall events was calculated using Equation (2). Figure 7 plots the percent reduction against peak flows for S1 to identify trends in the ability of GI practices to affect peak flows at the watershed outlet. S2 has overall higher percent reductions in peak flows as compared to S3 and S4 due to amount of retention volume available, especially for larger S1 peak flows. S3 shows better peak flow reduction as compared to S4 for S1 peak flows lower than 0.2 m3/s. However, for higher S1 peak flows, peak flow reduction for S3 decreases. It is also important to note that Korgaonkar et al. Case Study Scenarios Page 12 majority of the storms generate peak flows less than 1.0m3/s, and peak flows do not exceed 2.25 m3/s, except for the July 11, 2014 event. Front Built Environ. Author manuscript; available in PMC 2019 September 17. The AGWA Urban Tool These parcels can then be potential targets for installation of GI practices to capture rainwater using cisterns, or encourage infiltration by installing retention basins or permeable driveways. Potential impacts of GI applications can be analyzed for before-after scenarios using the differencing capability available in the AGWA Urban tool. Potential flood-prone streets and intersections can be identified using the accumulated runoff map (Figure 3, right). Street GI practices, such as curbside retention basins, swales, or traffic circles can then be designed to mitigate flood at these locations. Front Built Environ. Author manuscript; available in PMC 2019 September 17. Page 13 Page 13 Korgaonkar et al. Roof Runoff Analysis Roof runoff analysis estimated that on an average, 70% of the roof runoff captured every year was available for household use. Monthly rainfall volumes, averaged over a period of 10 years show seasonal trends with more rainfall occurring in the months of June, July, August, and September (Table 2). These months represent the monsoon season with short duration, high intensity thunderstorms caused due to convection. Rainfall volumes are lower in the winter months and are characterized by long duration, low intensity storms due to frontal lifting (Sheppard et al., 2002; Brooks et al., 2003). The roof runoff capture potential is high for the monsoon season, when more water is available via rainfall (Figure 6). This harvested rainwater can be stored and utilized during the drier months of the year. In a semi- arid climate, this harvested rainwater can augment scarce water supply sources in order to meet daily household water needs. City of Tucson Pima Country (2009) conducted an evaluation of the use of stormwater and rainwater as a supplemental water source. This study looked at potential harvestable rainwater at different scales ranging from a parcel to a tributary watercourse in the semi-arid climate of Southern Arizona. Figure 8 reveals that the greatest opportunity to harvest rainwater is at the parcel scale, with roofs being the primary contributor to rainfall excess, especially in the semiarid region of Southwestern USA. This supports that rainwater harvesting is a good alternate source of water for irrigation, toilet flushing, and consumption (Thomas et al., 2014; Silva et al., 2015; Campisano et al., 2017). However, roof runoff could be a source of chemical or microbial pollutants and could have health risks from its use as drinking water (Lye, 2009; Gwenzi et al., 2015). Hence, treatment of this water is essential before potable household use. EPA Author Manuscript Front Built Environ. Author manuscript; available in PMC 2019 September 17. AGWA Urban Tool Validation Validation results indicate that runoff volumes, on average, are predicted well by KINEROS2, while peak flows are generally over-predicted for the validation configuration. Over-prediction of volumes and peak flows may be due to a lower time of concentration on the parcels due to misrepresentation of physical reality. This misrepresentation may be a result of the assumption that all front yards (CP) and non-contributing (NC) areas on each parcel are equal to 10% of the lot area. For example, a parcel may have a smaller simulated pervious area (CP) that captures and retains roof runoff (ICI), as compared to the physical reality. As a result, the simulated volume of runoff reaching the streets is higher, resulting in higher peak flows for that particular event. Another reason could be that some of the parcels may have a larger non-contributing area, such as a backyard that stores and infiltrates most of the runoff, instead of contributing as overland flow to the street. In the La Terraza urban watershed, the yard area can be up to 4.5 times the footprint of the living area. This may result in lower observed runoff volumes and peak flows at the outlet, when compared to the simulated results. EPA Author Manuscript EPA Au EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript Korgaonkar et al. Page 14 Page 14 larger rainfall excess available for capture. Permeable driveways showed better volume and peak flow reductions for smaller events during the non-monsoon months of the year. During these months, rainfall intensity is generally low and could possibly be closer to the infiltration capacity of the permeable driveway. The capability of rainwater harvesting cisterns to reduce runoff volumes and peak flows is largely limited by the size of the cistern, and by the proportion of roof area available vs. the total parcel area. With larger cisterns, and roof areas, higher runoff volumes and peak reduction should be expected. EPA Author Manuscript GI design can play an important role in determining effectiveness in runoff and peak flow reduction (Liu et al., 2015). It is important to note that the GI designs used in this study, were “back-of-the-envelope” values in order to represent possible GI installation due to lack of physical implementations. For example, for an average parcel size of 1,780 m2, a retention basin would have a surface area of 178m2 and depth of 0.3m, with a retention volume of 53.4 m3. This retention volume may be considered on the higher side, possibly representing a best-case scenario, which may or may not be a practical implementation. However, a 3.785 m3 cistern is a reasonable assumption for implementation at a housing parcel level. Additionally, the case study assumed that basins and cisterns were empty at the start of each event, which may not be the case in real-world scenarios. If a cistern is not emptied between events, it may result in higher roof generated runoff. As a result, this assumption can lead to an under-estimation of overall runoff exiting from a parcel. Initial soil saturation in between rainfall events can affect predicted runoff in the KINEROS2 model (Yatheendradas et al., 2008). Therefore, these case study results are limited by the assumption of a 0.2 initial soil saturation. This value represents the soil saturation at permanent wilting point for sandy loam soils commonly found in this region (Woolhiser et al., 1990), simulating a best-case infiltration scenario. With variable initial soil saturation in between events, uncertainty in simulated runoff can be expected. In general, higher initial soil saturation will result in a decrease in the infiltration capacity of the soil, thereby resulting in larger simulated runoff and peak flows. Front Built Environ. Author manuscript; available in PMC 2019 September 17. Case Study Scenarios Case study scenarios indicated that retention basins were effective at volume and peak flow reduction as compared to permeable driveways and rainwater harvesting, particularly during the monsoon months. The monsoon months result in larger rainfall volumes, thereby causing CONCLUSIONS EPA Author Manuscript EPA Autho EPA Author Manuscript The AGWA Urban tool provides an easy-to-use GIS framework to prepare and execute the KINEROS2 model in order to simulate urban hydrology and green infrastructure. The KINEROS2 model is able to model the built environment with the help of the urban element that utilizes nine overland flow areas to simulate flow-on and flow-off processes for different areas. For a typical urban parcel, the roof, driveway, yards, and swimming pools, can be represented by indirectly connected impervious (ICI), directly connected impervious (DCI), directly connected pervious (DCP) and connecting pervious (CP), and non-contributing (NC) flow areas, respectively. GI practices such as retention basins, permeable driveways, and rainwater harvesting cisterns can be represented using the RB, DCI, and ICI urban element components respectively, by providing appropriate parameters. A combination of multiple urban elements can define an urban watershed. The AGWA urban tool utilizes spatial data, such as parcels, streets, land cover, precipitation, and soils, to extract input parameters required by the KINEROS2 model. Each individual parcel can be represented using the KINEROS2 urban element (Figure 2), and can be associated with a unique set of parameters. The AGWA Urban tool is able to extract these parameters from input spatial datasets to prepare and execute the KINEROS2 model. The tool also compiles and synthesizes the simulation results for visualization in the form of runoff and infiltration volume maps (Figure 3), as well as peak flow hydrographs (Figure 4). Infiltration and runoff volume maps can help identify parcels with lower infiltration and higher runoff volumes as potential GI implementation sites. Percent change analyses can be conducted to understand the impacts of GI implementation on a parcel-by-parcel basis. The AGWA Urban tool was validated using 47 rainfall events on the La Terraza subdivision in Sierra Vista, Arizona. Sixty-six parcels were identified and modeled as an urban watershed. Simulated runoff volumes and peak flows were compared with observed values at the outlet of the watershed (Figure 5). The regression equation for the runoff volumes comparison yielded a slope of 1.00 with an R2 value of 0.80, and yielded a slope of 1.12 with an R2 value of 0.83 for the peak flow comparison. In general, runoff volumes were well predicted, but peak flows were over-predicted by the model. EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript In the KINEROS2 urban element, initial soil saturation only affects areas modeled as directly connected pervious (DCP) and connecting pervious (CP). KINEROS2 assumes a constant seepage rate for retention basins and permeable driveways. Thus, initial soil saturation does not influence the amount of rainwater harvested off the roofs (scenario S4), nor the amount of rainfall directly infiltrated in retention basins (S2) or permeable driveways (S3). However, DCP and CP contribute runoff to the retention basins as shown in Figure 2. As a result, initial soil saturation in these pervious areas will affect the overall runoff and peak flows for scenario S3 and S5, simulating the retention basins. Other studies show similar results in volume and peak flow reductions using different combinations of GI practices (Lloyd et al., 2002; Ahiablame et al., 2013; Palla and Gnecco, 2015; Ahiablame and Shakya, 2016; Eckart et al., 2017; Fry and Maxwell, 2017; Hu et al., 2017). These studies conclude that there is scope for volume and peak flow reduction at various watershed scales using GI practices. The design criteria of the GI practices largely determines their effectiveness. Spatial variability in GI implementation could also influence level of effectiveness. These studies also provide an insight in the potential designs and locations of GI practices for their respective watersheds. Future research should be guided Korgaonkar et al. Page 15 toward spatial variability of GI implementation, to understand its effects on volume and peak flow reduction. Front Built Environ. Author manuscript; available in PMC 2019 September 17. CONCLUSIONS Seven-hundred and eighty-seven rainfall events were simulated on the same urban watershed over a period of 10 years, from January 2006 to December 2015 to analyze the potential to capture roof runoff via harvesting cisterns. Simulation results indicated a 15% capture of the average monthly rainfall volume with a volume capture rate of 0.28 m3/m2. Additionally, roof rainwater harvesting has the potential to provide up to 70% of the domestic annual per capita water use in Cochise County, Arizona. Five scenarios (Table 1) were simulated to analyze the impact and effectiveness of retention basins (S2, 53.31 m3 average volume), permeable driveways (S3), and rainwater harvesting (S4, 3.78 m3 capacity) on runoff volumes and peak flows at the watershed outlet. Retention Front Built Environ. Author manuscript; available in PMC 2019 September 17. Korgaonkar et al. Page 16 Page 16 basins in S2 reduced runoff volumes by almost 30%, permeable driveways in S3 reduced runoff volumes by 14%, and rainwater harvesting in S4 was successful in capturing around 6% of the runoff volume. A combination of all three GI practices in S5 resulted in identical volume and peak flow reduction as S2. Seasonal trends were also observed for all scenarios, with an increase in effectiveness in runoff reduction for S2, S4, and S5 for the monsoon months of June, July, August, and September, when high-intensity rainfall events are observed. However, permeable driveways in S3 showed lower runoff volume reduction for larger events during the monsoon season. basins in S2 reduced runoff volumes by almost 30%, permeable driveways in S3 reduced runoff volumes by 14%, and rainwater harvesting in S4 was successful in capturing around 6% of the runoff volume. A combination of all three GI practices in S5 resulted in identical volume and peak flow reduction as S2. Seasonal trends were also observed for all scenarios, with an increase in effectiveness in runoff reduction for S2, S4, and S5 for the monsoon months of June, July, August, and September, when high-intensity rainfall events are observed. However, permeable driveways in S3 showed lower runoff volume reduction for larger events during the monsoon season. EPA Author Manuscript For 786 rainfall events, the street network was the sole contributor to runoff and peak flow at the watershed outlet for S2 and S5 simulations. CONCLUSIONS This was because the retention basin was capable of retaining all of the overland runoff originating on the parcel, representing a best- case scenario. A rainfall event for July 11, 2014 was the only event for which the retention basins were overwhelmed, and the parcels contributed overland runoff to the street network. The magnitude of this rainfall event compares to a 3-h 200-year recurrence interval event determined from NOAA Atlas 14’s point precipitation estimates. For this event, scenarios S2 and S5 resulted in higher peak flow reduction at the watershed outlet as compared to S3 and S4 (Figure 4). Analysis of all peak flows at the watershed outlet for the 787 events revealed that S5 had the highest overall peak flow reduction followed by S2, S3, and S4, when compared to the base scenario S1 without any GI practices (Figure 7). Interestingly, permeable driveways in S3 were more effective than rainwater harvesting in S4 for lowering peak flows compared to no GI practices in S1, and percent reduction decreased as the magnitude of S1 peak flows increased. Limited driveway sizes and the divergence between rainfall intensity and infiltration capacity at larger events restrict the infiltration potential, which explains this trend. This study primarily focused on the potential of lot-level GI practices to capture rainfall excess in order to augment water supply in semi-arid regions. Roofs can have the highest potential to capture rainwater for domestic use. Rainwater captured during the larger monsoon events can be stored for use during the drier months. We can conclude from our analysis, that appropriate GI designs can be extremely effective at capturing rainfall excess on site for smaller events. It is important to note that these conclusions are based on specific GI designs, and will require future research to compare the impact of different GI designs for each of the practices, for a variety of return period events. However, we also advocate the use of the AGWA Urban tool for exploring GI construction options in arid and semi-arid environments, for the purpose of water conservation and reduced catastrophic surface flow during storm events. With the success of the various modeling scenarios presented in this study, we recommend the use of the AGWA Urban tool for modeling hydrology and GI practices in the built environment. EPA Author Manuscript EPA Author Manuscript This work was funded by NSF Sustainability Research Network (SRN) Cooperative Agreement 1444758. We would also like to acknowledge the USDA-ARS for funding in the early stages of the research. Basemap imagery used in all the maps was obtained from ESRI ArcMap (Source: Esri, DigitalGlobe, GeoEye, Earthstar Geographics, EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript Abdulla FA, and Eshtawi T (2007). Application of Automated geospatial watershed assessment (agwa) tool to evaluate the sediment yield in a semi-arid region: case study, kufranja basin-Jordan. Jordan J. Civil Eng 1, 234–244. 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Water infiltration in urban soils and its effects on the quantity and quality of runoff. J. Soils Sedim 11, 751–761. doi: 10.1007/s11368-011-0356-1 Yatheendradas S, Wagener T, Gupta H, Unkrich C, Goodrich D, Schaffner M, et al. (2008). Understanding uncertainty in distributed flash flood forecasting for semiarid regions. Water Resources Res. 44:W05S19. doi: 10.1029/2007WR005940 Zoppou C (2001). Review of urban storm water models. Environ. Model. Software 16, 195–231. doi: 10.1016/S1364-8152(00)00084-0 EPA Author Manuscript Korgaonkar et al. Page 21 Page 21 FIGURE 1 \|. The La Terraza urban watershed in Sierra Vista, Arizona. Inset: KINEROS2 assumption of the user-defined flow route. KINEROS2 assumes that overland flow is off the parcel into the street (dark green arrows), and accumulates along the street toward the watershed outlet (light green arrows). EPA Author Manuscript EPA Author Manuscript FIGURE 1 \|. The La Terraza urban watershed in Sierra Vista, Arizona. Inset: KINEROS2 assumption of the user-defined flow route. KINEROS2 assumes that overland flow is off the parcel into the street (dark green arrows), and accumulates along the street toward the watershed outlet (light green arrows). FIGURE 1 \|. The La Terraza urban watershed in Sierra Vista, Arizona. Inset: KINEROS2 assumption of the user-defined flow route. KINEROS2 assumes that overland flow is off the parcel into the street (dark green arrows), and accumulates along the street toward the watershed outlet (light green arrows). Front Built Environ. Author manuscript; available in PMC 2019 September 17. Page 22 FIGURE 2 \|. Parcel ID 9 in the La Terraza urban watershed (Left), KINEROS2 representation without GI (Center), and with retention basin (RB), permeable driveway (PD), and rainwater harvesting (RH) GI practices (Right). Front Built Environ. Author manuscript; available in PMC 2019 September 17. Front Built Environ. Author manuscript; available in PMC 2019 September 17. EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript Percent values of each of the overland flow areas are indicative of the percent of the total parcel area. DCP, directly connecting pervious; ICI, indirectly connected impervious; CP, connected pervious; DCI, directly connected impervious; NC, noncontributing area. Korgaonkar et al. Page 22 Page 22 Korgaonkar et al. FIGURE 2 \|. EPA Author Manuscript FIGURE 2 \|. Parcel ID 9 in the La Terraza urban watershed (Left), KINEROS2 representation without GI (Center), and with retention basin (RB), permeable driveway (PD), and rainwater harvesting (RH) GI practices (Right). Percent values of each of the overland flow areas are indicative of the percent of the total parcel area. DCP, directly connecting pervious; ICI, indirectly connected impervious; CP, connected pervious; DCI, directly connected impervious; NC, noncontributing area. EPA Author Manuscript FIGURE 3 \|. Visualization of results using the AGWA Urban tool with percent change in infiltration volumes between no GI practices in S1 and all GI practices in S5 (Left), runoff volumes for S1 (Center), and accumulated runoff from parcels and streets routed along the streets for S1 (Right). gaonkar et al. Page 23 Page 23 Page 23 Korgaonkar et al. EPA Author Manuscript EPA Author Manuscript Front Built Environ. Author manuscript; available in PMC 2019 September 17. Front Built Environ. Author manuscript; available in PMC 2019 September 17. Front Built Environ. Author manuscript; available in PMC 2019 September 17. FIGURE 3 \|. FIGURE 3 \|. Visualization of results using the AGWA Urban tool with percent change in infiltration volumes between no GI practices in S1 and all GI practices in S5 (Left), runoff volumes for S1 (Center), and accumulated runoff from parcels and streets routed along the streets for S1 (Right). FIGURE 3 \|. Visualization of results using the AGWA Urban tool with percent change in infiltration volumes between no GI practices in S1 and all GI practices in S5 (Left), runoff volumes for S1 (Center), and accumulated runoff from parcels and streets routed along the streets for S1 (Right). EPA Author Manuscript EPA Autho EPA Author Manuscript Korgaonkar et al. Page 24 FIGURE 4 \|. Hydrographs at the watershed outlet for the five scenarios for the July 11, 2014 event. S2- RB, with retention basin; S3-PD, with permeable driveways; S4-RH, with rainwater harvesting. EPA Author Manuscript EPA Autho EPA Author Manuscript FIGURE 4 \|. Hydrographs at the watershed outlet for the five scenarios for the July 11, 2014 event. S2- RB, with retention basin; S3-PD, with permeable driveways; S4-RH, with rainwater harvesting. EPA Author Manuscript FIGURE 5 \|. AGWA Urban tool validation results. Comparison of observed to simulated values for runoff volumes (Left) and peak flows (Right) at the watershed outlet. Korgaonkar et al. Page 25 Page 25 Page 25 Korgaonkar et al. Page 25 EPA Author Manuscript FIGURE 5 \|. AGWA Urban tool validation results. Comparison of observed to simulated values for runoff volumes (Left) and peak flows (Right) at the watershed outlet. EPA Author Manuscript EPA Autho EPA Author Manuscript EPA Author Manuscript Page 26 Page 26 Korgaonkar et al. FIGURE 6 \|. Average yearly roof runoff captured by a cistern and monthly per capita water usage for Cochise County estimated from a daily per capita usage rate of 0.41 m3/day. EPA Author Manuscript EPA Autho EPA Author Manuscript FIGURE 6 \|. Average yearly roof runoff captured by a cistern and monthly per capita water usage for Cochise County estimated from a daily per capita usage rate of 0.41 m3/day. FIGURE 6 \|. Average yearly roof runoff captured by a cistern and monthly per capita water usage for Cochise County estimated from a daily per capita usage rate of 0.41 m3/day. EPA Author Manuscript FIGURE 7 \|. Front Built Environ. Author manuscript; available in PMC 2019 September 17. Front Built Environ. Author manuscript; available in PMC 2019 September 17. Front Built Environ. Author manuscript; available in PMC 2019 September 17. FIGURE 3 \|. Trends in percent reduction of peak flows for each of the GI scenarios (S2, with retention basins; S3, with permeable driveways; and S4, with rainwater harvesting) as compared to S1 (without any GI practices) peak flows. gaonkar et al. Page 27 Korgaonkar et al. Page 27 orgaonkar et al. Page 27 EPA Author Manuscript EPA Autho EPA Author Manuscript FIGURE 7 \|. FIGURE 7 \|. Trends in percent reduction of peak flows for each of the GI scenarios (S2, with retention basins; S3, with permeable driveways; and S4, with rainwater harvesting) as compared to S1 (without any GI practices) peak flows. EPA Author Manuscript EPA Autho EPA Author Manuscript Page 28 Page 28 Korgaonkar et al. FIGURE 8 \|. Harvestable rainwater at different watershed scales (City of Tucson Pima Country, 2009). Figure created by Dr. Evan Canfield (used with permission). EPA Author Manuscript FIGURE 8 \|. FIGURE 8 \|. Harvestable rainwater at different watershed scales (City of Tucson Pima Country, 2009). Figure created by Dr. Evan Canfield (used with permission). EPA Author Manuscript Page 29 Korgaonkar et al. TABLE 1 \| Description of the five case study scenarios. Scenario GI practice Description S1 Base - No GI practices Validated model without any GI practices S2 Retention Basin (RB) S1 with retention basin on all 66 parcels. Retention basin area equals 10% of the parcel area, depth equals 0.3 m, and hydraulic conductivity of 8.3 mm/h. Average retention basin capacity equals 53.41 m3. S3 Permeable Driveways (PD) S1 with all 66 driveways considered permeable with a hydraulic conductivity of 8.3 mm/h S4 Rainwater Harvesting (RH) S1 with a 3.78 m3 cistern on each of the 66 parcels capturing roof runoff S5 All GI practices S1 with GI designs from S2, S3, and S4 in combination on each of the 66 parcels TABLE 1 \| Description of the five case study scenarios. EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript Korgaonk TABLE 2 \| Monthly runoff volumes at the watershed outlet for the five case study scenarios averaged over a period of 10 years (January 2006 to December 2015) and percent reduction in monthly runoff volumes for the scenarios as compared to the S1 base scenario (no GI) for the same period. Page 30 Page 30 Korgaonkar et al. EPA Author Manuscript EPA Author Manuscript EPA Author Manuscript TABLE 2 \| Monthly runoff volumes at the watershed outlet for the five case study scenarios averaged over a period of 10 years (January 2006 to December 2015) and percent reduction in monthly runoff volumes for the scenarios as compared to the S1 base scenario (no GI) for the same period. Month Rainfall (m3) Runoff volumes at watershed outlet (m3) and volume reduction compared to S1 (%) S1–Base No GI S2–RB S3–PD S4–RH S5–All GI m3 m3 % m3 % m3 % m3 % January 2,245 479 363 24 390 19 470 2 363 24 February 571 106 92 13 93 13 106 <1 92 13 March 1,349 271 218 20 229 16 264 3 218 20 April 392 77 63 18 64 17 77 1 63 18 May 383 79 62 21 63 20 78 1 62 21 June 2,944 1,057 476 55 993 6 953 10 476 55 July 14,274 4,706 2552 46 4,368 7 4,013 15 2,519 46 August 12,448 3,958 2013 49 3,669 7 3,344 16 2,013 49 September 8,064 2,287 1304 43 2,058 10 1,968 14 1,304 43 October 1,659 361 268 26 307 15 333 8 268 26 November 1,552 325 251 23 269 17 318 2 251 23 December 1,399 277 226 18 231 16 273 1 226 18 Average 3,940 1,165 658 30 1,061 14 1,016 6 655 30 Front Built Environ. Author manuscript; available in PMC 2 Front Built Environ. Author manuscript; available in PMC 2019 September 17.
https://openalex.org/W2956020399	https://dergipark.org.tr/tr/download/article-file/750308	Turkish	null	Püskürtme Betonda Kimyasal Katkı Miktarının Priz Süresine ve Beton Basınç Dayanımına Etkisi	Sinop Üniversitesi fen bilimleri dergisi	2,019	cc-by	4,690	* Sorumlu Yazar: ORCID ID: orcid.org/0000-0003-0084-1320 e-mail: celalettinbasyigit@sdu.edu.tr Sinop Üniversitesi Fen Bilimleri Dergisi Sinop Üniversitesi Fen Bilimleri Dergisi Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Araştırma Makalesi https://doi.org/10.33484/sinopfbd.494656 Püskürtme Betonda Kimyasal Katkı Miktarının Priz Süresine ve Beton Basınç Dayanımına Etkisi Ali Öztürka, Celalettin Başyiğitb* a1Süleyman Demirel Üniversitesi, Fen Bilimleri Enstitüsü, İnşaat Mühendisliği ABD, 32200, Isparta b2Süleyman Demirel Üniversitesi, Mühendislik Fakültesi, İnşaat Mühendisliği Bölümü, 32200, Isparta * Sorumlu Yazar: ORCID ID: orcid.org/0000-0003-0084-1320 Received: 10.12.2018 e-mail: celalettinbasyigit@sdu.edu.tr Accepted: 17.05.2019 Öz Bu çalışmada, tünellerde kullanılan püskürtme betona eklenen priz hızlandırıcı katkı malzemesi oranının optimum değeri araştırılmıştır. %5, %8, %10 oranlarında katkı kullanılarak ve katkı kullanılmadan, püskürtme betonu için çimento priz hızlandırıcı katkı uyum testi ve beton basınç dayanım deneyleri yapılmıştır. Yapılan deneylerde, su/çimento oranı, çimento miktarı ve tipi, agrega miktarı ve özellikleri sabit tutulmuştur. Priz hızlandırıcı katkı uyum testi için toplamda 8 adet numune hazırlanmıştır. Katkısız kontrol numuneleri, %5 katkı içeren numuneler, %8 katkı içeren numuneler ve %10 katkı içeren numuneler hazırlanarak elde edilen sonuçlar değerlendirilip katkı miktarının priz süresine ve beton basınç dayanımına etkisi incelenmiştir. Deneylerde kullanılan %10 priz hızlandırıcı katkı miktarı, priz süresini oldukça kısaltmaktadır ve bu denli bir kısalma uygulama zorluklarına yol açmaktadır. %5 priz hızlandırıcı katkı malzemesi kullanılması durumunda, priz süresi değerlendirme tablosuna göre ilk priz süresi kabul edilebilir olarak değerlendirilmiştir. %8 priz hızlandırıcı katkı miktarı kullanıldığında ise optimum sonuç elde edildiği gözlemlenmiştir. Anahtar Kelimeler: Tüneller, püskürtme beton, priz hızlandırıcı katkılar, basınç dayanımı. Abstract In this study, the optimum value of the ratio of added set accelerating admixture used in the tunnel spraying concrete was investigated. 5%, 8%, 10% of the use of the set accelerating admixture and additives without using set accelerating admixture, cement concrete accelerator admixture compliance test and concrete compressive strength tests were made. In the experiments, water / cement ratio, amount and type of cement, aggregate amount and properties were kept constant. A total of 8 samples were prepared for the fit acceleration additive compliance test. Pure control samples, samples containing %5, %8 and %10 set accelerator were prepared and the results obtained were evaluated and the effect on the concrete setting time and concrete compressive strength were investigated. The amount of 10% set accelerating additives used in the experiments significantly shortens the setting time, and such shortening leads to application difficulties. In case of using 5% set accelerating additives material, the initial setting time is considered to be acceptable according to the Received: 10.12.2018 Accepted: 17.05.2019 71 Sinop Üniversitesi Fen Bilimleri Dergisi Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 setting time table. When the% 8 accelerator additive amount is used, it is observed that the optimum result is obtained. Keywords: Tunnels, sprayed concrete, set accelerating admixture, compressive strength. yapılabilir olması, farklı çalışma ortamlarında farklı boyut ve kesitlerde istenilen şekilde uygulanabilir olması, kalıp gerektirmediğinden malzeme ve işçilik tasarrufu yapılması gibi etkenler de yaygın kullanılma sebeplerindendir [3]. Püskürtme beton, özellikle kalıp imalatının zor olduğu, kalıp maliyetlerinin ekonomik olmadığı, betonun yerleştirilme ve sıkıştırmasının güç olduğu yerlerde, betonun ince bir tabaka olarak uygulanması gereken yerlerde kullanılmasının uygundur [4]. Giriş Tüneller ve portaller inşa edilirken püskürtme beton (shotcrete) sıkça kullanılan bir malzemedir. Püskürtme beton; normal betona göre daha ince taneli ve daha yüksek çimento oranlı özel tasarım betonun, yüksek basınçlı pompa yardımı ile püskürtülmesiyle uygulanır. Amerikan Beton Enstitüsü (ACI: American Concrete Institue), 1966 yılında püskürtme betonun tanımını; “Bir hortumla taşınarak bir yüzey üzerine basınçlı hava yardımıyla yüksek hızla püskürtülen beton ya da harç karışımı” olarak nitelendirmiştir [1]. Türk standartları Enstitüsü’nün tanımlamasına göre ise püskürtme beton; onarım veya yapım amacı ile önceden hazırlanmış olan betonun hava basıncı ile yüksek hızla uygulama yüzeyi veya uygulama alanına püskürtülerek elde edilen betondur [2]. Püskürtme beton, kullanım alanına göre 2 kategoride değerlendirilebilir. Bunlar yeraltı ve yer üstü kullanımlardır. Yeraltı kullanımda en sık kullanılan yer tünel inşaatlarıdır. Metro, karayolu gibi inşaatlarda sıkça karşılaştığımız bir sanat yapısı olan tünellerde, oldukça önemli bir uygulamadır. Ayrıca yeraltı madenleri, yeraltı depoları gibi alanlarda da kullanılmaktadır. Püskürtme beton yer üstünde ise özellikle yol şevleri gibi son şeklini almış yapılarda duraylılığı sağlamak ve kaplamak amacıyla kullanılmaktadır. Püskürtme betonun kazı işlerinde yaygın kullanılmasının sebebi, kazı yapılan yüzeylerde kazının hemen Geleneksel betona göre kalıp gerektirmeden yerleştirilmesi ve özel katkılar yardımıyla hemen dayanım kazanmaya başlayarak taşıyıcılığını yerine getirmeye başlaması gibi özellikleri nedeniyle birçok alanda yaygın olarak kullanılmaktadır. Püskürtme betonda taşıma, yerleştirme, sıkıştırma aşamaların bir anda yapılması, uygulamanın hızlı 72 Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Öztürk ve Başyiğit yüksek dayanım kazandırmış ve bu metotta yaygın olarak kullanılmaya başlanmıştır [6]. Yaş püskürtme beton uygulamasında ise, karışım normal betonda olduğu gibi hazırlanarak beton pompasına beslenir, pompa ile hidrolik olarak iletim borusundan püskürtme ucuna kadar ilerletililir ve püskürtme beton yüzeye uygulanmaktadır. Yaş karışım yönteminde karışıma ilave edilen priz hızlandırıcı katkılar sıvı halde olduğundan püskürtme ucuna ilave edilmektedir. ardından kolayca uygulanabilir olmasıdır [5]. Geleneksel betondan farklı olarak, püskürtme betonun taşınması ve yerleştirilmesi işlemi basınçlı hava ile yapılmaktadır. Püskürtme beton kuru karışım ve yaş karışım olmak üzere 2 ayrı yöntem ile uygulanabilmektedir. Kuru yöntemde, tüm malzemeler kuru ortamda hazırlanıp basınçlı hava ile püskürtme ucuna iletilmektedir. Hidratasyon için gerekli su karışıma püskürtme ucundan basınçlı olarak verilmektedir. Kuru karışımda püskürtme beton karışımına eklenecek olan katkı malzemesi varsa; katkı malzemesi sıvı ise su ile beraber püskürtme ucundan, katı ise püskürtme beton makinesinin içerisinden karışıma eklenmektedir. Uygulama yapılırken çimentonun agregayı iyice sarması amacıyla kuru karışımdaki agrega ağırlığının %3-8’i kadar nem içermesi gerekmektedir [3]. Püskürtme beton 1970 yılına kadar yalnızca kuru sistem olarak uygulanmıştır. Giriş Bu tarihte yaş sistem devreye girmiş ancak yüksek su-çimento oranı kullanılması beton dayanımında düşme yarattığından yaygın uygulama görülmemiştir. 1975’de silikat ve plastisiteyi artırıcı katkıların püskürtme betonda başarıyla uygulanması yüksek kapasitede uygulanabilen yaş sisteme Püskürtme beton, normal betondaki gibi iri – ince agrega, çimento, su ve katkı maddeleri bileşenlerinden oluşan bir karışımdır. Püskürtme beton karışımlarında, beton bileşenleri betonun mekanik özellikleri ile doğrudan ilgili olduğu için malzeme bileşenleri ve bileşen özelliklerini bilmek oldukça önemli bir husustur. Püskürtme beton uygulamalarında genellikle Portland çimentosu kullanılmaktadır [3]. Hammaddeleri farklı kaynaklardan gelen çimentoların priz süreleri farklılık gösterir. Doğal olarak bu durum katlı maddesinin etkinliğinde de kendini gösterecektir [7]. Genel kural olarak püskürtme betonda kullanılan çimentonun trikalsiyum alüminat (C3A) oranı ve özgül yüzeyinin yüksek olması, böylece priz hızlandırıcılarla birlikte hidratasyonu hızlı 73 Öztürk ve Başyiğit Materyal ve Metot Püskürtme betonu için yapılan deneylerden bir tanesi çimento – priz hızlandırıcı katkı uyum testi deneyidir. Bu deney, priz hızlandırıcı katılmış çimento harcının TS EN 196-1 standardında açıklandığı şekilde, priz başlangıcı – priz bitişinin belirlenmesi amacıyla yapılan bir deneydir. Yapılış amacı çerçevesinde harcın hızlı hazırlanması gerekmektedir. Karışımda uygulanması öngörülen oranlardaki (su/çimento) ve katkı oranında suyun ve hızlandırıcının çimentoya ilave edilmesinden sonra harç 15 saniye karıştırılmakta ve 25. saniyeden sonra vicat iğnesiyle priz başlangıç ve priz bitiş süresi belirlenmektedir. Sıvı hızlandırıcı kullanılan deneylerde katkı suya katılmakta ve su/çimento oranı hesabında su olarak kabul edilmektedir. Priz başlangıcı 5 dakika, priz bitişi ise 13 dakikadan küçük ise priz hızlandırıcı katkı uygun olarak nitelendirilmektedir [15]. Püskürtme betonda, istenilen özellikler sağlanması amacıyla bazı katkı malzemeleri kullanılmaktadır. Kullanılacak olan katkı malzemeleri, betonu fiziksel ve mekanik özelliklerine etki edeceği için bu katkılı betonlar laboratuarda test edilmesi gerekmektedir. Priz hızlandırıcı katkılar, kuru ve yaş püskürtme beton uygulamalarında en sık kullanılan katkı malzemesidir. Bu katkılar priz süresini kısaltmak, erken dayanımı artırmak, püskürtme betonun yüzey yapışma kalitesini iyileştirmek ve geri sıçramayı azaltmak amacıyla kullanılmaktadır. Priz hızlandırıcılar toz veya sıvı şeklindedir. Katkı malzemesinin miktarı, çimento ağırlığının %3 - %10’u arasında olması önerilmektedir [13]. Priz hızlandırıcı katkılar karışımın priz başlangıcını kısalttığından aynı zamanda geri sıçramayı da azaltır ve bir defada kalın bir tabaka oluşturabilmeyi sağlar. Fakat priz hızlandırıcı katkılar betonun nihai (28 günlük) basınç dayanımını %35’e varan oranla azaltmaktadırlar [14]. Püskürtme betonda kullanılan priz hızlandırıcı katkı türleri; alkali içeren priz hızlandırıcılar ve alkali içermeyen priz hızlandırıcılardır Püskürtme betonda, istenilen özellikler sağlanması amacıyla bazı katkı malzemeleri kullanılmaktadır. Kullanılacak olan katkı malzemeleri, betonu fiziksel ve mekanik özelliklerine etki edeceği için bu katkılı betonlar laboratuarda test edilmesi gerekmektedir. Priz hızlandırıcı katkılar, kuru ve yaş püskürtme beton uygulamalarında en sık kullanılan katkı malzemesidir. Bu katkılar priz süresini kısaltmak, erken dayanımı artırmak, püskürtme betonun yüzey yapışma kalitesini iyileştirmek ve geri sıçramayı azaltmak amacıyla kullanılmaktadır. Priz hızlandırıcılar toz veya sıvı şeklindedir. Katkı malzemesinin miktarı, çimento ağırlığının %3 - %10’u arasında olması önerilmektedir [13]. Priz hızlandırıcı katkılar karışımın priz başlangıcını kısalttığından aynı zamanda geri sıçramayı da azaltır ve bir defada kalın bir tabaka oluşturabilmeyi sağlar. Fakat priz hızlandırıcı katkılar betonun nihai (28 günlük) basınç dayanımını %35’e varan oranla azaltmaktadırlar [14]. Püskürtme betonda kullanılan priz hızlandırıcı katkı türleri; alkali içeren priz hızlandırıcılar ve alkali içermeyen priz hızlandırıcılardır [15]. Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Teknik Şartnamesinde verilen değerlere uygun olmalıdır [11]. Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 beton uygulamasında agrega granülometresi hakkında dikkat edilmesi gereken noktalar aşağıda belirtilmiştir: gelişen ve erken dayanım kazanan tipte olması istenmektedir [8]. Özel kullanım isteklerine bağlı olarak sülfatlı su bulunan ortamda sülfata dirençli çimento veya kaplamanın erken dayanım kazanması istenen durumlarda hızlı sertleşen çimento gibi farklı tipte çimentolar kullanılabilir [3]. Kullanılan çimento miktarı, püskürtme betondan istenen mekanik özelliklere bağlı olarak değişebilmektedir [9]. Karışımda kullanılacak olan su için çok sıkı kurallar bulunmamaktadır. Bununla beraber yağ, asit, organik madde gibi suyun kalitesini bozan ve suyu kirleten maddeler içermemesi istenmektedir. İçme suyu olarak kullanılabilen su karışım için uygundur. Agrega, beton malzemesinin ana taşıyıcı iskeletini oluşturmaktadır. Dane çapı 4 mm’ye kadar olan “ince agrega”, 4 mm’den büyük olanı ise “iri agrega” olarak tanımlanmaktadır [10]. Kullanılan agrega malzemesi dayanımlı, ayrışma ve aşınmaya dirençli olmalı, içerisinde kil, silt, alkali ve organik madde bulundurmamalıdır. Agrega dane boyut dağılımı açısından sahip olması gereken özellikler ve bunlara dair sınır değerler Karayolları Teknik Şartnamesinde verilmiştir [11]. beton uygulamasında agrega granülometresi hakkında dikkat edilmesi gereken noktalar aşağıda belirtilmiştir:  Maksimum dane boyutu 8 – 10 mm aralığında olmalıdır. Agrega 12 mm’den büyük dane içermemeli, 8 mm’den büyük malzeme miktarı ise %10’u geçmemelidir.  Her elek aralığındaki malzeme miktarı, dane boyut dağılımında kesikliğe yol açabileceği için %30’u geçmemelidir.  İnce malzeme miktarı dayanım ve işlenebilirlik üzerinde etkilidir. 0,125 mm’den ince malzeme miktarı en az %4-5 olmalı, %8-9’u geçmemelidir [12]. Betonun kütlece yaklaşık %70-80’ini agregaların oluşturduğu göz önüne alındığında; agregaların sıcaklığı, sıcak ya da soğuk hava koşullarında beton üretiminde taze beton sıcaklığının en önemli belirleyicisidir. Bu nedenle taze beton sıcaklığı, plent çıkışında 30 °C’yi ve yerine yerleştirilirken de 32 °C’yi aşmayacak şekilde agreganın ısıtılması ya da soğutulması, karma suyuna buz katılması (beton içinde buz parçaları olmadan) gibi önlemler alınmalıdır.  Betonun kütlece yaklaşık %70-80’ini agregaların oluşturduğu göz önüne alındığında; agregaların sıcaklığı, sıcak ya da soğuk hava koşullarında beton üretiminde taze beton sıcaklığının en önemli belirleyicisidir. Bu nedenle taze beton sıcaklığı, plent çıkışında 30 °C’yi ve yerine yerleştirilirken de 32 °C’yi aşmayacak şekilde agreganın ısıtılması ya da soğutulması, karma suyuna buz katılması (beton içinde buz parçaları olmadan) gibi önlemler alınmalıdır. Püskürtme betonun granülometrik birleşimi ve maksimum agrega boyutu ise normal betonarme betonundan farklıdır. Püskürtme betonda kullanılacak agregaların granülometrisi Karayolları Püskürtme beton uygulamasında en büyük agrega çapı, kullanılan donanım ve iletim borusu çapı ile sınırlıdır. Püskürtme 74 Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Öztürk ve Başyiğit Materyal ve Metot Yapıdaki betonu karakterize etmesi amacıyla boy/çap oranı, 1,0’dan farklı olarak alınan karot numuneleri başlık yapıldıktan sonraki boy/çap= 1,0 olacak şekilde kesilerek düzeltildikten sonra basınç dayanım testine tabi tutulmuştur. Tablo 1’de püskürtme beton için basınç dayanım sınıfları ve kalite kontrol kriterleri verilmiştir. 75 Öztürk ve Başyiğit C 20/25 MPa sınıfındaki püskürtme betonun basınç dayanımının gelişim hızı, Tablo 1’e uygun yapılmıştır. Tablo1. Püskürtme beton için basınç dayanım sınıfları ve kalite kontrol kriterleri [11] Beton sınıfları Karakteristik basınç dayanımı (fckp) (MPa) Püskürtme betondan alınacak karot numunede dayanım yönünden (28 günlük) aranacak kalite kontrol kriterleri Karot numune Boy (H)= 100 mm, Çap (D)= 100 mm Silindir Basınç dayanım sınıfları D= 150 mm, H= 300 mm, Silindir A= 150 mm Küp Bireysel minimum dayanım (MPa) 3 adet numuneden oluşan grubun ortalama minimum dayanımı (MPa) C 20/25 20 25 19,0 21,5 C 25/30 25 30 22,5 25,5 C 30/37 30 37 28,0 31,5 C 35/45 35 45 34,0 38,5 C 40/50 40 50 37,5 42,5 C 45/55 45 55 41,5 47,0 C 50/60 50 60 45,0 51,0 Tablo 1’e uygun yapılmıştır. Yapılan deneylerde, tünellerde kullanılan püskürtme betonda alkali içermeyen sıvı priz hızlandırıcı katkılar değerlendirilmiştir. TS 11747 standardına göre, püskürtme beton için kullanılabilecek katkılar; priz hızlandırıcı, hava sürükleyici, su azaltıcı ve geciktirici katkılar olarak verilmiştir [2]. Bu deney çalışmasında, püskürtme betonlarda en çok kullanılan priz hızlandırıcı katkıların beton dayanımına ve priz süresine olan etkileri incelenmiştir. Püskürtme beton karışımı hazırlanırken ıslak ve kuru olmak üzere iki metot bulunmaktadır. Bu deney kapsamında uygulanan metot ıslak metottur. Alkali içermeyen priz hızlandırıcı katkı malzemesi üreticisinden alınan bilgiler doğrultusunda, su/bağlayıcı oranı 0,5’den küçük olmalı, tercihen 0,45 olmalıdır. Düşük su/bağlayıcı oranı, daha yüksek erken dayanım, daha iyi durabilite, daha düşük priz hızlandırıcı sarfiyatı ve baş üstü uygulamalarında daha kalın katmanlar halinde uygulama imkânı sağlamaktadır [13]. Yapılan deneyler 0,45 oranı kullanılarak yapılmıştır. Priz hızlandırıcı katkının dozajı, EFNARC Avrupa Püskürtme Beton Şartnamesi (1996) Ek 1, Madde 6.3’te belirtilen sınırlar dikkate alınarak belirlenmektedir [16]. Tablo 2’de priz değerlendirme değerleri verilmiştir. Priz hızlandırıcı katkı malzemesi kullanım miktarı, su/ bağlayıcı oranı, sıcaklık (betonun ve ortamın), çimento reaktivitesi, uygulanacak püskürtme beton kalınlığı, priz süresi ve 0,5’den küçük olmalı, tercihen 0,45 olmalıdır. Düşük su/bağlayıcı oranı, daha yüksek erken dayanım, daha iyi durabilite, daha düşük priz hızlandırıcı sarfiyatı ve baş üstü uygulamalarında daha kalın katmanlar halinde uygulama imkânı sağlamaktadır [13]. Yapılan deneyler 0,45 oranı kullanılarak yapılmıştır. Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 erken dayanım gelişimine bağlıdır. Katkı, üreticisinden alınan bilgiler kapsamında normal koşullarda bağlayıcı ağırlığının %4-10’u mertebesinde kullanılmaktadır. Tablo 2. Priz değerlendirme tablosu İlk Priz Son Priz 24 sa Dayanım Değerlendirme < 2 dakika < 6 dakika > 15 MPa İyi 2 - 5 dakika 8-13 dakika 10-15 MPa Kabul edilebilir > 5 dakika > 13 dakika < 10 MPa Kabul edilemez Tablo 2. Priz değerlendirme tablosu artırılırsa %50 mertebelerine ulaşabileceği gözlemlenmiştir [17]. Yapılan bazı çalışmalarda, püskürtme betonda kullanılan priz hızlandırıcı katkı malzemelerinin 28 günlük basınç dayanımlarında, referans karışımlara göre %20 - %25 oranında azalmaya neden olduğu görülmektedir. Bu dayanım azalışının, katkı miktarı daha da Tablo 3’te deneylerde kullanılan püskürtme betona ait karışım hesabı ve priz hızlandırıcı katkı malzemesinin özellikleri yer almaktadır. Tablo 3. Püskürtme Beton Karışım Hesabı ve Malzeme Özellikleri Çimento Cinsi CEM-I 42,5 Çimento Miktarı 475 kg/m³ Katkı Cinsi Priz Hızlandırıcı BASF SA-160 Katkı Miktarı %5 %8 %10 Su Miktarı 213 kg/m³ Agrega Taş Ocağı Karpuzkaya Taş Ocağı Maksimum Agrega Boyutu 12,5 mm Agrega yüzdesi ve Miktarı 0 – 5 mm %60 - 972,75 kg/m³ 5 – 12,5 mm %40 - 662,74 kg/m³ Tablo 3. Püskürtme Beton Karışım Hesabı ve Malzeme Özellikleri çimento miktarı ve tipi, agrega miktarı ve özellikleri sabit tutulmuştur. Materyal ve Metot Priz hızlandırıcı katkının dozajı, EFNARC Avrupa Püskürtme Beton Şartnamesi (1996) Ek 1, Madde 6.3’te belirtilen sınırlar dikkate alınarak belirlenmektedir [16]. Tablo 2’de priz değerlendirme değerleri verilmiştir. Priz hızlandırıcı katkı malzemesi kullanım miktarı, su/ bağlayıcı oranı, sıcaklık (betonun ve ortamın), çimento reaktivitesi, uygulanacak püskürtme beton kalınlığı, priz süresi ve 76 Öztürk ve Başyiğit Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Araştırma Bulguları %5, %8, %10 oranlarında katkı kullanılarak ve katkı kullanılmadan, püskürtme betonu için çimento priz hızlandırıcı katkı uyum testi ve beton basınç dayanım deneyleri yapılmıştır. Yapılan deneylerde, su/çimento oranı, Priz hızlandırıcı katkı uyum testi için toplamda 6 adet numune hazırlanmıştır. Tablo 4’te farklı yüzdelerde kullanılan priz hızlandırıcı yaş püskürtme betonu için çimento priz hızlandırıcı katkı uyum test değerleri verilmiştir. Şekil 1’de 77 Öztürk ve Başyiğit Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 farklı yüzdelerde priz hızlandırıcı katkı kullanılarak elde edilen numunelerin priz süreleri verilmiştir. Tablo 4. Farklı yüzdelerde kullanılan priz hızlandırıcı yaş püskürtme betonu için çimento priz hızlandırıcı katkı uyum testi Püskürtme betonu için çimento priz hızlandırıcı katkı uyum testi Numune No Katkı Miktarı % Priz Başlangıcı (sn) Priz Bitişi (sn) Priz Başlangıcı Değerlendirme Priz Bitiş Değerlendirme 1 5 131 269 Kabul edilebilir İyi 2 5 127 261 Kabul edilebilir iyi 3 8 81 158 İyi İyi 4 8 83 164 İyi İyi 5 10 69 144 İyi İyi 6 10 67 140 İyi İyi o 4. Farklı yüzdelerde kullanılan priz hızlandırıcı yaş püskürtme betonu için çimento pr hızlandırıcı katkı uyum testi Şekil 1. Farklı yüzdelerde priz hızlandırıcı katkı kullanılarak elde edilen numunelerin priz süreleri (sn) Şekil 1. Farklı yüzdelerde priz hızlandırıcı katkı kullanılarak elde edilen numunelerin priz süreleri (sn) Elde edilen değerler Tablo 4 ile değerlendirildiğinde, %5 oranında priz hızlandırıcı katkı kullanıldığında priz başlangıç süresi kabul edilebilir olarak değerlendirilmektedir. %5 katkı kullanımı için priz bitiş süresi, %8 ve %10 katkı kullanımındaki priz başlangıç ve priz bitiş süreleri ise iyi olarak değerlendirilmektedir. 150150150 mm boyutlarında küp katkısız kontrol numuneleri ve %5, %8 ve %10 oranlarında priz hızlandırıcı katkı kullanılarak elde edilen numuneler üzerinde 3, 7 ve 28 günlük basınç dayanımı deneyi yapılmıştır. Tablo 5’te katkısız kontrol numuneleri için 3, 7 ve 28 günlük basınç 78 Öztürk ve Başyiğit Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Tablo 6. %5 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) % 5 3 26,1 27,0 27,5 27,4 7 34,9 36,7 37,8 37.3 28 41,7 43,5 44,8 44,0 Tablo 6. %5 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri dayanım değerleri yer almaktadır. Tablo 6’da %5 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri yer alırken, Şekil 2’de katkısız numune ile %5 katkılı numune basınç dayanımı gün grafiği yer almaktadır. Tablo 7’de, %8 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri yer alırken, Şekil 3’te katkısız numune ile %8 katkılı numune basınç dayanımı gün grafiği yer almaktadır. Tablo 8’de %10 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri yer alırken, Şekil 4’te katkısız numune ile %10 katkılı numune basınç dayanımı gün grafiği yer almaktadır. Şekil 2. Katkısız numune ile %5 katkılı numune basınç dayanımı gün grafiği almaktadır. Tablo 5. Katkısız kontrol numuneleri için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) % 0 3 35,1 36,7 38,8 36,7 7 41,0 43,2 44,8 43,9 28 50,3 53,1 55,1 54,0 Şekil 2. Katkısız numune ile %5 katkılı numune basınç dayanımı gün grafiği Tablo 5. Katkısız kontrol numuneleri için 3, 7 ve 28 günlük basınç dayanım değerleri Tablo 5. Katkısız kontrol numuneleri için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) % 0 3 35,1 36,7 38,8 36,7 7 41,0 43,2 44,8 43,9 28 50,3 53,1 55,1 54,0 Tablo 5. Katkısız kontrol numuneleri için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) % 0 3 35,1 36,7 38,8 36,7 7 41,0 43,2 44,8 43,9 28 50,3 53,1 55,1 54,0 Şekil 2. Katkısız numune ile %5 katkılı numune basınç dayanımı gün grafiği 79 Öztürk ve Başyiğit Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 ISSN: 2536 4383 Tablo7. Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 %8 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) % 8 3 23,8 23,3 22,5 23,6 7 32,6 31,5 30,2 31,7 28 38,7 37,9 36,7 38,4 Şekil 3. Katkısız numune ile %8 katkılı numune basınç dayanımı gün grafiği Tablo 8. %10 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) %10 3 20,4 20,1 20,8 19,4 7 28,0 28,0 28,8 27,2 28 32,3 32,5 33,9 31,4 Şekil 4. Katkısız numune ile %10 katkılı numune basınç dayanımı gün grafiği Farklı katkı yüzdeleriyle yapılan Tablo7. %8 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) % 8 3 23,8 23,3 22,5 23,6 7 32,6 31,5 30,2 31,7 28 38,7 37,9 36,7 38,4 Şekil 3. Katkısız numune ile %8 katkılı numune basınç dayanımı gün grafiği Tablo 8. %10 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) %10 3 20,4 20,1 20,8 19,4 7 28,0 28,0 28,8 27,2 28 32,3 32,5 33,9 31,4 Şekil 4. Katkısız numune ile %10 katkılı numune basınç dayanımı gün grafiği F kl k tk ü d l i l l Tablo 8. %10 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) %10 3 20,4 20,1 20,8 19,4 7 28,0 28,0 28,8 27,2 28 32,3 32,5 33,9 31,4 Tablo7. %8 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) % 8 3 23,8 23,3 22,5 23,6 7 32,6 31,5 30,2 31,7 28 38,7 37,9 36,7 38,4 Tablo7. %8 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) % 8 3 23,8 23,3 22,5 23,6 7 32,6 31,5 30,2 31,7 28 38,7 37,9 36,7 38,4 Tablo7. %8 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Tablo 8. %10 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Tablo 8. Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 grubun ortalama minimum değeri standartlara uygun olmalıdır. Kullanılan tüm katkı yüzdelerinde Karayolu Teknik şartnamesi değerine uyulmaktadır. Bu değerler Tablo 9’da verilmiştir. Tablo 9. Farkı yüzdelerde kullanılan katkı miktarı için 28 günlük basınç dayanım değerleri değerlendirmesi kı yüzdelerde kullanılan katkı miktarı için 28 günlük basınç dayanım değerleri esi Tablo 9. Farkı yüzdelerde kullanılan katkı miktarı için 28 günlük basınç dayanım değerleri değerlendirmesi Katkı Miktarı % Süre (Gün) Basınç Dayanımı (N/mm²) Karayolu Teknik Şartnamesi (C 20/25) [10] Ortalama Basınç Dayanımı (N/mm²) Karayolu Teknik Şartnamesi (C 20/25) [10] %5 28 41,7 > 19,0 43,5 > 21,5 44,8 44,0 %8 28 38,7 > 19,0 37,9 > 21,5 36,7 38,4 %10 28 32,3 > 19,0 32,5 > 21,5 33,9 31,4 göz önüne alındığında uygunluk sağlamamaktadır. Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 %10 katkı miktarı için 3, 7 ve 28 günlük basınç dayanım değerleri Katkı Miktarı % Süre (Gün) Basınç Dayanım Değerleri (N/mm²) Ortalama Basınç Dayanım Değerleri (N/mm²) %10 3 20,4 20,1 20,8 19,4 7 28,0 28,0 28,8 27,2 28 32,3 32,5 33,9 31,4 Şekil 4. Katkısız numune ile %10 katkılı numune basınç dayanımı gün grafiği Şekil 4. Katkısız numune ile %10 katkılı numune basınç dayanımı gün grafiği Şekil 4. Katkısız numune ile %10 katkılı numune basınç dayanımı gün grafiği Şekil 3. Katkısız numune ile %8 katkılı numune basınç dayanımı gün grafiği Farklı katkı yüzdeleriyle yapılan deneylerde elde edilen 28 günlük basınç dayanım değerleri, Tablo 1’deki değerlere uygun olmalıdır. C 20/25 püskürtme betonu olarak tasarlandığı için bu beton sınıfının 28 günlük bireysel minimum dayanım ve 3 adet numuneden oluşan 80 Öztürk ve Başyiğit Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 Kaynaklar [1] ACI Committe 506, 1966. Recommended Practice for Shotcrete (ACI 506 – 66). Shotcreting, ACI SP-14. [2] TS 11747, 1995. Püskürtme Beton (Shotcrete) Yapım, Uygulama ve Bakım Kuralları. Türk Standartları Enstitüsü, Birinci Baskı, ICS 91.100.30. Ankara. [2] TS 11747, 1995. Püskürtme Beton (Shotcrete) Yapım, Uygulama ve Bakım Kuralları. Türk Standartları Enstitüsü, Birinci Baskı, ICS 91.100.30. Ankara. [3] Arıoğlu, E., Yüksel, A., Yılmaz, A. O., 2008. Püskürtme Beton Bilgi Föyleri – Çözümlü Problemler. TMMOB Maden Mühendisleri Odası Yayın No: 142, İstanbul.  %8 oranında kullanılan priz hızlandırıcı katkı kullanımı, EFNARC Avrupa Püskürtme Beton Şartnamesi (1996) Ek 1, Madde 6.3’te ve Karayolu Teknik Şartnamesinde (2013) [11] belirtilen priz başlama ve priz sona erme sürelerinde belirtilen priz değerlendirme koşullarına göre, 2 dakikanın altında olan 1 dakika 52 saniye değeri elde edilen ilk priz değeri ideal bir sonuçtur. 1 dakika 52 saniyeden kısa olacak ilk priz süreleri, beton dayanımını daha da azaltacak ve şantiyede püskürtme beton uygulamasını zorlaştıracaktır. Son priz değeri olarak elde edilen 2 dakika 35 saniye değeri de, 6 dakikadan düşük bir zamandır ve “iyi” olarak değerlendirilmektedir. [19]  %8 oranında kullanılan priz hızlandırıcı katkı kullanımı, EFNARC Avrupa Püskürtme Beton Şartnamesi (1996) Ek 1, Madde 6.3’te ve Karayolu Teknik Şartnamesinde (2013) [11] belirtilen priz başlama ve priz sona erme sürelerinde belirtilen priz değerlendirme koşullarına göre, 2 dakikanın altında olan 1 dakika 52 saniye değeri elde edilen ilk priz değeri ideal bir sonuçtur. 1 dakika 52 saniyeden kısa olacak ilk priz süreleri, beton dayanımını daha da azaltacak ve şantiyede püskürtme beton uygulamasını zorlaştıracaktır. Son priz değeri olarak elde edilen 2 dakika 35 saniye değeri de, 6 dakikadan düşük bir zamandır ve “iyi” olarak değerlendirilmektedir. [19] [4] Aka, İ., ve Celep, Z., 1978. Püskürtme Beton ve Uygulaması. İstanbul Teknik Üniversitesi Mühendislik ve Mimarlık Fakültesi, Betonarme, Ahşap ve Çelik Yapılar Kürsüsü, İstanbul. [5] Özdoğan, M. V., 2009. Yer altı Yapılarında Püskürtme Beton ve Dolgu Dizaynı. Dokuz Eylül Üniversitesi Fen Bilimleri Enstitüsü Yüksek Lisans Tezi, İzmir. [6] Sümer, T., 1994. Püskürtme Beton Malzeme, Ekipman, Katkı Seçim Kriterleri ve Maliyet Optimizasyonu, TMMOB İnşaat Mühendisleri Odası, İzmir Şubesi Haber Bülteni, Sayı: 56, 18-21s., İzmir. [7] ACI Committe 116, 1990. Cement and Concrete Terminology, ACI 116R-90, SP- 19 (90), p.54. American Concrete Institute, Detroit. [8] Yi S T, Heo G, 2010. Experimental study on the setting time of cement paste mixed with accelerating admixtures based on C(3)A, Advances in cement research 22(3): 149-155. Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 bir basınç dayanımı azalmasına neden olmuştur. Birimi Başkanlığı tarafından desteklenmiştir.  Elde edilmiş olan bu sonuç, yapılan araştırma sonuçları ile kıyaslanmıştır. Priz Hızlandırıcı katkılar, katkının dozaj oranına, çimento kimyasına ve kendi kimyasına bağlı olarak farklı etkilere sahiptir. Birçok hızlandırıcı katkı, çimentonun ve hızlandırıcının uyumuna bağlı olarak 28 günlük dayanımı %25-40 oranında azaltmaktadır [18]. Sonuç Çalışmada, tünellerde kullanılan priz hızlandırıcı katkılar için yapılan deneysel çalışmalar ve değerlendirmeler incelenmiştir. Deneysel çalışmalar sonucunda elde edilen verilerin değerlendirilmesi aşağıda yer almaktadır. Çalışmada, tünellerde kullanılan priz hızlandırıcı katkılar için yapılan deneysel çalışmalar ve değerlendirmeler incelenmiştir. Deneysel çalışmalar sonucunda elde edilen verilerin değerlendirilmesi aşağıda yer almaktadır.  %5 priz hızlandırıcı katkı malzemesi kullanılması durumunda, priz süresi değerlendirme tablosuna göre ilk priz süresi kabul edilebilir olarak değerlendirilmiştir.  Tüm yüzdeler incelendiğinde yapılan deney sonuçlarına göre %8 priz hızlandırıcı katkı miktarının en uygun sonucu verdiği gözlemlenmiştir.  Deneylerde kullanılan %10 priz hızlandırıcı katkı miktarı, priz süresini oldukça kısaltmaktadır. Priz süresinin bu denli kısalması, uygulama açısından zorluklar oluşturmaktadır. Bu sebepten %10 katkı miktarı, elde edilen priz süreleri  Optimum olarak belirlenen %8’lik priz hızlandırıcı katkı, katkısız kontrol numunesine göre değerlendirilirse %29’luk 81 Öztürk ve Başyiğit Teşekkür Bu çalışma, 4383-YL1-15 numaralı proje ile Süleyman Demirel Üniversitesi Bilimsel Araştırma Projeleri Yönetim 82 Öztürk ve Başyiğit Sinop Uni J Nat Sci 4 (1): 71-83(2019) ISSN: 2536-4383 [9] Bourchier, F., B., 1990. Practical Considerations of Shotcrete in an Underground Mining Enviroment. Toronto Üniversitesi Doktora Tezi, Toronto. [19] Erdoğan T Y, 2016. Beton, ODTÜ Geliştirme Vakfı Yayıncılık ve İletişim A.Ş., Ankara. [10] TS 706 EN 12620, 2006. Beton Agregaları. Türk Standartları Enstitüsü, Ankara. [11] Karayolu Teknik Şartnamesi, 2013. T.C. Ulaştırma Denizcilik ve Haberleşme Bakanlığı, Karayolları Genel Müdürlüğü, Ankara. [12] EFNARC, 1999. European Specification for Sprayed Concrete Guidelines, ISBN. 0952248360, p28. [13] Melbye, T., 2006, Sprayed Concrete for Rock Support. UGC International. Division of Degussa Construction Chemicals (Switzerland) Ltd. 11th Edition, Switzerland. [14] Levent, Y., Kalmış, M., 2014. Püskürtme Beton Uygulamalarında Yaşanan Sorunlar ve Çözümleri, İksa Kurs Notları, İstanbul. [15] Neville A M, 2011. Properties of concrete, Pearson Education Limited, England. [16] EFNARC, 1996. European Specification for Sprayed Concrete, UK. [17] Yıldırım, H., Uyan, M., Kemerli, K., 1996. “Priz Hızlandırıcılı Püskürtme Beton Katkılarının Dayanıma Etkisi”, 4. Ulusal Beton Kongresi, TMMOB İnşaat Mühendisleri Odası, İstanbul. [18] U.S. Army Corps of Engineers, Engineering and Design Standard Practice for Shotcrete. Department of the Army U.S, 2005. Army Corps of Engineers, Distribution Restriction Statement. CECW-EG Engineering Manual No: 1110- 2-2005, Washington. 83
https://openalex.org/W3100525054	https://www.nature.com/articles/s41467-020-15389-4.pdf	English	null	Cavity electromechanics with parametric mechanical driving	Nature communications	2,020	cc-by	11,061	ARTICLE ARTICLE Cavity electromechanics with parametric mechanical driving https://doi.org/10.1038/s41467-020-15389-4 OPEN D. Bothner1✉, S. Yanai1, A. Iniguez-Rabago 1, M. Yuan1,2, Ya.M. Blanter1 & G.A. Ste Microwave optomechanical circuits have been demonstrated to be powerful tools for both exploring fundamental physics of macroscopic mechanical oscillators, as well as being pro- mising candidates for on-chip quantum-limited microwave devices. In most experiments so far, the mechanical oscillator is either used as a passive element and its displacement is detected using the superconducting cavity, or manipulated by intracavity ﬁelds. Here, we explore the possibility to directly and parametrically manipulate the mechanical nanobeam resonator of a cavity electromechanical system, which provides additional functionality to the toolbox of microwave optomechanics. In addition to using the cavity as an interferometer to detect parametrically modulated mechanical displacement and squeezed thermomechanical motion, we demonstrate that this approach can realize a phase-sensitive parametric ampliﬁer for intracavity microwave photons. Future perspectives of optomechanical systems with a parametrically driven mechanical oscillator include exotic bath engineering with negative effective photon temperatures, or systems with enhanced optomechanical nonlinearities. 1 Kavli Institute of Nanoscience, Delft University of Technology, PO Box 5046, 2600 GA Delft, The Netherlands. 2Present address: Paul-Drude-Institut für Festkörperphysik Leibniz-Institut im Forschungsverbund Berlin e.V., Hausvogteiplatz 5-7, 10117 Berlin, Germany. ✉email: daniel.bothner@gmail.com; g.a.steele@tudelft.nl NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 S uperconducting microwave circuits have been demonstrated to be extremely powerful tools for the ﬁelds of quantum information processing1–3, circuit quantum electrodyna- mics4–8, astrophysical detector technologies9 and microwave optomechanics10–12. In the latter, microwave ﬁelds in super- conducting cavities are parametrically coupled to mechanical elements such as suspended capacitor drumheads or metallized nanobeams, enabling high-precision detection and manipulation of mechanical motion. Milestones achieved in the ﬁeld include sideband-cooling of mechanical oscillators to the quantum ground state11, strong coupling between photons and phonons13, the generation of non-Gaussian states of motion14–16 or the entanglement between two mechanical oscillators17. mechanical element in an microwave electromechanical cavity using a combination of DC ﬁelds and electrical ﬁelds resonant with the lower frequency mechanical device have been used in the past for probing mechanical resonators in cavity devices10,29,30. These schemes also allow tuning of the mechanical frequency in an optomechanical cavity29–31 and enable direct parametric driving of the mechanical resonator32. Using this electrostatic tuning for parametric driving in an electromechanical system, however, has until now not been explored. Results The device. Figure 1 shows an image of a superconducting coplanar waveguide (CPW) quarter-wavelength (λ∕4) resonator used as a microwave cavity. The cavity is patterned from a 50 μm 400 μm Ground Ground Ground Feedline Cavity center line a b 50 μm 400 μm 1 μm Ground Ground Ground Feedline Cavity center line MoRe SiN Si 15 mK 4 K MW in MW out DC in Frequency (GHz) 6.429 6.432 6.438 6.435 Magnitude of S21 (dB) 0 –10 –20 1.43145 1.43150 1.43155 Mechanical amplitude (arb. units) Frequency (MHz) DC Voltage (V) –5 –2.5 0 2.5 5 1.35 1.40 1.45 1.50 Frequency (MHz) d e f g a b c Fig. 1 Superconducting circuit nano-electromechanical system with electrostatic and low-frequency access. a False-color scanning electron microscopy image of a superconducting quarter-wavelength cavity (here for ωc = 2π ⋅7.5 GHz), capacitively side-coupled to a coplanar waveguide feedline. The molybdenum-rhenium (MoRe) metallization is shown in blue and the silicon (Si) substrate in gray. b Zoom into the coupling capacitance region, where the mechanical nanobeam as part of the coupling capacitance is visible. The dimensions of the beam, which consists of MoRe on top of high-stress silicon- nitride (Si3N4), are 100 μm × 150 nm × 143 nm. c A magniﬁed view of the suspended nanobeam. d Simpliﬁed circuit and measurement scheme, showing a lumped element circuit representation of the device as well as the microwave (MW) input and output lines (including a DC block and high electron mobility transistor ampliﬁer shown as triangle) and the DC (directed current) input line connected to the microwave lines via a bias-tee. A more detailed version of the setup is given in Supplementary Note 1. e Cavity resonance data (black) and ﬁt curve (orange). From the ﬁt, we extract the cavity resonance frequency ωc = 2π ⋅6.434 GHz and the internal and external linewidths κi = 2π ⋅370 kHz and κe = 2π ⋅5.7 MHz, respectively. f Resonance curve of the mechanical oscillator readout via the superconducting cavity. Data are shown as black dots, a Lorentzian ﬁt as orange line. From the ﬁt we extract the mechanical resonance frequency Ωm = 2π ⋅1.4315 MHz and a quality factor Qm = 195,000. g Optomechanically detected excitation spectrum of the nanobeam vs. applied DC voltage. The bright line resembling an inverted parabola represents the resonance of the in-plane mode, which was used everywhere throughout this paper. Cavity electromechanics with parametric mechanical driving https://doi.org/10.1038/s41467-020-15389-4 OPEN Here, we present measurements of a superconducting micro- wave optomechanical device in which we use direct electro- static driving to achieve strong parametric modulation of the mechanical resonator. By modulating the mechanical reso- nance frequency, we generate phase-sensitive parametric ampli- tude ampliﬁcation and thermomechanical noise squeezing of the mechanical motion, both detected using optomechanical cavity interferometry10. Furthermore, we demonstrate how parametric modulation of the mechanical resonance frequency can be used to generate phase-sensitive ampliﬁcation of a microwave probe tone, which is three orders of magnitude larger in frequency than the parametric pump tone itself. For the operation of the microwave ampliﬁer, the optomechanical system can be driven on the red cavity sideband, which allows for simultaneous mechanical cooling and microwave ampliﬁcation. The experi- mental implementation presented here provides an optomecha- nical platform for further exploration of phase-sensitive quantum-limited ampliﬁcation and photon bath engineering using mechanical parametric driving. Recently, there are increasing efforts taken towards building passive and active quantum-limited microwave elements for quantum technologies based on microwave optomechanical cir- cuits, connecting the ﬁelds of microwave optomechanics, circuit quantum electrodynamics and quantum information science18–20. Among the most important developments into this direction are the demonstration of microwave ampliﬁcation by blue sideband driving in simple optomechanical circuits21, and the realization of direc- tional microwave ampliﬁers22 as well as microwave circulators23,24 in more complex multimode systems25. Recent theoretical work26–28 on optomechanical systems with a parametrically driven mechanical oscillator proposed the use of mechanical parametric driving to enable parametric ampliﬁcation with enhanced bandwidth and reduced added noise, compared to the case of an optomechanical ampliﬁer using a blue-sideband drive26. Furthermore, the authors predict that there is a para- meter regime that results in an effective density of states, which can be interpreted as an effective negative temperature for cavity photons26. Other related recent works have predicted enhance- ments of the optomechanical coupling27 and the generation of non-Gaussian microwave states28. Direct electrostatic driving of a Results The thin second line around 1.48 MHz corresponds to the mechanical out-of-plane mode. The red dashed line at Vdc = −4 V indicates the voltage operation point we chose to use. Frequency (GHz) 6.429 6.432 6.438 6.435 Magnitude of S21 (dB) 0 –10 –20 e f 15 mK 4 K MW in MW out DC in d g d e a DC Voltage (V) –5 –2.5 0 2.5 5 1.35 1.40 1.45 1.50 Frequency (MHz) g Frequency (MHz) Frequency (GHz) 1.43145 1.43150 1.43155 Mechanical amplitude (arb. units) Frequency (MHz) f f c DC Voltage (V) DC Voltage (V) Frequency (MHz) Frequency (MHz) Fig. 1 Superconducting circuit nano-electromechanical system with electrostatic and low-frequency access. a False- o-electromechanical system with electrostatic and low-frequency access. a False-color scanning electron microscopy Fig. 1 Superconducting circuit nano-electromechanical system with electrostatic and low-frequency access. a False-color scanning electron microscopy image of a superconducting quarter-wavelength cavity (here for ωc = 2π ⋅7.5 GHz), capacitively side-coupled to a coplanar waveguide feedline. The molybdenum-rhenium (MoRe) metallization is shown in blue and the silicon (Si) substrate in gray. b Zoom into the coupling capacitance region, where the mechanical nanobeam as part of the coupling capacitance is visible. The dimensions of the beam, which consists of MoRe on top of high-stress silicon- nitride (Si3N4), are 100 μm × 150 nm × 143 nm. c A magniﬁed view of the suspended nanobeam. d Simpliﬁed circuit and measurement scheme, showing a lumped element circuit representation of the device as well as the microwave (MW) input and output lines (including a DC block and high electron mobility transistor ampliﬁer shown as triangle) and the DC (directed current) input line connected to the microwave lines via a bias-tee. A more detailed version of the setup is given in Supplementary Note 1. e Cavity resonance data (black) and ﬁt curve (orange). From the ﬁt, we extract the cavity resonance frequency ωc = 2π ⋅6.434 GHz and the internal and external linewidths κi = 2π ⋅370 kHz and κe = 2π ⋅5.7 MHz, respectively. f Resonance curve of the mechanical oscillator readout via the superconducting cavity. Data are shown as black dots, a Lorentzian ﬁt as orange line. From the ﬁt we extract the mechanical resonance frequency Ωm = 2π ⋅1.4315 MHz and a quality factor Qm = 195,000. g Optomechanically detected excitation spectrum of the nanobeam vs. applied DC voltage. Results where m is the effective nanowire mass, x is the effective nanowire displacement, k0 = km + kdc, kp ∝VdcV2Ω and F0 ∝VdcV0. From an approximate solution of this equation of motion, the parametric amplitude gain Gp = ∣x∣on∕∣x∣off can be derived to be given by where m is the effective nanowire mass, x is the effective nanowire displacement, k0 = km + kdc, kp ∝VdcV2Ω and F0 ∝VdcV0. From an approximate solution of this equation of motion, the parametric amplitude gain Gp = ∣x∣on∕∣x∣off can be derived to be given by Gp ¼ cos2ðϕp þ φÞ 1 þ V2Ω Vt 2 þ sin2ðϕp þ φÞ 1 V2Ω Vt 2 2 64 3 75 1=2 : ð2Þ ð2Þ y g The mechanical nanobeam oscillator has a resonance frequency of its fundamental in-plane mode of Ωm0 = 2π ⋅1.475 MHz. It can be signiﬁcantly tuned by applying a DC voltage Vdc between center conductor and ground of the CPW feedline, adding an electrostatic spring constant to the intrinsic spring (cf. Supple- mentary Note 4). The measured functional dependence of the resonance frequency on DC voltage is shown in Fig. 1g. Throughout this whole article, we bias the mechanical resonator with Vdc = −4 V, leading to a resonance frequency Ωm = 2π ⋅ 1.4315 MHz and a linewidth Γm ≈2π ⋅7.5 Hz. A resonance curve of the mechanical oscillator at Vdc = −4 V is shown in Fig. 1f. The detuning-dependent threshold voltage Vt for parametric instability in this relation is given by Vt ¼ Vt0 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ 4Δ2 m Γ2 m s ð3Þ ð3Þ with the threshold voltage on resonance Vt0 and the detuning from mechanical resonance Δm = Ω −Ωm. The phase φ ¼ arctanð2Δm=ΓmÞ considers the detuning-dependent phase difference between the near-resonant driving force and the mechanical motion. Details on the theoretical treatment of the device are given in Supplementary Note 6. g The device is operated in a dilution refrigerator with a base temperature of Tb = 15 mK, which corresponds to a thermal cavity occupation of kBTb _ωc 0:05 photons. Assuming the mode temperature of the nanobeam being the fridge base temperature, we expect an average occupation of the mechanical mode with nm ¼ kBTm _Ωm 220 thermal phonons. g pp y Figure 2 summarizes our results on the phase and detuning- dependent parametric frequency modulation. Results When we excite the mechanical resonator exactly on resonance, apply a parametric modulation with twice the resonance frequency and sweep the phase ϕp, we ﬁnd an oscillatory behavior between amplitude ampliﬁcation and de-ampliﬁcation with a periodicity of Δϕp = π (cf. Fig. 2b). To explore the dependence of the ampliﬁcation on the parametric modulation amplitude V2Ω, we repeat this experiment for different voltages V2Ω and extract maximum and minimum gain by ﬁtting the data with Eq. (2) for Vt = Vt0 and φ = 0. The extracted values follow closely the theoretical curves up to a voltage V2Ω ≈0.9Vt0, above which we are limited by resonance frequency ﬂuctuations of the mechanical resonator. The maximum gain we achieve by this is about ~22 dB. Parametric mechanical amplitude ampliﬁcation. When the resonance frequency Ωm of a harmonic oscillator is modulated with twice the resonance frequency Ωp = 2Ωm, then a small starting amplitude of the oscillator motion can be increased or reduced, depending on the relative phase between the oscillator motion and the frequency modulation34,35. To modulate the resonance frequency of a mechanical oscillator, one of the relevant system parameters like the oscillator mass m or the restoring spring force constant k can be modulated. Here, we follow the latter approach and modulate the effective spring constant of the nanobeam by applying a combination of a static voltage Vdc and an oscillating voltage V2Ω sin 2Ωt with roughly twice the mechanical resonance frequency Ω ~ Ωm. The static voltage adds an electrostatic spring contribution kdc to the intrinsic spring constant km and the oscillating part modulates the total spring constant with ~2Ωm. The capacitive modulation of the mechanical resonance frequency is a natural choice for superconducting cavity electromechanics36, but other possibilities have been explored as well, mainly in the optical domain with nonmetallized mechanical oscillators. It has been demonstrated that the time-varying dyna- mical backaction of modulated laser beams37,38 and switching of trapping frequencies for levitated dielectric particles39 can also be utilized for mechanical parametric ampliﬁcation. g y In order to characterize the device response also for drive frequencies detuned from resonance, we repeat the above measurements for different detunings and extract the maximum and minimum gain for each of these data sets. Hereby, we always keep the parametric drive frequency twice the excitation frequency and not twice the resonance frequency. NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications Results The bright line resembling an inverted parabola represents the resonance of the in-plane mode, which was used everywhere throughout this paper. The thin second line around 1.48 MHz corresponds to the mechanical out-of-plane mode. The red dashed line at Vdc = −4 V indicates the voltage operation point we chose to use. NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 voltage V0 cosðΩt þ ϕpÞ and characterize its steady-state dis- placement amplitude depending on the parametric modulation amplitude V2Ω and on the relative phase difference between resonant drive and parametric modulation ϕp. The mechanical amplitude is detected by monitoring the optomechanically generated sidebands to a microwave drive tone sent into the cavity, which is constant in amplitude and frequency with ω ~ ωc (cf. Fig. 2a). ~60-nm-thick ﬁlm of 60∕40 molybdenum-rhenium alloy (MoRe, superconducting transition temperature Tc ~ 9 K33) on a 10 × 10 mm2 and 500 μm-thick high-resistivity silicon substrate; cf. Methods section and Supplementary Fig. 1. For driving and readout, the cavity is capacitively side-coupled to a transmission feedline by means of a coupling capacitance Cc = 16 fF. The cavity has a fundamental mode resonance frequency ωc = 2π ⋅ 6.434 GHz and internal and external linewidths κi = 2π ⋅370 kHz and κe = 2π ⋅5.7 MHz, respectively. The transmission spectrum of the cavity around its resonance frequency is shown in Fig. 1e; for details on the device modeling and ﬁtting, see Supplementary Note 2. We operate the nanobeam in a regime of voltages where it can be modeled by the equation of motion €x þ Γm _x þ 1 m k0 þ kp sin 2Ωt h i x ¼ F0 m cos Ωt þ ϕp ; ð1Þ ð1Þ The superconducting cavity is parametrically coupled to a MoRe-coated high-stress Si3N4 nanobeam, which is electrically integrated into the transmission feedline. The nanobeam has a width w = 150 nm, a total thickness t = 143 nm (of which ~83 nm are Si3N4 and 60 nm are MoRe) and a length r = 100 μm. It is separated from the center conductor of the cavity by a ~200-nm- wide gap (cf. Fig. 1c) and we estimate the electromechanical coupling strength to be g0 = 2π ⋅0.9 Hz. More design and fabrication details are described in the Methods section and Supplementary Fig. 1. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 a c+ Ωm 2Ωm Ωm c– Ωm c –5 10 Mechanical gain (dB) π −π −π/2 π/2 0 Normalized parametric drive voltage 0.2 0.4 0.6 0.8 1.0 –5 0 5 10 15 20 25 Drive voltage offset phase Mechanical gain (dB) Detuning (Hz) 0 5 10 –10 –5 b c d –4 –2 0 2 4 6 8 10 12 Mechanical gain (dB) 5 0 Instability regime Fig. 2 Electromechanical detection of parametric, phase-sensitive mechanical amplitude ampliﬁcation. a Experimental scheme. The a narrow bandwidth can be described by34 a narrow bandwidth can be described by34 ð4Þ xthðtÞ ¼ XðtÞ cos Ωmt þ YðtÞ sin Ωmt: ð4Þ Here, X(t) and Y(t) are random variable quadrature amplitudes, which vary slowly compared to Ω1 m . Similarly to the coherently driven mechanical amplitude detection discussed above, this thermal motion or thermomechanical noise can be measured by optomechanical sideband generation in the output ﬁeld of a microwave signal sent into the superconducting cavity (cf. the inset schematic in Fig. 3a). –5 10 Mechanical gain (dB) π −π −π/2 π/2 0 Drive voltage offset phase b 5 0 Mechanical gain (dB) Detuning (Hz) 0 5 10 –10 –5 d –4 –2 0 2 4 6 8 10 12 Mechanical gain (dB) g We measure the thermomechanical noise quadratures X(t) and Y(t) with and without parametric pump. An exemplary result is shown in Fig. 3b. As we have demonstrated above by ampliﬁcation and de-ampliﬁcation of a coherent excitation, one of the quadrature amplitudes, here Y(t), is getting ampliﬁed while the other, here X(t), is simultaneously reduced, when the mechanical resonance frequency is parametrically modulated with 2Ωm. This puts the mechanical nanobeam into a squeezed thermal state. From the time traces of the quadratures, we reconstruct by means of a Fourier transform the power spectral density (PSD) of the noise as shown in Fig. 3a. With parametric driving, the total PSD is larger than without, in particular close to Ωm, as the additional energy pumped into the ampliﬁed quadrature Y(t) is larger than the energy reduction in X(t) and at the same time the total linewidth decreases for the same reason. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 In summary, we have achieved an excellent experimental control and theoretical modeling regarding the parametric ampliﬁcation of the coherently driven nanobeam in both parameters, the relative phase between the drives and the detuning from mechanical resonance a c+ Ωm 2Ωm Ωm c– Ωm c –5 10 Mechanical gain (dB) π −π −π/2 π/2 0 Normalized parametric drive voltage 0.2 0.4 0.6 0.8 1.0 –5 0 5 10 15 20 25 Drive voltage offset phase Mechanical gain (dB) Detuning (Hz) 0 5 10 –10 –5 b c d –4 –2 0 2 4 6 8 10 12 Mechanical gain (dB) 5 0 Instability regime Fig. 2 Electromechanical detection of parametric, phase-sensitive mechanical amplitude ampliﬁcation. a Experimental scheme. The mechanical oscillator is coherently driven by a combination of DC and alternating voltage with frequency Ω ~ Ωm, while the electrostatic spring constant is modulated with twice this frequency 2Ω ~ 2Ωm. Via the optomechanical coupling, the mechanical oscillations generate sidebands to a microwave pump tone sent to the cavity with frequency ω = ωc, which are used for homodyne detection of the mechanical amplitude. b Mechanical amplitude gain 20log 10Gp vs. offset phase ϕp between resonant drive and parametric modulation. When the phase is swept, the amplitude is oscillating between ampliﬁcation or de-ampliﬁcation with a periodicity of π. Circles show data and the line shows a ﬁt with the theoretical expression Eq. (2). c Maximum and minimum gain on resonance vs. parametric modulation strength. The maximum (ϕp = π∕2) and minimum (ϕp = 0) gain values on resonance follow the theoretical curves (lines) up to a maximum gain of ~22 dB. For stronger parametric modulation amplitudes close to the instability threshold (indicated as vertical line), the gain in our experiments is limited by resonance frequency ﬂuctuations of the mechanical resonator. d Maximum and minimum gain vs. detuning from resonance. For a driving frequency slightly detuned from resonance, the maximum gain gets reduced compared to the resonant case. Points are extracted from phase-sweep curve ﬁts. Lines show the corresponding theoretical curves and the shaded area contains all gain values achievable by changing ϕp. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 Mechanical gain (dB) Normalized parametric drive voltage 0.2 0.4 0.6 0.8 1.0 –5 0 5 10 15 20 25 g p c Mechanical gain (dB) Instability regime Mechanical gain (dB) From the time traces, we can also generate quadrature amplitude histograms, shown in the bottom panels of Fig. 3b. In the histograms the squeezing of the thermal noise is apparent as a deformation from a circular, two-dimensional (2D) Gaussian distribution in the case without parametric pump to a cigar-like- shaped overall probability distribution, when the parametric modulation is applied. To determine the squeezing factor we achieve by this, we integrate the 2D-histograms along the Y- quadrature and extract the variance σ2 X of the X-quadrature from a Gaussian ﬁt to the resulting data (cf. Fig. 3c). Analogously, we obtain the variance σ2 Y for the Y-quadrature. To calibrate out the noise of the HEMT ampliﬁer, we substract the independently measured variance of the ampliﬁer noise σ2 amp and deﬁne the bare variances ΔX2 ¼ σ2 X σ2 amp;X and ΔY2 ¼ σ2 Y σ2 amp;Y. Normalized parametric drive voltage Detuning (Hz) Normalized parametric drive voltage Detuning (Hz) Fig. 2 Electromechanical detection of parametric, phase-sensitive Fig. 2 Electromechanical detection of parametric, phase-sensitive mechanical amplitude ampliﬁcation. a Experimental scheme. The mechanical oscillator is coherently driven by a combination of DC and alternating voltage with frequency Ω ~ Ωm, while the electrostatic spring constant is modulated with twice this frequency 2Ω ~ 2Ωm. Via the optomechanical coupling, the mechanical oscillations generate sidebands to a microwave pump tone sent to the cavity with frequency ω = ωc, which are used for homodyne detection of the mechanical amplitude. b Mechanical amplitude gain 20log 10Gp vs. offset phase ϕp between resonant drive and parametric modulation. When the phase is swept, the amplitude is oscillating between ampliﬁcation or de-ampliﬁcation with a periodicity of π. Circles show data and the line shows a ﬁt with the theoretical expression Eq. (2). c Maximum and minimum gain on resonance vs. parametric modulation strength. The maximum (ϕp = π∕2) and minimum (ϕp = 0) gain values on resonance follow the theoretical curves (lines) up to a maximum gain of ~22 dB. For stronger parametric modulation amplitudes close to the instability threshold (indicated as vertical line), the gain in our experiments is limited by resonance frequency ﬂuctuations of the mechanical resonator. d Maximum and minimum gain vs. detuning from resonance. NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications Results The maximum and minimum values of gain we ﬁnd for V2Ω ≈0.75Vt0 are shown in Fig. 2d and are in good agreement with theoretical curves shown as lines. We note that the dependence of maximum and minimum gain of detuning is not Lorentzian lineshaped, as the threshold voltage is detuning dependent itself and the deviations between experimental data and theoretical lines mainly occur due to slow and small resonance frequency drifts of the nanobeam. Moreover, the phase between near-resonant excitation drive and parametric modulation for maximum/minimum gain does not have a constant value; it follows an arctan-function as is discussed in more detail in Supplementary Note 6. In addition to the parametric driving, we slightly excite the mechanical oscillator by adding a near-resonant oscillating 3 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 units) 0 0.2 0.4 –0.4 –0.2 Pump on Pump off Pump off Pump off Pump on Pump on 2Ωm c Ωm c– Ωm c+ Ωm Fig. 3 Interferometric detection of squeezed thermomechanical noise in a nanomechanical oscillator. a The thermal displacement ﬂuctuations a b Time (s) 0 1 2 3 4 5 Detuning (Hz) 0 50 –50 X(t), Y(t) PSD (arb. units) 10 1 100 0 1 –1 –1 0 1 0 Time (s) 0 1 2 3 4 5 0 1 –1 X quadrature (arb. units) X quadrature (arb. units) Y quadrature (arb. units) Pump off Pump off Pump on Pump on 2Ωm c Ωm c– Ωm c+ Ωm Parametric microwave ampliﬁcation. In a cavity optomechanical system, the mechanical oscillator can not only be coherently driven by a directly applied resonant force, but also by amplitude mod- ulations of the intracavity ﬁeld. Such a near-resonant amplitude modulation can be generated by sending two microwave tones with a frequency difference close to the mechanical resonance into the cavity. Here, we apply a strong microwave drive tone on the red sideband of the cavity, i.e., at ωd = ωc −Ωm, and add a small probe signal around the cavity resonance frequency at ωp ~ ωc. This experimental scheme generates a phenomenon called optomecha- nically induced transparency (OMIT), where by interference a narrow transparency window opens up in the center of the cavity absorption dip45,46. The width of the transparency window is given by the sum of intrinsic mechanical linewidth Γm and the additional linewidth due to the red-sideband-drive-induced optical damping Γo. The effect of OMIT effect can be understood as follows. The amplitude beating between the two microwave tones coherently drives the nanobeam by an oscillating radiation pressure force, which transfers energy from the cavity ﬁeld to the nanobeam. The resulting mechanical motion with frequency Ω = ωp −ωd mod- ulates the cavity resonance frequency and hereby generates side- bands to the intracavity drive tone at ωd ± Ω, with a well-deﬁned phase relation to the probe tone. The sideband generated at ωd + Ω interferes with the probe signal and generates OMIT (cf. Fig. 4a for vanishing parametric modulation and Fig. 4b). In Fig. 4b, the transparency window can be seen in the center of the cavity transmission spectrum as extremely narrow spectral line and a zoom into this region, shown in Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 White pixels correspond to no recorded counts. The blue circles in the histogram plots are guides to the eye. In (c) we plot the distribution of X-quadrature values for the histograms shown in (b) as dots and Gaussian ﬁts as lines. When the parametric modulation is switched on, the variance of the X-quadrature gets signiﬁcantly decreased and the squeezing factor is approximately s = 0.49. The histograms are normalized to the total number of ~13,000 data points. Data were taken for V2Ω∕Vt = 0.67. Fig. 3 Interferometric detection of squeezed thermomechanical noise in a nanomechanical oscillator. a The thermal displacement ﬂuctuations generate sidebands at ω = ωc + Ωm and ω = ωc −Ωm to a microwave tone sent to the cavity at ω = ωc as schematically shown in the inset. After down-conversion, we detect these sidebands and the corresponding power spectral density is shown for the parametric modulation switched off as blue line and with the parametric modulation switched on as red line. The black line is a Lorentzian ﬁt to the data without parametric modulation. b shows the quadratures of the thermal displacement ﬂuctuations vs. time in the top panels and as histograms (taken for 300 s of measurement time) in the bottom panels. Without parametric modulation, the thermal ﬂuctuations are distributed equally in both quadratures (left side) and the quadrature histogram is a rotational symmetric Gaussian curve; with a parametric modulation applied, as shown on the right side, the ﬂuctuations in one quadrature get ampliﬁed while the ﬂuctuations in the second quadrature get de-ampliﬁed. The result is a squeezed thermal state. The colorscale represents histogram counts from low (dark) to high (orange) values. White pixels correspond to no recorded counts. The blue circles in the histogram plots are guides to the eye. In (c) we plot the distribution of X-quadrature values for the histograms shown in (b) as dots and Gaussian ﬁts as lines. When the parametric modulation is switched on, the variance of the X-quadrature gets signiﬁcantly decreased and the squeezing factor is approximately s = 0.49. The histograms are normalized to the total number of ~13,000 data points. Data were taken for V2Ω∕Vt = 0.67. Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 For a driving frequency slightly detuned from resonance, the maximum gain gets reduced compared to the resonant case. Points are extracted from phase-sweep curve ﬁts. Lines show the corresponding theoretical curves and the shaded area contains all gain values achievable by changing ϕp. p; p; For the parametric modulation amplitude V2Ω∕Vt ≈0.67 used here, we ﬁnd the squeezing factor s ¼ ΔX2 on ΔX2 off ¼ 0:49; ð5Þ ð5Þ where ΔX2 on and ΔX2 off are the X-quadrature variances with the parametric drive on and off, respectively. This squeezing factor is below the usually mentioned 3 dB limit due to the ﬁnite analysis bandwidth (cf. discussion in Supplementary Note 7). Using more advanced squeezing schemes with feedback40–42 or based on measurement43,44, it has been demonstrated that the variance of the X-quadrature can be squeezed by even more than 3 dB, but these approaches typically operate in the instability regime and suppress the corresponding ampliﬁcation of the Y-quadrature. The variance σ2 amp is the quadrature noise originating from the cryogenic ampliﬁer in our detection chain and is measured by monitoring the noise slightly detuned from the mechanical resonance. In summary, we have achieved an excellent experimental control and theoretical modeling regarding the parametric ampliﬁcation of the coherently driven nanobeam in both parameters, the relative phase between the drives and the detuning from mechanical resonance. From an analysis of the individual quadrature power spectral densities and variances, based on ref. 41, we estimate the effective temperatures of the quadratures to be increased by about 18% for X and about 40% for Y due to the parametric drive. We believe that this excess noise as compared to an ideal parametric ampliﬁer is induced by resonance frequency ﬂuctuations of the mechanical oscillator and could be reduced by a device with higher frequency stability. Thermomechanical noise squeezing. Due to a large residual occupation of the mechanical mode with 102−103 thermal pho- nons, its displacement is subject to thermal ﬂuctuations, which in 4 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 ARTICLE a c b Time (s) 0 1 2 3 4 5 Detuning (Hz) 0 50 –50 X(t), Y(t) PSD (arb. units) 10 1 100 0 1 –1 –1 0 1 0 Time (s) 0 1 2 3 4 5 0 1 –1 Normalized counts 0.0 0.1 X quadrature (arb. units) X quadrature (arb. units) X quadrature (arb. units) Y quadrature (arb. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 4c, reveals the Lorentzian lineshape with a width Γeff ≈2π ⋅12 Hz. c b Time (s) 0 1 2 3 4 5 Detuning (Hz) 0 50 –50 X(t), Y(t) PSD (a 1 0 1 –1 –1 0 1 0 Time (s) 0 1 2 3 4 5 0 1 –1 Normalized counts 0.0 0.1 X quadrature (arb. units) X quadrature (arb. units) X quadrature (arb. units) Y quadrature (arb. units) 0 0.2 0.4 –0.4 –0.2 Pump on Pump off Pump off Pump off Pump on m c m c m Fig. 3 Interferometric detection of squeezed thermomechanical noise in a nanomechanical oscillator. a The thermal displacement ﬂuctuations generate sidebands at ω = ωc + Ωm and ω = ωc −Ωm to a microwave tone sent to the cavity at ω = ωc as schematically shown in the inset. After down-conversion, we detect these sidebands and the corresponding power spectral density is shown for the parametric modulation switched off as blue line and with the parametric modulation switched on as red line. The black line is a Lorentzian ﬁt to the data without parametric modulation. b shows the quadratures of the thermal displacement ﬂuctuations vs. time in the top panels and as histograms (taken for 300 s of measurement time) in the bottom panels. Without parametric modulation, the thermal ﬂuctuations are distributed equally in both quadratures (left side) and the quadrature histogram is a rotational symmetric Gaussian curve; with a parametric modulation applied, as shown on the right side, the ﬂuctuations in one quadrature get ampliﬁed while the ﬂuctuations in the second quadrature get de-ampliﬁed. The result is a squeezed thermal state. The colorscale represents histogram counts from low (dark) to high (orange) values. White pixels correspond to no recorded counts. The blue circles in the histogram plots are guides to the eye. In (c) we plot the distribution of X-quadrature values for the histograms shown in (b) as dots and Gaussian ﬁts as lines. When the parametric modulation is switched on, the variance of the X-quadrature gets signiﬁcantly decreased and the squeezing factor is approximately s = 0.49. The histograms are normalized to the total number of ~13,000 data points. Data were taken for V2Ω∕Vt = 0.67. b Time (s) 0 1 2 3 4 5 Detun X(t), Y(t) 0 1 –1 –1 0 1 0 X quadrature (arb. units) Y quadrature (arb. units) Pump off b Y quadrature (arb. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 units) p When we perform the OMIT protocol with a parametric modulation applied to the nanobeam, the mechanical oscillations get modiﬁed according to the previously shown results, i.e., dependent on the relative phase between the cavity ﬁeld-induced mechanical oscillation and the parametric modulation, the mechanical amplitude gets ampliﬁed or de-ampliﬁed. By choosing the optimal phase for each detuning Δm = Ω −Ωm, the transpar- ency window amplitude can be increased to values above 1, i.e., the microwave probe tone is ampliﬁed by parametrically pumping the mechanical resonator, which is three orders of magnitude smaller in frequency than the probe signal (cf. Fig. 4c). With an ampliﬁed mechanical motion, the motion-induced sideband of the drive tone gets ampliﬁed as well, such that the total cavity output ﬁeld at the probe frequency can be enhanced to values larger than 1. Here, we achieve an intracavity ﬁeld gain of about 14 dB, which corresponds to a net gain of about 7 dB due to the unampliﬁed OMIT signal being signiﬁcantly below unity transmission. A schematic of OMIT and the ampliﬁcation mechanism is shown in Fig. 4a. c Normalized counts 0.0 0.1 X quadrature (arb. units) 0 0.2 0.4 –0.4 –0.2 Pump on Pump off X quadrature (arb. units) Fig. 3 Interferometric detection of squeezed thermomechanical noise in a nanomechanical oscillator. a The thermal displacement ﬂuctuations generate sidebands at ω = ωc + Ωm and ω = ωc −Ωm to a microwave tone sent to the cavity at ω = ωc as schematically shown in the inset. After down-conversion, we detect these sidebands and the corresponding power spectral density is shown for the parametric modulation switched off as blue line and with the parametric modulation switched on as red line. The black line is a Lorentzian ﬁt to the data without parametric modulation. b shows the quadratures of the thermal displacement ﬂuctuations vs. time in the top panels and as histograms (taken for 300 s of measurement time) in the bottom panels. Without parametric modulation, the thermal ﬂuctuations are distributed equally in both quadratures (left side) and the quadrature histogram is a rotational symmetric Gaussian curve; with a parametric modulation applied, as shown on the right side, the ﬂuctuations in one quadrature get ampliﬁed while the ﬂuctuations in the second quadrature get de-ampliﬁed. The result is a squeezed thermal state. The colorscale represents histogram counts from low (dark) to high (orange) values. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 a 6.429 0 Frequency (GHz) b –5 –10 –15 –20 –25 6.432 6.435 6.438 c Magnitude of S21 (dB) Magnitude of S21 (dB) Magnitude of S21 (dB) Magnitude of S21 (dB) –10 0 10 Microwave amplification Microwave amplification Microwave amplification π 2π –2π –π 0 Parametric drive offset phase d –10 0 10 Normalized parametric drive voltage 0.5 1.0 1.5 V t2 Vt1 Vt3 Increased damping Detuning from resonance Δm/2π (Hz) –80 –60 –40 –20 0 20 Drive off 40 60 80 10 0 –10 –20 Drive on e 1 2 1 2 Vt0 eff/Vt0 Γeff/Γm 2Ωm c c– Ωm IV. Ωm III. I. II. Fig. 4 Phase-sensitive and tunable microwave ampliﬁcation by parametric mechanical driving. a Experimental scheme. The cavity is coherently driven on the red sideband ωd = ωc −Ωm. In addition, a small probe tone is swept through the cavity resonance. At the same time, the resonance frequency of the mechanical oscillator is parametrically modulated with 2Ω. b Optomechanically induced transparency (OMIT) without parametric modulation V2Ω = 0. Data are shown in blue, black line is a ﬁt, the dashed box indicates the zoom-in region shown in panel (c). In addition to the data without parametric modulation, we show the highest achieved transmission with parametric driving as orange circles. Close to the mechanical resonance we observe intracavity gain of the probe signal up to ~14 dB and net transmission gain of ~7 dB. The orange line shows a theoretical curve calculated with independently obtained parameters. The schematic in (a) visualizes the ampliﬁcation mechanism. By the beating of the two cavity tones, energy from the cavity ﬁeld is converted into mechanical motion, which is ampliﬁed by parametric modulation. The hereby increased energy is upconverted back to the probe tone frequency as sideband of the red-detuned drive tone. d The microwave gain is phase-sensitive; it depends on the phase between the parametric modulation and the intracavity amplitude beating. The three data sets (black lines are ﬁts) show the gain for different detunings from ωp −ωd = Ωm (0 Hz, 2π ⋅7 Hz and 2π ⋅12 Hz). e Probe-tone gain vs. parametric drive voltage for three different red-sideband drive powers. The parametric drive voltage is normalized to its value obtained in Fig. 2 using a resonant drive for amplitude detection. Lines are calculations based on independently extracted parameters. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 Close to the mechanical resonance we observe intracavity gain of the probe signal up to ~14 dB and net transmission gain of ~7 dB. The orange line shows a theoretical curve calculated with independently obtained parameters. The schematic in (a) visualizes the ampliﬁcation mechanism. By the beating of the two cavity tones, energy from the cavity ﬁeld is converted into mechanical motion, which is ampliﬁed by parametric modulation. The hereby increased energy is upconverted back to the probe tone frequency as sideband of the red-detuned drive tone. d The microwave gain is phase-sensitive; it depends on the phase between the parametric modulation and the intracavity amplitude beating. The three data sets (black lines are ﬁts) show the gain for different detunings from ωp −ωd = Ωm (0 Hz, 2π ⋅7 Hz and 2π ⋅12 Hz). e Probe-tone gain vs. parametric drive voltage for three different red-sideband drive powers. The parametric drive voltage is normalized to its value obtained in Fig. 2 using a resonant drive for amplitude detection. Lines are calculations based on independently extracted parameters. The parametric instability threshold, indicated by dashed vertical lines, is shifted to higher values with increasing red-sideband drive power, partly due to optical damping, partly due to a power-dependent intrinsic mechanical damping rate. The inset shows the extracted threshold voltage vs. effective mechanical linewidth and as dashed line the theoretical prediction. The cooperativity for (b−d) is C 0:5 and C 0:16; 0:28; 0:5 for (e). data for drive powers corresponding to cooperativities C1 0:16, C2 0:28 and C3 0:5 or intracavity photon numbers nc1 = 2.25 ⋅106, nc2 = 4.5 ⋅106, and nc3 = 9 ⋅106. The functional depen- dence of the maximum transmitted power is formally identical to the case without parametric driving gain increases with increasing sideband drive power, as the baseline (the peak height of the transparency window) is shifted up as well and because the gain in this experiment was limited by the mechanical nonlinearity, cf. Supplementary Note 9, which gets less signiﬁcant for a larger total mechanical linewidth. g g So far, our current device is far from being optimized for large gain, large bandwidth and low added noise for several reasons. Due to the small maximally achieved cooperativity of C 0:5, not all intracavity gain is translated to net output gain. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 The parametric instability threshold, indicated by dashed vertical lines, is shifted to higher values with increasing red-sideband drive power, partly due to optical damping, partly due to a power-dependent intrinsic mechanical damping rate. The inset shows the extracted threshold voltage vs. effective mechanical linewidth and as dashed line the theoretical prediction. The cooperativity for (b−d) is C 0:5 and C 0:16; 0:28; 0:5 for (e). a 6.429 0 Frequency (GHz) b –5 –10 –15 –20 –25 6.432 6.435 6.438 Magnitude of S21 (dB) 2Ωm c c– Ωm IV. Ωm III. I. II. Magnitude of S21 (dB) –10 0 10 Microwave amplification π 2π –2π –π 0 Parametric drive offset phase d d b a Parametric drive offset phase Parametric drive offset phase c Magnitude of S21 (dB) Magnitude of S21 (dB) Microwave amplification Microwave amplification –10 0 10 Normalized parametric drive voltage 0.5 1.0 1.5 V t2 Vt1 Vt3 Increased damping Detuning from resonance Δm/2π (Hz) –80 –60 –40 –20 0 20 Drive off 40 60 80 10 0 –10 –20 Drive on e 1 2 1 2 Vt0 eff/Vt0 Γeff/Γm Magnitude of S21 (dB) Microwave amplification –10 0 10 Normalized parametric drive voltage 0.5 1.0 1.5 V t2 Vt1 Vt3 Increased damping e 1 2 1 2 Vt0 eff/Vt0 Γeff/Γm c Magnitude of S21 (dB) Microwave amplification Detuning from resonance Δm/2π (Hz) –80 –60 –40 –20 0 20 Drive off 40 60 80 10 0 –10 –20 Drive on e Magnitude of S21 (dB) Magnitude of S21 (dB) 1.5 Normalized parametric drive voltage Fig. 4 Phase-sensitive and tunable microwave ampliﬁcation by parametric mechanical driving. a Experimental s Fig. 4 Phase-sensitive and tunable microwave ampliﬁcation by parametric mechanical driving. a Experimental scheme. The cavity is coherently driven on the red sideband ωd = ωc −Ωm. In addition, a small probe tone is swept through the cavity resonance. At the same time, the resonance frequency of the mechanical oscillator is parametrically modulated with 2Ω. b Optomechanically induced transparency (OMIT) without parametric modulation V2Ω = 0. Data are shown in blue, black line is a ﬁt, the dashed box indicates the zoom-in region shown in panel (c). In addition to the data without parametric modulation, we show the highest achieved transmission with parametric driving as orange circles. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 3 Interferometric detection of squeezed thermomechanical noise in a g The observed microwave ampliﬁcation is, similarly to the bare mechanical amplitude gain, phase-sensitive and modulates between ampliﬁcation and de-ampliﬁcation when sweeping the phase of the parametric drive, with a periodicity of 2π. This phase-sensitivity of the microwave gain is shown in Fig. 4d for three different detunings from the mechanical resonance. We note that the phase periodicity here is equivalent to the case of the mechanical amplitude ampliﬁcation, but due to the details of our theoretical analysis of the system (see Supplementary Note 8) the phase is given for the parametric drive instead of the resonant force here, which doubles its value. Similar to the mechanical amplitude ampliﬁcation, the micro- wave gain depends on the parametric drive voltage, which has a threshold value above which the parametric instability regime begins. When we plot the maximally achievable transmission ∣S21∣ exactly on the mechanical resonance vs. the parametric excitation voltage, we ﬁnd a monotonously increasing behavior as shown in Fig. 4e for three different red-sideband drive powers. Shown are 5 NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications Methods D i f b Device fabrication. The device fabrication starts with the deposition of a 100-nm- thick layer of high-stress Si3N4 on top of a 500-μm-thick 2-inch silicon wafer by means of low pressure chemical vapour deposition. Afterwards, 60-nm-thick gold markers on a 10-nm chromium adhesion layer were patterned onto the wafer using electron beam lithography (EBL), electron beam evaporation of the metals and lift- off. Then, the wafer was diced into individual 10 × 10 mm2 chips, which were used for the subsequent fabrication steps. Thermomechanical noise squeezing—data processing. As ﬁrst step, we manually rotate the measured quadratures X0ðtÞ and Y0ðtÞ by ~ π∕36 to obtain the ampliﬁed and de-ampliﬁed quadratures X(t) and Y(t). We calculate the total PSD by S = ∣X(Ω) + iY(Ω)∣2, where X(Ω) and Y(Ω) are the Fourier transforms of the recorded X(t) and Y(t), respectively. The obtained spectra are smoothed by applying a 100-point bin averaging and the smoothed spectra are divided by the smoothed background noise spectrum to remove the lock-in ampliﬁer ﬁlter function. The result is shown in Fig. 3. By using a three-layer mask (S1813, tungsten and ARN-7700-18), EBL and several reactive ion etching (RIE) steps with O2 and an SF6/He gas mixture, the Si3N4 was thinned down everywhere to ~10 nm on the chip surface except for rectangular patches (124 × 9 μm large) around the future locations of the nanobeams. After resist stripping in PRS3000, the remaining ~10 nm of Si3N4 were removed in a buffered oxide etching step, which also thinned down the Si3N4 in the rectangular patch areas to ~ 83 nm. This two-step removal of Si3N4 by dry and wet etching was performed in order to avoid over-etching with RIE into the silicon substrate. The histogram data in Fig. 3 were obtained by ﬁrst applying a 40-point moving average and plot each ﬁfth datapoint of the resulting dataset into the histograms. The variances were calculated from Gaussian ﬁts to the Y-integrated histograms. Immediately afterwards, a ~60-nm-thick layer of superconducting molybdenum-rhenium alloy (MoRe, 60/40) was sputtered onto the chip. By means of another three-layer mask (S1813, W, PMMA 950K A6), EBL, O2 and SF6/He RIE, the microwave structures were patterned into the MoRe layer. The remaining resist was stripped off in PRS3000. Parametric microwave ampliﬁcation—measurement routine and data pro- cessing. Both the measurement routine and the data processing are done in full analogy with the mechanical amplitude ampliﬁcation. Discussion I hi k In this work, we have demonstrated an electromechanical cavity with mechanical parametric driving. By means of an opto- mechanical, interferometric readout scheme of a high-quality factor mechanical nanobeam oscillator, we have demonstrated phase-sensitive mechanical amplitude ampliﬁcation, and observed thermomechanical noise squeezing. We demonstrated that this parametric mechanical drive can be used to implement a phase- sensitive microwave ampliﬁcation, in a regime where dynamical backaction can simultaneously cool the mechanical resonator. Using the presented experimental platform in an optimized device, it should be possible to cool the mechanical oscillator into its quantum ground state and perform a near-quantum-limited ampliﬁcation scheme for microwave photons. Furthermore, this approach will allow to explore exotic regimes of bath engineering for microwave cavities26 and enable other applications of mechanical parametric driving and mechanical squeezing, that have been proposed and discussed in the recent years27,28. where ± is chosen for minimum and maximum gain, respectively, and βi are the ﬁt parameters. By this method, we determine the maximum gain on resonance with higher ﬁdelity than just setting the excitation frequency to the resonance frequency due to small mechanical resonance frequency drifts and ﬂuctuations of unknown origin. We note that ultimately and for parametric excitation voltages close to the threshold voltage, these frequency shifts also limit the observable gain, as it where ± is chosen for minimum and maximum gain, respectively, and βi are the ﬁt parameters. By this method, we determine the maximum gain on resonance with higher ﬁdelity than just setting the excitation frequency to the resonance frequency due to small mechanical resonance frequency drifts and ﬂuctuations of unknown origin. We note that ultimately and for parametric excitation voltages close to the threshold voltage, these frequency shifts also limit the observable gain, as it becomes more and more sensitive to frequency ﬂuctuations as can also be seen in Supplementary Fig. 8, where the range of largest gain gets narrower with increased V2Ω∕Vt. Thermomechanical noise squeezing—measurement routine. To characterize the thermomechanical noise of the nanobeam, we send a resonant microwave tone to the cavity and detect the cavity output ﬁeld after mixing it with the drive tone as local oscillator. This down-converts the motional sidebands to the original mechanical frequency, similar to the protocol for mechanical amplitude ampliﬁ- cation. Discussion I hi k To detect the quadratures of the sidebands X0ðtÞ and Y0ðtÞ, we measure the voltage with a lock-in ampliﬁer set to the mechanical resonance frequency with a sample rate of 225 samples/s. The total sampling time was 300 s. This measurement scheme was repeated for different parametric modulation strengths of the mechanical resonance frequency V2Ω∕Vt, including V2Ω∕Vt = 0. To characterize also the (white) background noise ﬂoor originating from the detection ampliﬁer chain, we repeat the measurement for a lock-in center frequency sufﬁciently detuned from the mechanical resonance such that there is no signature of the mechanical thermal noise included. Mechanical amplitude ampliﬁcation—measurement routine and data pro- p g ( pp y ) In terms of ampliﬁer performance, such an optimized device would be on par with other, recently developed multimode or multitone optomechanical ampliﬁcation schemes20,22,25,47, but provides a simpliﬁed setup as it does not require multiple circuit modes or frequency conversion. In contrast, however, to most previously realized optomechanical ampliﬁcation schemes21,22,25,48, our system provides phase-sensitive ampliﬁcation, enabling for example mechanically mediated squeezing of microwave signals36. Compared to other cavity-based parametric ampliﬁers such as Josephson-based circuits49,50, optomechanical ampliﬁers suffer from a reduced bandwidth, but with the beneﬁt of a large dynamic range25. Considering the additional possibilities arising from the presented scheme such as enhancing optomechanical nonlinea- rities27, photon bath engineering26 and force sensing in hybrid devices with a Bose−Einstein condensate51–53, our platform offers rich and exciting perspectives for quantum-limited optomechanical device engineering. p p We ﬁtted the resulting power curves with f ðtÞ ¼ α1 cos2ðα2t þ α3Þ ð1 þ α4Þ2 þ sin2ðα2t þ α3Þ ð1 α4Þ2 " # ð8Þ ð8Þ and ﬁt parameters αi, from where we get Gmin ¼ 1 1 þ α4 ; Gmax ¼ 1 1 α4 : ð9Þ ð9Þ Repeating this procedure for different detunings allows to determine the maximum and minimum gain dependent on Δm. Finally, we ﬁt the detuning-dependent maximum and minimum gain points with the corresponding theoretical expression Repeating this procedure for different detunings allows to determine the maximum and minimum gain dependent on Δm. Finally, we ﬁt the detuning-dependent maximum and minimum gain points with the corresponding theoretical expression f ðΔmÞ ¼ β1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ β2ðΔm β3Þ2 q ± β4 2 ; ð10Þ ð10Þ NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunicati NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 At the same time, the cooperativity limits the ampliﬁcation bandwidth, which is given by the effective mechanical linewidth Γeff. Finally, it would be desirable to operate in the sideband-resolved limit Ωm > κ to enable ground-state cooling in contrast to the slightly bad cavity limit in our present device, where even for large cooperativities the lowest possible phonon occupation is given by nmin ¼ κ 4Ωm 1. The residual phonon occupation, however, directly translates to input-referenced added noise26. In an optimized device, operated in the resolved sideband regime and with cooperativities C > nth, all intracavity ﬁeld gain corresponds to net microwave gain, the bandwidth will be increased by several orders of magnitude compared to the current value and the jS21j2 ¼ κ2 i κ2 þ Cp Γm Γ2 eff 2 κiκe κ2 Γeff þ κ2 e κ2 CpΓm ð6Þ ð6Þ with a parametrically enhanced cooperativity with a parametrically enhanced cooperativity Cp ¼ C 1 V2Ω Veff t0 ; ð7Þ Cp ¼ C 1 V2Ω Veff t0 ; ð7Þ where the effective threshold voltage is given by Veff t0 ¼ Vt0Γeff=Γm. From ﬁts to the data, shown as lines, we can extract the instability threshold voltages, indicated as dashed vertical lines and plotted in the inset vs. effective mechanical linewidth. The threshold gets shifted towards higher values due to an increase of mechanical linewidth, which is partly due to the optical spring and partly due to a microwave power-dependent intrinsic linewidth (see Supplementary Note 5). At the same time, the net microwave 6 ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 ampliﬁer will be near-quantum limited, as has been extensively discussed in ref. 26. The most straightforward way to achieve these realistic numbers would be a signiﬁcant increase of the single- photon coupling rate g0 by about a factor of ~10 and a simultaneous decrease of the cavity linewidth by ~10 through a weaker coupling to the feedline (cf. also Supplementary Note 10). nanobeam was etched away by this measure and the beam was released from the substrate. After the nanobeam release, the remaining resist was stripped using an O2 plasma. p A simpliﬁed schematic of the fabrication is shown in Supplementary Fig. 1, omitting the patterning of the electron beam markers. Mechanical amplitude ampliﬁcation—measurement routine and data pro- cessing. To measure the mechanical amplitude ampliﬁcation, we sweep the phase between the drive tone and the parametric pump. In order to sweep the phase, we add a small detuning δ on the order of ~0.1 Hz to the parametric drive tone, i.e., modulate with 2Ω + δ, and measure a time trace of the down-converted cavity sideband signal at Ω. Then, the parametric phase is given by ϕp = δt + γ with an arbitrary offset term γ. For the down-conversion, we send a resonant microwave tone to the cavity and detect the cavity output ﬁeld at Ω after mixing it with the drive tone as local oscillator. This protocol provides us with a voltage signal pro- portional to the mechanical amplitude (cf. also Supplementary Note 3). We ﬁtted the resulting power curves with NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 References 1. Wallraff, A. et al. Strong coupling of a single photon to a superconducting qubit using circuit quantum electrodynamics. Nature 431, 162–167 (2004). pp y 34. Rugar, D. & Grütter, P. 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After the MoRe-Si3N4 bilayer was completely etched by the SF6/He gas mixture, the etching was continued for several minutes. As we had chosen the RIE parameters to achieve slight lateral etching, the silicon underneath the narrow 7 TURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications Data availability 30. Singh, V. et al. Optomechanical coupling between a multilayer graphene mechanical resonator and a superconducting microwave cavity. Nat. Nanotechnol. 9, 820–824 (2014). y The data presented in this study will be available on Zenodo with the identiﬁer https:// doi.org/10.5281/zenodo.3713207 upon publication of this work. 31. Andrews, R. W., Reed, A. P., Cicak, K., Teufel, J. D. & Lehnert, K. W. Quantum-enabled temporal and spectral mode conversion of microwave signals. Nat. Commun. 6, 10021 (2015). Received: 5 August 2019; Accepted: 24 February 2020; Received: 5 August 2019; Accepted: 24 February 2020; Author contributions Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints A.I.-R., S.Y. and G.A.S. designed and fabricated the device. D.B., S.Y. and M.Y. performed the measurements. D.B., S.Y. and G.A.S. analyzed the data. D.B. and Y.M.B. developed the theoretical treatment. G.A.S. conceived the experiment and supervised the project. D.B. wrote the manuscript with input from G.A.S. All authors discussed the results and the manuscript. 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Acknowledgements g This research was supported by the Netherlands Organisation for Scientiﬁc Research (NWO) in the Innovational Research Incentives Scheme—VIDI, project 680-47-526. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 681476—QOMD) and from the European Union’s Horizon 2020 research and innova- tion program under grant agreement No. 732894—HOT. 28. Qvarfort, S. et al. Time-evolution of nonlinear optomechanical systems: Interplay of mechanical squeezing and non-Gaussianity. J. Phys. A 53, 075304 (2020). 29. Weber, P., Güttinger, J., Tsioutsios, I., Chang, D. E. & Bachtold, A. Coupling graphene mechanical resonators to superconducting microwave cavities. Nano Lett. 14, 2854–2860 (2014). 8 NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15389-4 Competing interest p g The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-15389-4. Correspondence and requests for materials should be addressed to D.B. or G.A.S. Peer review information Nature Communications thanks Aurelien Dantan, Anja Metelmann and Marc-Antoine Lemonde for their contribution to the peer review of this work. Peer reviewer reports are available. © The Author(s) 2020 NATURE COMMUNICATIONS \| (2020) 11:1589 \| https://doi.org/10.1038/s41467-020-15389-4 \| www.nature.com/naturecommunications
https://openalex.org/W3083619750	https://ruc.udc.es/dspace/bitstream/2183/26259/3/Pineiro_Isabel_Predictive%20variables%20of%20sexual%20inactivity%20in%20the%20elderly.pdf	English	null	Predictive variables of sexual inactivity in the elderly	Anales de psicología	2,020	cc-by	8,243	Introduction dom (Mercer et al., 2013; Mitchell et al., 2013) which note that older people engage in intimate relationships and believe sexuality to be an important component of their lives, lead- ing to the conclusion, also in Spanish samples, that sexual ac- tivity can be a predictor of general health and quality of life in these older people (Palacios et al., 2012). It is worth plac- ing the importance of this current research in this context, the aim of which is to understand the factors that can poten- tially explain sexual inactivity in older populations. Despite the importance that society generally places on sexu- ality, the sexual behaviour of over the 65s has not been ex- tensively explored in the scientific literature. One thing that has probably contributed to that is the fact that until twenty or thirty years ago, it was an age group with little demo- graphic representation. However, from the middle of the 20th century, longer lifespans, leading to significant demo- graphic changes, has provided an important space for older people, and their sexuality. Today, most of the world’s popu- lation lives longer than previous generations, and continue sexual activity into advanced ages (Freixas & Luque, 2009). Sexuality, not just intercourse but including expressions and behaviours such as gestures of tenderness, kissing, fondling and caressing, does change but is maintained throughout life (Sáez, 2010) and is an integral part of an individual’s devel- opment (Cayo, Flores, Perea, Pizarro & Aracena, 2003). The little tradition of research on the topic of sexuality in old age has focused on sexual dysfunction in both men and women, and on the link between health and sexual activity. Since the beginning of the 21st century, and as the availabil- ity and efficacy of drugs to treat male erectile dysfunction have increased, men’s active sex lives and that of their part- ners have been extended (Lindau & Gavrilova, 2010). In light of the development of the literature about sexual dys- function, research has addressed the link between health, in- dividual wellbeing and sexual activity in older populations. The results of various studies have contributed to the claim that, in effect, sex, especially linked to a sexual partner, is re- lated to health (Palacios et al., 2012). Predictive variables of sexual inactivity in the elderly Isabel Piñeiro1, Marcia Ullauri1, Susana Rodríguez1, Bibiana Regueiro2, and Iris Estévez1 1 Universidade da Coruña (Spain) 2 Universidade de Santiago de Compostela (Spain) 1 Universidade da Coruña (Spain) 2 Universidade de Santiago de Compostela (Spain) Título: Variables predictoras de la inactividad sexual en las personas mayo- res. Título: Variables predictoras de la inactividad sexual en las personas mayo- res. Abstract: Extensive research in recent decades has noted the importance of sexual activity to older people’s health, well-being and quality of life. This study aims to understand the factors that potentially explain sexual in- activity in older populations. In addition to the sociodemographic factors that have traditionally been associated with sexual activity, we examined the explanatory potential and sensitivity of the perception of health and at- titudes towards sexuality in the estimation of sexual inactivity in the previ- ous year in a sample of 200 subjects between 62 and 91 years old (M = 71.30, SD = 5.48). The results suggest significant percentages of variance explained by the regression model which included variables such as having a partner, age and place of origin (R2 = .295). Our results also point to the fact that both perception and concern for sexual health, and opinions about sex before marriage or sex without love, would explain sexual inac- tivity in the elderly. These results suggest the potential of interventions centred on stereotypes and attitudes towards sexuality. Resumen: En las últimas décadas se han llevado a cabo varios estudios que evidencian la relevancia de la actividad sexual para la salud, el bienestar y la calidad de vida de las personas mayores. En este contexto se ubica esta investigación, que tiene por objeto conocer los factores que potencialmente pueden explicar la inactividad sexual en las poblaciones mayores. Además de los factores sociodemográficos, que tradicionalmente se han asociado a la actividad sexual, exploramos el potencial explicativo y la sensibilidad en la estimación de la inactividad sexual en el último año de las percepciones de salud y las actitudes hacia la sexualidad en una muestra de 200 sujetos de entre 62 y 91 años (M = 71.30; DT = 5.48). Los resultados obtenidos sugie- ren importantes porcentajes de varianza explica de la inactivad sexual por el modelo de regresión donde se incorporan variables sociodemográficas (R2 = .295). anales de psicología / annals of psychology 2020, vol. 36, nº 3 (october), 512-520 https://doi.org/10.6018/analesps.381951 anales de psicología / annals of psychology 2020, vol. 36, nº 3 (october), 512-520 https://doi.org/10.6018/analesps.381951 © Copyright 2020: Editum. Servicio de Publicaciones de la Universidad de Murcia. Murcia (Spain) ISSN print edition: 0212-9728. ISSN online edition (http://revistas.um.es/analesps): 1695-2294. Online edition License Creative Commons 4.0: BY-SA Predictive variables of sexual inactivity in the elderly Nuestros resultados apuntan también, a que tanto la percepción y preocupación por la salud sexual como las consideraciones en torno a las relaciones sexuales antes del matrimonio o al sexo sin amor, explicarían la inactividad sexual de las personas mayores. Estos resultados permiten suge- rir el potencial de la intervención centrada en los estereotipos y actitudes hacia la sexualidad. yp y Keywords: Sexual inactivity; Elderly; Self-perceived health; Attitudes to- wards sexuality; Sociodemographic factors. Palabras clave: Inactividad sexual; Personas mayores; Salud percibida; Ac- titudes hacia la sexualidad; Factores sociodemográficos. Correspondence address [Dirección para correspondencia]: Isabel Piñeiro Aguín. Departamento de Psicología. Facultade de Ciencias da Educación. Universidade da Coruña. Campus Elviña, 15071, A Coruña, Ga- licia (Spain). E-mail: isabel.pineiro.aguin@udc.es (Article received: 31-05-2019; revised: 08-09-2019; accepted: 30-10-2019) Introduction These cultural differ- ences in the sexual sphere may mould older people’s sexual expression and behaviour (Sandfort, Hubert, Bajos & Bos, 1998). We have comparative studies which demonstrate dif- ferences between countries and regions in terms of permis- siveness related to sexual behaviour (Baumeister & Mendo- za, 2011; Haavio-Mannila & Kontula, 2003) which lead us to hypothesise the impact of the sociocultural environment on sexuality (Fischer, Traen, & Martin, 2018). Because of that, the place of origin may be an explanatory variable for older people’s sexual attitudes and behaviour. The level of education has also been shown to be one of the variables to explore when explaining sexual activity in older people, with the understanding that sexual activity may be positively correlated with older individuals’ educational levels (Palacios et al., 2012; Wang, Lu, Chen & Yu, 2008). Despite sexuality being an important part of most peo- ple’s lives, stereotypes about older people generally disregard the value of sexual activity for quality of life and emotional wellbeing (Lee et al., 2016). The attitudes older people have towards sexuality may be one of the variables which affect their sexual activity. Various studies have indicated a very stigmatised view of sexuality on the part of older people, in women (García, 2000, 2005; Leyva-Moral, 2008; Rodríguez- Llorente, Piñeiro, Rodríguez, Regueiro, Estévez & Freire, 2018) and in men (Syme & Cohn, 2016). Our overall objective with this study is to understand the factors that may potentially explain sexual inactivity in a sample of older people. We are particularly interested in dis- covering: - Whether various sociodemographic factors (place of origin and geographic region, educational and economic level, civil status/partner status, and age) are associated with sexual inactivity in the previous year. ) ( y ) Modern society maintains many stereotypes about sexual behaviour in old age, and these are also reflected in the atti- tudes of older people, affecting their sexual practices. Leyva- Moral (2008) stated that the sexual behaviour of many older women could be affected if they were convinced of the truth of certain ideas such as older women who were interested in sex were those who were addicted to it in youth, or that sex before marriage or sex outside of a stable relationship were bad. Introduction An individual’s health problems, or problems in either one of a couple, are report- ed as the greatest obstacle to sexual activity in the population aged between 50 and 92 (Gott & Hinchliff, 2003). Apart from its reproductive side, sexuality as a relational, hedonistic, ego-integrative aspect of humanity makes up a plan of personal growth that doubtlessly will contribute to older people’s quality of life (Cayo et al., 2003). On these lines we can refer to large studies performed in the United States of America (Lindau et al., 2007) and the United King- Assuming an increase with age in the umbers of sexually active men and women reporting sexual health problems (Lee, Nazroo, O’Connor, Blake & Pendleton, 2016), re- search seems to suggest that suffering from various chronic - 512 - 513 Predictive variables of sexual inactivity in the elderly et al., 2009; Palacios et al., 2012; Smith, Mulhall, Deveci, Monaghan & Reid, 2007) or that age may be one of the vari- ables with most explanatory weight for this inactivity (Gins- berg et al., 2005; Palacios et al., 2012). ailments common in the older population (e.g. arthritis, CVA and diabetes) and the perception of poor general health are associated with reduced sexual functioning and activity, es- pecially in men. Sexual activity is related to the perception of physical and sexual health in those over 65 such that both men and women who believe themselves to be in poor health are less likely to be sexually active (Palacios et al., 2012; Schick et al., 2010). Low levels of sexual activity and problems of sexual functioning, especially in men, have been associated with poor perceived health (Steptoe, Breeze, Banks & Nazroo, 2013). While men in good self-perceived health are predominantly sexually active with their partner, those in perceived poor health report higher frequencies of masturbation (Schick et al., 2010). In women there is also an association between reduced frequency of oral sex and inter- course and the perception of poor health (Schick et al., 2010). This can be interpreted as the link between sexual in- activity and the perception of health which we hypothesise in this study (Ekström et al., 2018; Ginsberg, Pomerantz & Kramer-Feeley, 2005). Given that sexual behaviour is learned from the specific sociocultural surroundings (Gagnon, 1990), it would not be unusual for certain cultural differences to be explanatory fac- tors for this activity in older people. Design This study followed a quantitative empirical-analytical approach, with a correlational-descriptive design via survey. It is transversal, as the data were collected at a single point in time. Introduction Even when it comes to evaluating the capacity to enjoy sex, many studies have reported a stereotypical belief in women that men have naturally higher sexual drive than women (e.g. Rodríguez-Llorente et al., 2018). Research has shown that sexual activity is still important to older people, and despite society having ever more permissive attitudes, there continues to be a stigma about sexual activity in old age. It is important, therefore, to thoroughly examine these types of variables related to beliefs and attitudes, and how they affect sexual activity as people age. p - Whether self-perceived health is an explanatory factor in sexual inactivity in the previous year. - Whether attitudes towards sexuality are also an explana- tory factor for a lack of sexual activity in the previous year. anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) Participants Finally, in this study we also explore a series of socio- demographic variables such as age, civil/partnership status, educational qualifications and place of origin in order to identify the extent to which these conditions predict sexual inactivity in the previous year in a sample of older people. Previous research leads us to hypothesise, for example, that the lack of a partner could be a key factor in explaining sexu- al activity (Ekström et al., 2018; Ginsberg et al., 2005; Huang The sample was non-probabilistic by convenience. It was made up of 200 older people (men and women) resident in the city of Santa Rosa (El Oro, Ecuador) who were members of the Association of Pensioners and Retirees of the city of Santa Rosa. Inclusion criteria were set at being over 64 years old and having the evident capacity to respond to the ques- tions coherently. Most of the men and women in the sample (79.5%) were from the coastal region (la costa) and the re- anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) 514 Isabel Piñeiro et al. Significant differences were found between men and women in terms of civil status, current partnership status and educational level (χ2 = 43.495, p < .000; d = 1.02; χ2 = 29.804, p < .000, d =.78 and χ2 = 16.157, p = .003, d = 0.56, respectively). While 63% of men were married, only 29% of women were, and 39% of the women were widowed com- pared to 9% of men. These differences are consistent with the 67.7% of the men reporting having a sexual partner, while 71% of the women reported the opposite (see Table 1). mainder (20.5%) were from the highlands (la sierra) of Ecua- dor. Sociodemographic studies have characterised people from the coastal region as more open and uninhibited than those from the highlands, who are more reserved and con- servative in many areas such as sexuality. The mean age of the women in the sample was 71.8 (SD = 5.52), with ages ranging from 65 to 91. The mean age of the men in the sample was 70.7 (SD = 5.42), ranging from 64 to 90. As Table 1 shows, there were no significant differ- ences in the proportions of men and women in the age rang- es in terms of economic level, origin, zone or sector. Table 1. Participants Frequencies and proportions in the interaction between sociodemographic variables and gender. Total Men Women Statistical significance and magnitude of differences n (%) n (%) n (%) χ2 p d Age ≤70 119(59.5) 65(65) 54(59.5) 2.511 .113 -- >70 81(40.5) 35(35) 46(46) Economic level Medium 125(62.5) 65 (65) 60(60) .533 .467 -- Low 75(37.5) 35(35) 40(40) Sector North 159(79.5) 83(52.2) 76(76) 1.806 .405 -- Centre 19(9.5) 7(7) 12(12) South 22(11) 10(10) 12(12) Origin Coast 159(79.5) 85(85) 74(74) 3.712 .054 --- Highlands 41(20.5) 15(15) 26(26) Zone Rural 39(19.5) 16(16) 23(23) 1.561 .212 -- Urban 161(80.5) 84(84%) 77(77) Civil status Married 92(47.2) 63(66.3) 29(29) 43.495 .000 1.02 Single 11(5.6) 9 (9.5) 2(2) Free Partnership 15(7.7) 7(7.4) 8(8) Divorced/separated 29(14.9) 7(7.4) 22(14.9) Widowed 48(24.6) 9(9.5) 39(39) Partnership status With partner 96(48) 67(67.7) 29(29) 29.804 .000 0.78 Without partner 103(51.8) 32(32.3) 71(71) Educational level Uneducated/did not finish primary 50(25.1) 16(16) 34(34.3) Primary 51(25.6) 25(25) 26(26.3) Did not finish secondary 30(15.1) 14(14) 16(16.2) Secondary 41(20.6) 24(24) 17(17.2) University 27(13.6) 21(21) 6(6.1) Previous partners Yes 111(58.1) 68(70.1) 43(45.7) 11.636 .001 0.51 No 80(41.9) 29(40) 51(54.3) Table 1. Frequencies and proportions in the interaction between sociodemographic variables and gender. rtions in the interaction between sociodemographic variables and gender. University of A Coruña (Spain). This translation modified the original items, designed for British populations, for Spanish-speakers. The original instrument was created using other validated instruments (Mitchell, Ploubidis, Datta & Wellings, 2012; O’Connor et al., 2008; Waite, Laumann, Das & Schumm, 2009) and ensures gender specificity by offering one questionnaire for men and another for women. In total the men’s scale has 50 items, the women’s scale has 44. The men’s scale asks about ability to achieve and maintain erec- tions, the women’s scale asks about ability to be sexually aroused. Both versions include items about orgasm and the respondent’s overall sex life in recent months as well as their sexual experiences during their lives. Given our study objec- tives, we only used questions related to sexual activity and at- titudes towards sex. Significant differences were also noted in the educational levels of the men and women in the sample. Almost half (45%) of the men reported finishing secondary education or university, only 23.2% of the women reported the same. There were also significant differences between men and women regarding previous partners (χ2 = 43.495, p = .001; d = 0.51). Participants Almost three-quarters (70%) of the men had part- ners in the past, but less than 50% of the women reported having previous partners. anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) Results p y g p y g For the evaluation of perception of health, which is also of interest in this study, we used two questions: (a) My general health is currently… and (b) My sexual health is currently…; in both cases we used a Likert scale with five response options from 1 = excellent to 5 = very poor (α=. 60). Concern about sexual health was evaluated with the SRA-Q item: Even if you have not had any sexual activity in the last three months, how concerned are you about your sex life overall? with a five-point Likert scale response from 1 = not concerned at all to 5 = extremely worried. Once we had determined that 60.5% of the sample had not had any sexual activity in the previous year, the next step was to explore the impact of sociodemographic factors, per- ceived health, and attitudes towards sexuality on this sexual inactivity. Procedure Table 2. Frequencies and coding of parameter (1) for the sociodemograph- ic variables included in the regression equation. Table 2. Frequencies and coding of parameter (1) for the sociodemograph- ic variables included in the regression equation. Frequency Parameter coding (1) Origin Coast 153 0 Highlands 40 1 Zone Urban 155 0 Rural 38 1 Economic level Medium 120 1 Low 73 0 Educational level Secondary or higher 92 1 Primary or lower 101 0 Civil/ Partnership status Married or with a partner 106 0 Single / widowed /divorced / without partner 87 1 Age group ≤70 119 0 > 70 81 1 Firstly, the management of the Retiree’s Association of Santa Rosa (El Oro, Ecuador) signed their informed con- sent. Then the participants were informed of the aims of the study, confidentiality and the ethical management of the da- ta. Once it was confirmed that they met the inclusion crite- ria, the self-report form was presented and the instructions for completing it were read aloud. The questionnaire was completed individually, anony- mously and voluntarily. The researchers were present during the administration of the test instruments to answer any questions and ensure that the subjects completed their ques- tionnaires independently. No subjects participated without their express agreement once they were aware of the objec- tive of the research. Impact of place of origin, zone, economic level, ed- ucational level, civil status / partner status and age on sexual inactivity Finally, in order to examine various sociodemographic as- pects of the sample, we included a series of questions about place of origin, zone or sector, civil status or current partner status, edu- cational level and economic level. For the sociodemographic variables, a logistical regres- sion model was produced to make estimations about sexual inactivity in the previous year (sexual activity = 0 / sexual in- activity = 1). The dichotomous categorical predictor varia- bles for the regression equations are codified as in Table 2. Instruments The instrument chosen for this study was a translation of the SRA-Q (Lee et al., 2016) which was carried out by the Educational Psychology Research Group (GIPED) at the anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) 515 Predictive variables of sexual inactivity in the elderly Predictive variables of sexual inactivity in the elderly activity YES = 0, sexual activity NO = 1) in response to the item: In the previous twelve months, have you engaged in any sexual activity (intercourse, masturbation, petting or fondling)? activity YES = 0, sexual activity NO = 1) in response to the item: In the previous twelve months, have you engaged in any sexual activity (intercourse, masturbation, petting or fondling)? To determine sexual inactivity in the sample we used the item “In the last twelve months, have you engaged in sexual activity (intercourse, masturbation, fondling or petting)? from the SRA-Q, with a dichotomous yes/no response that we coded as sexual activity YES = 0 and sexual activity NO = 1. Two indicators were used to assess model fit: (a) Nagelkerke’s R2, which indicates the percentage of variance explained by the models (Nagelkerke, 1991), and (b) the per- centage of correctly classified cases, which lets us determine how useful the predictor variable is in estimating the criteri- on in each proposed model. We used 13 items (α=.62) from the SRA-Q (Lee et al., 2016) to examine attitudes towards sexuality. These were pre- sented as a series of statements such as: “It is wrong to have sexual relations before marriage”; “Men naturally have a higher sex drive than women”; “Two adults of the same sex having sexual rela- tionships is wrong”; and “There is too much sex in the media nowa- days”, with responses on a five-point Likert-type scale from 1 = completely disagree to 5 = completely agree. All of the statistical analyses were performed using SPSS (Version 20). anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) Data Analysis First, we examined the properties of the items used to measure attitudes towards sexuality and perception of health. The predictor equations for sexual inactivity in the previous year were established via logistical regression, following the for- ward stepwise regression process based on the Wald statistic. Three logistical regressions were performed taking socio- demographic variables, perceived health and attitudes to- wards sexuality as predictors and including in each case as the criterion, the dichotomous sexual activity variable (sexual The final sociodemographic model correctly classified 71% of the sample (χ2 = 47.150, p = .000) with a high level of sensitivity when estimating sexual inactivity in the previ- ous year (81.5%) (see Table 3). 516 Isabel Piñeiro et al. Table 3. Omnibus tests for the coefficients of the sociodemographic mod- l Table 3. Omnibus tests for the coefficients of the sociodemographic mod- el Table 3. Omnibus tests for the coefficients of the sociodemographic mod- el level) do not seem to provide predictive information about sexual inactivity. el. χ2 df Sig. Step 1 Step 35.105 1 .000 Block 35.105 1 .000 Model 35.105 1 .000 Step 2 Step 7.690 1 .006 Block 42.795 2 .000 Model 42.795 2 .000 Step 3 Step 4.355 1 .037 Block 47.150 3 .000 Model 47.150 3 .000 The forward stepwise regression procedure based on the Wald statistic shows that not having a partner is the variable that most explains sexual inactivity during the previous year (W = 25.503; p < .01), although age and place of origin also ap- pear in the model (W = 5.891; p = .015 and W = 4.074; p = .044, respectively) (See Table 4). With a percentage of ex- plained variance around 30% (Nagelkerke’s R2 = .295), look- ing at the codification of the parameters, we can say that not having a partner -being single, widowed, or divorced-, being over 70, and coming from the highlands would increase the likelihood of sexual inactivity in the sample in the previous year. Analysis of the final step suggests the inclusion of only three variables: civil status, age and place of origin, giving us to understand that the other sociodemographic variables added at the beginning (zone, economic level, educational Table 4. Results of stepwise logistical regression for the probability of sexual inactivity. anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) Data Analysis B SE Wald df p Exp(B) Step 1 Civil/Partnership status 1.917 .351 29.752 1 .000 6.801 Constant -2.183 .489 19.957 1 .000 0.113 Step 2 Age group 0.960 .352 7.424 1 .006 2.613 Civil/Partnership status 1.793 .358 25.062 1 .000 6.010 Constant -3.326 .671 24.544 1 .000 0.036 Step 3 Origin 0.915 .453 4.074 1 .044 2.497 Age group 0.870 .358 5.891 1 .015 2.387 Civil/Partnership status 1.835 .363 25.503 1 .000 6.266 Constant -4.345 .864 25.307 1 .000 0.013 Impact of perceived health on sexual inactivity mate sexual inactivity during the previous year (sexual activi- / Table 4. Results of stepwise logistical regression for the probability of sexual inactivity. Impact of perceived health on sexual inactivity mate sexual inactivity during the previous year (sexual activi- ty = 0 / sexual inactivity = 1). With a good success rate (73.5%), the model of perceived health would correctly clas- sify 64.2% of the sample (χ2 = 26.163, p < .000) (see Table 6). Having confirmed that the distribution of the items was normal (see Table 5), considering the perception of overall health, current sexual health and concern about sexual health, a logistical regression model was produced to esti- Table 5. Descriptive statistics of the items used to measure perceived health. M SD Asymmetry Kurtosis My overall health is currently… (1) 2.74 0.80 0.380 0.962 My sexual health is currently… (1) 3.08 1.01 0.224 -0.521 In the last three months, how concerned have you been about your sexual health? (2) 1.39 0.71 1.773 2.394 (1) 1=Excellent, 2= Good, 3= Neither good nor bad, 4= Poor, 5= Very poor (2) 1=Not at all, 2=A little, 3=Moderately, 4=Quite a lot, 5= Extremely Table 5. Descriptive statistics of the items used to measure perceived health. M SD Asymmetry Kurtosis My overall health is currently… (1) 2.74 0.80 0.380 0.962 My sexual health is currently… (1) 3.08 1.01 0.224 -0.521 In the last three months, how concerned have you been about your sexual health? (2) 1.39 0.71 1.773 2.394 (1) 1=Excellent, 2= Good, 3= Neither good nor bad, 4= Poor, 5= Very poor (2) 1=Not at all, 2=A little, 3=Moderately, 4=Quite a lot, 5= Extremely Table 6. Omnibus tests for the coefficients of the model of perceived health. χ2 df Sig. Influence of attitudes towards sexuality on sexual inactivity B SE Wald df p Exp(B) Step 1 Sex before marriage is wrong (1) .440 .106 17.135 1 .000 1.552 Constant -.977 .360 7.387 1 .007 0.376 Step 2 Sex before marriage is wrong (1) .395 .109 13.056 1 .000 1.484 There is nothing wrong with sex without love (1) -.294 .126 5.468 1 .019 0.745 Constant -.186 .489 0.145 1 .704 0.830 (1) On a scale of 1= completely disagree to 5= completely agree and the attitude towards sexuality affect sexual inactivity in older people. The levels of inactivity in our sample were sim- ilar to those found in other research (Palacios et al., 2012; Smith et al., 2007). Our results confirm the lack of a partner, and the attitude towards sexuality affect sexual inactivity in older people. The levels of inactivity in our sample were sim- ilar to those found in other research (Palacios et al., 2012; Smith et al., 2007). Our results confirm the lack of a partner, and the attitude towards sexuality affect sexual inactivity in older people. The levels of inactivity in our sample were sim- ilar to those found in other research (Palacios et al., 2012; Smith et al., 2007). Our results confirm the lack of a partner, Data Analysis Step 1 (1) 1=Excellent, 2= Good, 3= Neither good nor bad, 4= Poor, 5= Very poor (2) 1=Not at all, 2=A little, 3=Moderately, 4=Quite a lot, 5= Extremely ality were linked to inactivity a stepwise regression model was produced looking at the measures used in this study. We can confirm that beliefs about sex before marriage and sex with- out love can affect current sexual inactivity to some extent (Nagelkerke’s R2 = .161). Data Analysis We see that the combination of current poor perceived health (W = 16.635; p < .01), together with low concern about sexual health (W = 5 056; p = 025) can explain to a certain extent Table 5. Descriptive statistics of the items used to measure perceived health. M SD Asymmetry Kurtosis My overall health is currently… (1) 2.74 0.80 0.380 0.962 My sexual health is currently… (1) 3.08 1.01 0.224 -0.521 In the last three months, how concerned have you been about your sexual health? (2) 1.39 0.71 1.773 2.394 (1) 1=Excellent, 2= Good, 3= Neither good nor bad, 4= Poor, 5= Very poor (2) 1=Not at all, 2=A little, 3=Moderately, 4=Quite a lot, 5= Extremely Table 6. Omnibus tests for the coefficients of the model of perceived health. χ2 df Sig. Step 1 Step 20.996 1 .000 Block 20.996 1 .000 Model 20.996 1 .000 Step 2 Step 5.167 1 .023 Block 26.163 2 .000 Model 26.163 2 .000 We see that the combination of current poor perceived health (W = 16.635; p < .01), together with low concern about sexual health (W = 5.056; p = .025), can explain to a certain extent (Nagelkerke’s R2 = .178) sexual inactivity in the sample in the previous year (see Table 7). Table 5. Descriptive statistics of the items used to measure perceived health. Table 6. Omnibus tests for the coefficients of the model of perceived health. χ2 df Sig. Step 1 Step 20.996 1 .000 Block 20.996 1 .000 Model 20.996 1 .000 Step 2 Step 5.167 1 .023 Block 26.163 2 .000 Model 26.163 2 .000 Table 6. Omnibus tests for the coefficients of the model of perceived health. We see that the combination of current poor perceived health (W = 16.635; p < .01), together with low concern about sexual health (W = 5.056; p = .025), can explain to a certain extent (Nagelkerke’s R2 = .178) sexual inactivity in the sample in the previous year (see Table 7). anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) 517 Predictive variables of sexual inactivity in the elderly Table 7. Results of the stepwise logistical regression for the probability of sexual inactivity. anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) Influence of attitudes towards sexuality on sexual inactivity Once the properties of the items had been examined (see Table 8), in order to explore whether attitudes towards sexu- Table 8. Descriptive statistics of the items used to measure attitudes towards sexuality Table 8. Descriptive statistics of the items used to measure attitudes towards sexuality M SD Asymmetry Kurtosis 1. Sex before marriage is wrong 3.23 1.49 -0.263 -1.371 2. A married person having sex with someone other than their spouse is wrong 3.67 1.47 -0.742 -0.953 3. Two same sex adults having a sexual relationship is wrong 3.70 1.56 -0.765 -1.057 4. Having one-night stands is wrong 3.44 1.48 -0.445 -1.270 5. Good sexual relations are essential for good long term relationships 3.88 1.23 -1.052 0.184 6. There is nothing wrong with sex without love 2.15 1.27 0.897 -0.384 7. Nowadays people are under too much pressure to have sex 3.43 1.29 -0.410 -0.996 8. There is too much sex in the media nowadays 4.08 1.11 -1.279 1.071 9. Nowadays, young people start having sex too early 4.18 1.18 -1.561 1.491 10. Men naturally have a higher sex drive than women 3.81 1.23 -0.938 -0.115 11. The capacity for sex is reduced as people get older 3.88 1.19 -1.225 0.720 12. Sexual changes that come with age are not important for older people 3.03 1.42 -0.034 -1.348 13. Being sexually active is physically and psychological beneficial for older people 3.28 1.32 -0.348 -0.972 Table 9. Omnibus tests for the coefficients of the model attitudes towards sexuality. This model of attitudes towards sexuality would correctly classify 68.3% of the sample (χ2 = 24.036, p < .000) with a high level of sensitivity (83.2%) when estimating sexual inac- tivity in the previous year (see Table 9). sexuality. χ2 df Sig. Step 1 Step 18.489 1 .000 Block 18.489 1 .000 Model 18.489 1 .000 Step 2 Step 5.547 1 .019 Block 24.036 2 .000 Model 24.036 2 .000 Similarly, considering sex before marriage wrong (W = 13.056; p < .000), or sex without love wrong (W = 5.468; p = .019) can also limit a subject’s sexual activity (see Table 10). No significant relationships were found between other be- liefs about sexuality and sexual inactivity. Table 10. Results from the stepwise logistical regression for the probability of sexual inactivity. Discussion A study by Huang et al. (2009) concluded that, in effect, not having a sexual partner contributed more to sexual inactivity than, for example, health problems. Finally, when it comes to attitudes towards sexuality, we should bear in mind that the stereotypes of an asexual old age are still present in older people’s perspectives. Research has found that older people interiorise sexual stereotypes re- lated to their age which become significant obstacles to them being able to freely express and enjoy their sexuality (Gewirtz-Meydan et al., 2018; Snyder & Zweig, 2010). De- spite starting to be included in research agendas, and despite stigmatised views of sexuality being explanatory factors in sexual inactivity, the attitudes of older people about the role and value of sex in their lives continues to be a little- explored topic. Although not having a partner is the sociodemographic variable that explains sexual inactivity in the previous year best, our results show that age and origin also enter into the explanatory equation for sexual inactivity. This is also in line with other research. Age was one of the reasons with most explanatory weight in sexual inactivity in studies by Ginsberg et al. (2005) and Palacios et al. (2012). In terms of origin, our results show that coming from the highland region of Ecua- dor rather than the coastal region can be associated with greater sexual inactivity in the previous year. It is likely that the reserved, conservative nature usually evident in those from the highlands, with a more negative view of sex, would have an impact on sexual activity. Looking at our results, negative views about sex before marriage and sex outside loving relationships can be specifically as- sociated with sexual inactivity to the same extent as having a partner and age. In fact, the sensitivity of the attitude model (negative beliefs about sex before marriage and sex without love) esti- mating the probability of sexual inactivity is similar to that of the sociodemographic model (not having a partner -being single, widowed, or divorced, being over 70, and coming from the mountains). Thus we see the potential of our results as a basis for health interventions as well as socioeducational initiatives, as peo- ple’s beliefs and attitudes about their sexuality may be a more parsimonious predictor of sexual inactivity than the traditional sociodemographic factors. Discussion The aim of this study was to understand the extent to which various sociodemographic variables, self-perceived health, anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) 518 Isabel Piñeiro et al. Isabel Piñeiro et al. age, and place of origin to be the variables that best predict sexual inactivity in older people. It is also worth highlighting that sexual activity may also be affected by the perception of and concern about sexual health, as well as by beliefs about premarital sex, and sex outside loving relationships. show that perceived poor sexual health, together with low concern about it, enter into the equation explaining sexual inactivity in the previous year, albeit with criterion sensitivity slightly lower than the models related to attitude or socio- demographics. Having a partner, age and origin had significant explana- tory power in sexual inactivity in the previous year (R2 = .295). The percentages of variance of sexual inactivity ex- plained by the model of perceived heath (R2 = .178) and the model of attitude towards sexuality (R2 = .161) were similar and both lower than the variance explained by sociodemo- graphic conditions. Our findings are in line with previous research indicating that those who reported not having engaged in sexual activi- ties in the previous year reported worse sexual health and worse overall health, while more sexually active men and women reported better health (Lindau & Gavrilova, 2010; Ginsberg et al., 2005; Ekström et al., 2018; Palacios et al., 2012). In addition, research by Rodríguez-Llorente et al. (2018) with a Spanish sample aged between 65 and 90 found that the perception of health in those who had engaged in any kind of sexual activity in the previous year was signifi- cantly better than those who had not. The predictive capacity of the lack of a partner on sexual inactivity confirms the findings of a large number of previ- ous studies (Ginsberg et al., 2005; Huang et al., 2009; Ekström et al., 2018). In Smith et al. (2007), for example, the reported reasons for sexual inactivity in older women includ- ed a lack of desire and/or not having a partner, and in older men they included erectile dysfunction and not having a partner. In a sample of older Spanish people, Palacios et al. (2012) also reported that not having a partner was a predic- tor of sexual inactivity in both men and women. Discussion Culture is no doubt an important framework for the un- derstanding of sexuality (Agocha, Asencio & Decena, 2014) and while there are various studies that have looked at dif- ferences in permissiveness and acceptance of sexuality be- tween countries and regions (e.g., Baumeister & Mendoza, 2011; Haavio-Mannila & Kontula, 2003), they are susceptible to change in currently highly globalised societies. In this re- spect, despite expecting to find substantial differences be- tween older people form the North, centre, and South of Europe, a recent study by Fischer et al. (2018) did not find origin to be a predictor variable of sexual activity in male- female couples aged between 60 and 75 in Norway, Den- mark, Belgium and Portugal. Older people who recognise sexual activity as an im- portant indicator of their wellbeing and quality of life, and those with positive attitudes towards sexuality in old age, are more likely to continue being sexually active with the passage of time (Fischer et al., 2018; Kontula & Haavio-Mannila 2009). However, beliefs that associate sexuality with loving partnerships and matrimony would tend to explain sexual in- activity in older populations and could also underlie recur- rent differences research has found in sexual activity be- tween older men and women. Our study also looked at the relationship between percep- tion and concern about sexual health and sexual inactivity in the previous twelve months, as other studies have done before (e.g., Dominguez & Barbagallo, 2016; Lee et al., 2016; Lindau et al., 2007; Lindau & Gavrilova, 2010). Our results In the light of these results, we believe that the challenge for health professionals in this area is to recognise the im- portance of sexuality in old age through fluent, effective communication with older people about their concerns and worries about their sexual lives (Bauer, Haesler & Fether- anales de psicología / annals of psychology, 2020, vol. 36, nº 3 (october) 519 Predictive variables of sexual inactivity in the elderly Predictive variables of sexual inactivity in the elderly and had been more fully representative of the population it came from. 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https://openalex.org/W4283205531	http://sjce.journals.sharif.edu/article_22730_14a4f772eaf7f08233e723c300d548b2.pdf	English	null	ارزیابی پارامترهای مقاومت برشی سطح مشترک بتن و آسفالت بازیافتی و ژئوگرید با استفاده ازآزمایش برش مستقیم	Muhandisī-i ̒umrān-i Sharīf/Muhandisī-i ̒umrān-i Sharīf	2,022	cc-by	12,303	heiratyhosein.1993@gmail.com hassanlou@eng.ikiu.ac.ir a.ardakani@eng.ikiu.ac.ir pwUt xOvU}wv "1400 12 1 VQ}PB '1400 11 16 x}LqY= '1400 5 31 Ci=}QO %M}Q=D DOI:10.24200/J30.2022.59660.3065 Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; 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w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt 1401 u=DU@=D 'h}QW u=Qta \|UOvyt \|WywSB '4027 "X '2/1 \|xQ=tW '38 2 \|xQwO OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; 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[3]"OQ=Ov =yCWO w =yxO=H Q=vm '1xr=@R Ov=tUB uD Q=Ry 35 =D 33 u}@ xv=RwQ u=QyD QyW QO xm CU= u; R= \|m=L Ov=tUB R= \|v=tDN=U \|xr=Nv \UwDt O}rwD u=R}t '1 pwOH QO "OwW\|t O}rwD \|v=tDN=U \|Di=}R=@ Kr=Yt u}QDRQ=@ R= [4]"CU= xOW x=Q= u=QyD QyW QO 99 =D 95 p=U \|Di=}R=@ Cr=iU; \|=yxv=OnvU w 2RCA xOWOQN uD@ \|=yxv=OnvU x@ u=wD\|t '=yx=oOwQiOv=@'\|vD@\|=yxO=H?}QND=}\|R=UR=@R=xmOQmxQ=W= 3RAP\|R=UwQ [1]"Ov};\|t CUO x@ \|R=UwQ Cr=iU; w =yu=tDN=U 'pB \|=yxR=U swta CW=OQ@ '\|Di=}R=@ Kr=Yt R= OOHt \|xO=iDU= `v=wt u}QDsyt R= \|m} s=wO w ha[ '\|a}@] \|=ynvU =@ xU}=kt QO \|Di=}R=@ Kr=Yt xm CU= u}= R= u}=Q@=v@ &OW=@ K}LY \|OL =D CU= umtt xrUt u}= xm \|r=L QO "OvQ=O \|QDtm Kr=Yt OQmrta Ow@y@ \|=Q@ \|OQm}wQ '\|Di=}R=@ Kr=Yt R= xO=iDU= R= p@k O}=@ \|y=oW}=tR; \|=yV}=tR; s=Hv= 'xOW xQ=W= \|=yOQm}wQ R= \|m} "OwW P=ND= QwmPt uQOt `t=wH Q=O}=B \|xaUwD \|U=U= u=mQ= R= \|m} '\|a}@] `@=vt R= K}LY \|xO=iDU= ^iL=@x]@=QQO \|t=O\|v=Qovl}'\|a}@]Q}PB=vO}OHD`@=vtG=QNDU=Vy=m"CU= ?}QND'xr=UQy"Ovm\|tj}wWD=Q\|Di=}R=@Kr=YtR=xO=iDU=wCU=CU}R\}Lt "Ovvm\|t O}rwD =Q \|D=a}=[ Kr=Yt R= \|O=}R sHL '=yx=Q w =yu=tDN=U \|R=Uwv w R= w OvDUy CU}R\}Lt \|oOwr; 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\|Di=}R=@ Kr=Yt R= QDtm O}QowS x@ xOW KrUt \|Di=}R=@ Kr=Yt T=tD K]U w \|m}v=mt X=wN '2018 u=Q=mty w \|o}@ u=t}rU [11]"CU= xOvvmKrUt uwO@ \|=yQ=w}OQOR}Qm=Nu=wvax@xO=iDU=\|=Q@=Q\|Di=}R=@uD@\|=yxv=OnvU\|m}rwQO}y VWm uwQ}@ x=oDUO 'T=}ktnQR@ s}kDUt VQ@ x=oDUO =@ MSE KrUt l=N \|m}v=mt C=}YwYN uOQw; CUOx@ \|=Q@ xOW \|WmyR \|QwLtxU u}vJty w xm OvDi=}QO w OvO=O s=Hv= hrDNt \|=yl}DDvUwS =@ RCA Kr=Yt VvmQOv= "CU=xDW=O=QT=tDK]Ul=m]Y=\|x}w=R u}QDq=@'xQwLtlDO}QowS RCA KrUt \|xU=t w uW =@ \|}xU}=kt p@=k VWm uwQ}@ C}iQ_ 'xOW KrUt RCA \|m}v=mt X=wN '2019 u=Q=mty w \|o}@ u=t}rU [12]"CU= xO=O u=Wv xOW R}Qm=N u=wvax@ xO=iDU= \|=Q@ =Q \|Di=}R=@ Cr=iU; w \|Di=}R=@ uD@ \|=yxv=OnvU \|QwLtxU \|=yV}=tR; u=W}= "OvOQm \|@=}RQ= xOW C}@FD \|m}v=mt \|=yQ=w}O QO QwmPt Kr=Yt VvmQOv= \|UQQ@ \|=Q@ =Q VWm uwQ}@ w s}kDUt VQ@ 'T=}ktnQR@ \|xvwtv \|=Q@ VRN p}Uv=DB \|@=}RQ= \|=Q@ =Q \|QwLtxU V}=tR; R}v w l}DDvUwS =@ O}QowS `wv R= QwmPt VywSB QO xOW xO=iDU= l}DDvUwS "OvO=O s=Hv= RAP "Ow@ p}=DUmDwS `wv l} w xQwLtwO w xQwLtlD \|=Q@ ?U=vt w u=RQ= u}Ro}=H l} '4T=}ktnQR@ s}kDUt VQ@ V}=tR; [2]"CU= \|xQwLtxU \|=yuwtR; =@ xU}=kt QO \}Lt l} QO xOW KrUt \|=yl=N V}=tR; VQ@ V}=tR;"OyO\|t x=Q= =Q \|@wN G}=Dv xm CU= \|v=O}t \|=yuwtR; =} T=}ktnQR@ CN=wvm}Q}e \|WQ@ VvQm 'VQ@ \|xLiY uOw@ \|Q=@H= %xrtH R= '\|@}=at 's}kDUt [5]"OQ=O VQ@ \|xLiY QO =yVvD CN=wvm}Q}e `}RwD w xvwtv `=iDQ= QO hrDNt \|=yxR=Ov= R= xm OvDUy l}DDvUwS \|xOvvmKrUt Kr=Yt '5=yO}QowS \|DNU '=yO}QowS "OvQ=O \|rw] w \|[Qa \|Qt}rB \|=yQ=wv w Ov=xOW p}mWD xtWJ "OvWN@\|t Ow@y@ O}QowS \|=yxtWJ QO C=QP \|oDiQwQi syQO j}Q] R= =Q l=N V}=Ri= =Q T=tD K]U VQ@ Q@=Q@ QO Ctw=kt 'sm=QDt Kr=Yt w O}QowS u}@ T=tD \|Ov@xv=O %xrtH R= '\|O=}R pt=wa x@ l=N C=QP O}QowS u}@ K]U Q=DiQ "OyO\|t sm=QD \|=Q@ \|SQv= 'O}QowS C=}YwYN w `wv '=ypmW C=}YwYN w =yxv=OnvU \|O}rm syt C=aw[wt R= \|m} [6]"OQ=O \|oDU@ \|Q=PoQ=@ \}=QW w ?Yv s=ovy QO xm CU= xOvvmKrUt w l=N u}@ 6VvmQOv= 'KrUt l=N \|=yR=U \|L=Q] QO xm \|v=tR "OwW\|t u}}aD s}kDUt VQ@ w VWm uwQ}@ V}=tR; R= xO=iDU= =@ ?re= =@ O}QowS Kr=Yt \|oDN}Uo \|=yOt 'OvwW\|t KrUt O}QowS =@ \|}xv=O Kr=Yt \|oQ=B \|oDN}Uo "CU= x=Qty VWm uwQ}@ \|oDN}Uo w \|WRer \|oDN}Uo '\|oQ=B R= "OwW QDW}@ u; \|WWm VvD R= O}QowS QO \|WWm VvD xm OyO\|t MQ \|v=tR ODi=\|t j=iD= O}QowS \|wQ Q@ C=QP CmQL p}rO x@ \|WRer \|oDN}Uo 'Qo}O hQ] 'VWm uwQ}@ \|oDN}Uo QO "OwW\|t pQDvm T=tD K]U \|WQ@ Ctw=kt \UwD w u=wD\|t '1 pmW QO [7]"OwW\|t xO}Wm l=N \|xOwD R= uwQ}@ CtU x@ O}QowS u}}aD \|=Q@ =Q \|y=oW}=tR; uwtR; u}QD?U=vt w OvD ?}W l} \|oDN}Uo sU}v=mt [8]"OQm xOy=Wt VvmQOv= C=}YwYN ?}DQDx@ xOWsm=QDt RAPw RCA\|Di=}R=@Kr=YtxmO=Ou=Wvu=W}=G}=Dv VQ=Ro \|xOwOLt QO xm 'Ov=xOw@ xHQO 29 w 49 \|rN=O l=m]Y= \|x}w=R \|=Q=O \|QDq=@ VvmQOv= l=m]Y= \|x}w=R 'QwmPt Kr=Yt u}vJty "CU= C=}@O= QO xOW p}=DUmDwS u}@ \|WRer I}y "OvDW=Po V}=tv x@ p}=DUmDwS x@ C@Uv O}QowS =@ p}=DUmDwS \|oQ=B p}rO x@ =y\|oDN}Uo w xOWv xOy=Wt RCA O}QowS w RCA C@Uv RCA VvmQOv= ?}Q[ "CU= xOw@ VWm uwQ}@ V}=tR; pw] QO O}QowS w VvD V}=Ri= =@ VvmQOv= ?}=Q[ u}vJty w xOw@ QDW}@ =yl}DDvUwS =@ RAP x@ sm=QDt RAP xm O=O u=Wv VRN V}=tR; R= pY=L G}=Dv "CU= xDi=} V}=Ri= p=tQv xO=O u=Wv OwN R= xOW ptLDt \|i=QLv= VvD CLD =Q \|O=}R \|i=QLv= VvQm 'xOW MSE KrUt l=N \|=yQ=w}O R}Qm=N QO RCA w RAP R= xO=iDU= u=W}= "CU= R= \|Q}owrH \|=Q@ "OwW KrUt O}QowS =@ Qo= \ki w Qo= xD@r= 'Ov=xOQm x}YwD =Q Kr=Yt\|WQ@Ctw=kt\|=y\|oS}w\|wQQ@\|D=ar=]t'2002u=Q=mtyw7=}t=ywD =@ T=}ktnQR@ s}kDUt VQ@ x=oDUO R= xO=iDU= =@ =yu; OQmrta \|UQQ@ w \|Di=}R=@ s=Hv= xOvvmKrUt uwO@ w =@ \|Di=}R=@ Kr=Yt \|wQ Q@ QDt\|r}t 305 305 O=a@= xOW OQN uD@ Ovv=t \|Di=}R=@ Kr=Yt QO =yxOvvmKrUt OwHw xm OvDi=}QO w OvO=O VQ@ Q@=Q@ QO Ctw=kt xHwD p@=k V}=Ri= x@ QHvt xOW OQN \|v=tDN=U \|=}=k@ w \|x}w=R"CU=xOWpQDvm\|OwOL=DxvwtvpmWQ}}eD'u}=Q@xwqa"CU=xOWKr=Yt lWN w ?w]Qt Cr=L QO xOvvmKrUt uwO@ xOW OQN \|Di=}R=@ Kr=Yt l=m]Y= lWNw?w]QtCr=LQO O}QowS=@uOWKrUts=ovyw xHQO39w35 ?}DQDx@ 28 80x@O}=@R}Qm=NQO\|i=QLv=VvD\|xv}W}@'xOWsm=QDt RAPVRN\|oDN}Uo \|D=ar=]t '2020 u=Q=mty w \|OQ=HQyt [13]"OwW OwOLt RAP \|Q=Wi Ctw=kt QO xO=iDU= \|=Q@ 16 CDW Kr=Yt \|m}v=mt w \|m}R}i C=}YwYN \|@=}RQ= \|=Q@ RCA \|Di=}R=@ uD@ \|xv=OovU w 17xr=Nv R= u=W}= "OvO=O s=Hv= =yxO=H T=U=Q}R CUOx@ O=R Kr=Yt \|R=U=OH R= TB u=QyD u=DU= QO \|ra@; \|xr=@R uiO pLt R= xm \|Q@Q=@ C@Uv 'nQ@QD= OwOL '\|Ov@xv=O uwJ \|}=yV}=tR; "OvOQm xO=iDU= 'Ow@ xOt; \|=Q@ \|rm}U \|}xNQJ \|Q=PoQ=@ V}=tR; u}OvJ R}v w s}kDUt VQ@ w '=}vQi}r=m s=Hv= CDW Kr=Yt ptLD C}iQ_ QO pUwS \|xOvvmKrUt \|WN@QF= \|@=}RQ= QDW}@ =@ CDW Kr=Yt \|m}v=mt w \|m}R}i C=}YwYN xm OvDi=}QO w OvO=O CN=U QO T=U=Q}R Kr=Yt u=wva x@ xO=iDU= \|=Q@ =yOQ=Ov=DU= \|=yQ=}at w \@=w[ [14]"CU= pw@kp@=k =yxO=H DU@=D h}QW u=Qta \|UOvyt CQ=@a x@ "CU= VvmQOv= ?}Q[ w =yl}DDvUwS w Kr=Yt lQDWt T=tD K]U u}Ro}=H Kr=Yt u=wvax@ \|Di=}R=@ Cr=iU; w uD@ Kr=Yt R= xO=iDU= u=mt= 'QDj}kO "O};\|t Q=tWx@ Q[=L VywSB \|=y\|Qw;wv R= KrUt l=N \|=yQ=w}O R}Qm=N QO xOW xO=iDU= Kr=Yt "2 \|m=N Kr=Yt "1"2 \|Di=}R=@uD@\|=yxv=OovU%\|Di=}R=@Kr=Yt`wvwOR= Q[=LVywSBs=Hv=Qw_vtx@ Kr=Yt R= u}vJty w 'RAP \|R=UwQ Ci=}R=@ Cr=iU; \|=yxv=OovU w RCA Kr=Yt Q}wYD '2 pmW QO "CU= xOW xO=iDU= xU}=kt \|=Q@ 20NA \|a}@] \|vD@ \|=y?amt R= \|Di=}R=@ uD@ \|=yxv=OovU "OwW\|t xOy=Wt xOW xO=iDU= u}wRk u=DU= QO `k=w uD@ s=mLDU= x=oW}=tR; R= =yu=tDN=U R= \|Q}oxvwtv R= pY=L =@ p=mU=B=ot 25 \|Q=Wi Ctw=kt \|=Q=O 'QwmPt \|vD@ \|=y?amt "Ov=xOW x}yD Cr=iU;\|xv=NQ=mQOumOQNx=oDUOQO\|vD@\|=y?amt"OvOw@xRwQ90\|Qw;pta Cr=iU; \|=yxv=OovU "OvOW OQN =QyRu}w@ \|xO=H \|QDtwr}m 15 QO u}wRk u=DU= R= =yu; "OvOW x}yD u}wRk u=DU= \|}xO=H pkvwptL w \|Q=Oy=Q \|xQ=O= R= \|Di=}R=@ V=QDCr=iU; x=oDUO \UwD \|rY= \|=yxO=H '=yx=QoQR@ =yxO=H \|R=UwQ uO}W=QD \|a}@] Kr=Yt "OvOW xDW=OQ@ QDt\|Dv=U 20 \|xv}W}@ jta =D u}W=t Qw@a Q=@l} =@ \|=yV}=tR; "Ov=xOW x}yD xQ \|v}tN s=t= prtr=u}@ x=oWv=O uD@ x=oW}=tR; R= xHQO '?; ?PH 'Gs Ot=H \|=yxv=O \|r=oJ u}}aD '\|Ov@xv=O %uwJty '\|m}R}i XwYNt uRw u}}aD \|=Q@ u}vJty w Kr=Yt \|=y\|oS}w u}}aD Qw_vt x@ pH \@DQt \|=yOQ=Ov=DU= j@] xOW KqY= sm=QD V}=tR; 'xv}y@ C@w]Q w xv}W}@ Qw_vt x@ "Ov=xOW x=Q= 2 pwOH QO Kr=Yt \|m}R}i C=}YwYN "Ov=xOW s=Hv= xOW xDiQo Q_v QO u=Um} ,=@}QkD \|Ov@xv=O 'Kr=Yt xU Qy QDy@ \|xU}=kt w \|@=}RQ= "CU= u}@ T=tD K]U \|WQ@ Q=DiQ \|@=}RQ= \|=Q@ '2020 u=Q=mty w 18nvBOwU V}=tR; R= xO=iDU= =@ '\|R=UwQ \|=yOQ@Q=m QO O}QowS w \|Di=}R=@ uD@ \|=yxv=OnvU 'xtWJ \|xR=Ov= QF= 'QDt\|r}t 305 305 204 T=}ktnQR@ s}kDUt VQ@ uD@ \|Di=}R=@ Kr=Yt w O}QowS VvmQOv= QO =Q \|Ov@xv=O w O}QowS \|WWm Ctw=kt w xtWJ hrDNt \|=yxR=Ov= =@ xQwLtwO O}QowS `wv xU R= u=W}= "OvOQm \|UQQ@ Cw=iDt \|=y\|Ov@xv=O =@ \|Di=}R=@ uD@ \|xvwtv `wv wO w Cw=iDt \|WWm Ctw=kt Ctw=kt QO \|tm Q}F-=D '=yO}QowS \|WWm Ctw=kt 1 :xm OvDi=}QO w OvOQm xO=iDU= \|oDN}Uo Cr=L x@ VQ@ V}=tR; pw] QO =yO}QowS =Q}R 'OQ=O T=tD K]U \|WQ@ QO O}QowS \|xtWJ \|xR=Ov= w \|Di=}R=@ uD@ Kr=Yt C=QP \|xR=Ov= 2 "OvUQ\|tv Ctw=kt uOW QDW}@ Ea=@ QDnQR@ \|xtWJ ZQa "CU= Q=PoQ}F-=D \|WQ@ Ctw=kt 'Ovm\|t \|Q}owrH C=QP C=mQL R= O}QowS 3 "OwW\|t T=tD K]U QO \|WQ@ =@ Qt= u}= xm OwW\|t QDW}@ RCA C=QP uDiQ wQi sy pN=O w uOQwN Q )U =Pr xOy=Wtp@=k \|WQ@ \|}=Hx@=H p@=kt QO \|Owta \|}=Hx@=H QO C=QP \|a=UD= Q=DiQ [7]"CU= Cr=iU; \|=yxv=OnvU \|xR=Ov= \|@Uv QF= R}v '2021 u=Q=mty w 19\|=JtwO= x@ lQDWt K]U \|WQ@ Ctw=kt \|=yQDt=Q=B QO =Q O}QowS \|=yxtWJ w \|Di=}R=@ C=aq]= QOkJ Qy [15]"OvOQm \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; ltm C=ar=]t 'CU= TQDUO QO \|a}@] l=N w =yl}DDvUwS T=tD K]U OQwt QO \|xOW OQN uD@ \|Di=}R=@ Kr=Yt T=tD K]U \|WQ@ Q=DiQ OQwt QO xOW s=Hv= OwOLt KrUt l=N R}Qm=N OQ@Q=m \|=Q@ \|R=UwQ \|Di=}R=@ Cr=iU; w \|Di=}R=@ OQwt QO Rwvy 'l}vmDwS \|UOvyt pwY= QO O=}R \|=yCiQW}B seQ\|ra "CU= QwmPt Kr=Yt =}; xmv}= w p}@k u}= R= \|}=yxOwD w =yxr=Nv 'xOQwNCUO \|=yl=N Vr=J 'Q}N =} Ovvm\|t xOQw;Q@ =Q \|m}vmDwS \|=yQ=m QO R=}vOQwt \|m}vmDwS C=t=Rr= uD@ xrtH R= '\|R=UwQ w \|v=tDN=U \|=yxr=Nv OQwt QO \|O=}R C=ar=]t "OQ=O OwHw xO=iDU= \|=Q@ =yxv=OnvU O}rwD x@ \w@Qt ,=DOta xm CU= xOW s=Hv= Cr=iU; w w [19w18]'=yxO=H T=U= w T=U=Q}R \|x}q \|=yxv=OnvU \|x}yD w [17w16]'uD@ QO "Ov=xOw@ \|}xR=UQ}e KrUt uD@ <=RH= CN=U "xOW xO=iDU= \|m=N Kr=Yt Q}wYD "2 pmW \|R=UwQw\|v=tDN=U\|xr=Nvs}_asHLR==};xmCU=u}=`w[wt\|rmC}ty= w OwW\|t CU}R\}Lt \|oOwr; Ea=@ u; CW=@v= w Ca}@] QO u; \|R=U=yQ xm u=wD\|t 'xOW QmP OQ=wt \|xO=iDU= Q@ xwqa 'OwW\|t \|krD xr=@R u=wva x@ ,=twta Kr=Yt u=wva x@ =} w \|m=N \|=y\|v=wQ}W \|=yxSwQB QO Q@Q=@ \|m=N Kr=Yt u=wvax@ VywSB \|rY= hOy u}=Q@=v@ #Q}N =} OQm xO=iDU= KrUt l=N \|=yxR=U R}Qm=N u=wvax@ xOW OQN Cr=iU; w uD@ \|Di=}R=@ Kr=Yt R= xO=iDU= u=mt= \|UQQ@ 'Q[=L \|WQ@ Ctw=kt OQmrta \|UQQ@ OQm}wQ =@ KrUt l=N \|=yQ=w}O QO R}Qm=N Kr=Yt 29 "V}=tR;OQwt \|=yO}QowS \|m}R}i C=}YwYN "3 pwOH GP3 GP2 GP1 O}QowS PET PET PET O=wt PVC PVC PVC VWwB x}wUlD x}wUlD x}wUlD `wv 60 60 110 QO \|}=yv \|WWm Ctw=kt (kN=m) \|rw] CyH 20 20 30 QO \|}=yv \|WWm Ctw=kt (kN=m) \|[Qa CyH 12 12 12 \|WWm Ctw=kt QO VvQm () \|rw] CyH QO \|tU= 560 340 860 O}QowS \|rw] \|DNU 5 VvQm QO 20 25 25 QO O}QowS xtWJ \|xR=Ov= (mm) \|[Qa CyH 20 33 33 QO O}QowS xtWJ \|xR=Ov= (mm) \|rw] CyH 2 2 2 (mm) Ct=N[ PolyVinyl Chloride "R}Qm=N \|Ov@xv=O \|xOwOLt w Kr=Yt \|Ov@xv=O \|vLvt "4 pmW QO xOW xO=iDU= R}Qm=N [21]'101 \|x}QWv w [20]'FHWA OQ=Ov=DU= j@] KrUt l=N \|=y?}W w MSEW xOW C}@FD \|m}v=mt \|m=N \|=yQ=w}O OQ=Ov=DU= j@] \|Ov@xv=O V}=tR; "CU= \|Ov@xv=O \|xOwOLt l} \|=Q=O 21RSS xOW xO=iDU= l=N \|Ov@xv=O \|vLvt "CU= xOW s=Hv= [22] ASTM42287 USCSOQ=Ov=DU=T=U=Q@"OwW\|txOy=Wt4pmWQO\|Ov@xv=OV}=tR;R=pY=L j@=]t"OW\|Q=Pos=v SWu=wvax@xOWxO=iDU=l=N'O}=i}vw}\|Ov@xk@]sDU}U xt=vu}}; Q_vOQwt \|xOwOLt QO 'xOW xO=iDU= Kr=Yt xU Qy \|Ov@xv=O 'Q}N= pmW KrUt l=N \|=yQ=w}O \|=Q@ FHWA \|Ov@xv=O \|xOwOLt \|=Q@ =t= 'CU= xOw@ j@] "CU= xOw@ GQ=N xOwOLt R= \|RH \|Q=Okt Kr=Yt \|Ov@xv=O 'MSE V}=Ri= Ov=wD\|t QDt\|Dv=U 10 =D R}Qm=N \|xv=O u}QDnQR@ \|xR=Ov= '101 \|x}QWv Kr=Yt R= xO=iDU= QF= QO =yl}DDvUwS \|oO}O?}U; sOa O}=@ 'Cr=L u}= QO "O@=} 38 R= QDnQR@ \|=yxv=OovU CU= QDy@ "OwW \|UQQ@ \|W}=tR; C=a]k QO xv=OCWQO \|xt=vu}}; "OvyO p}mWD =Q \|R}Qm=N Kr=Yt 20 R= QDtm 'Iv}= 1=5 QDt\|r}t "\|m=N Kr=Yt \|m}R}i C=}YwYN "2 pwOH RAP RCA NA xvwtv 0=33 0=8 0=51 D10(mm) 1=7 2=4 1=98 D30(mm) 3=77 4=15 4=08 D50(mm) 4=28 5=18 5=26 D60(mm) 14=39 6=45 10=21 Cu 1=8 1=0 1=43 CC 2=44 2=6 2=68 GS SW SW SW USCS l=N \|Ov@xk@] 0=8 0=7 0=8 QDR}Q C=QP OYQO 200 lr= R= 1=1 4=1 3=2 ?; ?PH 7=82 10=7 7=36 pH "\|iQYt O}QowS Q}wYD "3 pmW O}QowS "2"2 \|Q=HD \|=ys=v =@ PET x}wUlD O}QowS `wv xU 'Q[=L VywSB QO KrUt \|=Q@GPGRID60=20 w GPGRIR60=30 'GPGRID110=30 s}kDUt VQ@ V}=tR; s=Hv= \|=Q@ u}vJty w \|a}@] \|Di=}R=@ Kr=Yt uOQm \|Qt}rB VmwQ l} \UwD q=@ \|WWm Ctw=kt =@ QDU=\|rB \|=yMv R= xm T=}ktnQR@ \|WWm \|=yCtw=kt 'QwmPt O}QowS `wv xU "CU= xOW xO=iDU= 'xOW xOv=WwB x=}U Qy QO q=@ Ctw=kt =@ =yO}QowS "Ovvm\|t O=H}= lJwm \|=yVvQm QO =Q \|yHwDp@=k =Q u;,qwtat xm x=oDUO ZQa O=ODt= w MD x=oDUO CyH pwQ pw] CyH wO CmQW R= \|iQYt \|=yO}QowS "OvwW\|t xDi=@ Ovt=v\|t CD \|[Qa CyH s=v =@ VywSB QO xOW xO=iDU= \|=yO}QowS Q}wYD '3 pmW QO "Ov=xOW x}yD u=}UQ=BwS w =yxtWJ w u=Um} \|UOvy Q=DN=U '\|iQYt \|=yO}QowS "OwW\|t xOy=Wt Q[=L \|m}v=mt w \|m}R}i C=}YwYN '3 pwOH QO "OvQ=O Cw=iDt \|WWm \|=yCtw=kt CrwyU\|=Q@"CU=xOWx=Q='CU=xOWx}yDu=}UQ=BwSCmQW\UwDxm=yO}QowS 'GPGRID110=30 O}QowS \|=Q@ ?}DQD x@ GP3 w 'GP2 'GP1 \|=ys=v R= "CU= xOW xO=iDU= GPGRID60=30 w GPGRID60=20 "V}=tR;OQwt \|=yO}QowS \|m}R}i C=}YwYN "3 pwOH GP3 GP2 GP1 O}QowS PET PET PET O=wt PVC PVC PVC VWwB x}wUlD x}wUlD x}wUlD `wv 60 60 110 QO \|}=yv \|WWm Ctw=kt (kN=m) \|rw] CyH 20 20 30 QO \|}=yv \|WWm Ctw=kt (kN=m) \|[Qa CyH 12 12 12 \|WWm Ctw=kt QO VvQm () \|rw] CyH QO \|tU= 560 340 860 O}QowS \|rw] \|DNU 5 VvQm QO 20 25 25 QO O}QowS xtWJ \|xR=Ov= (mm) \|[Qa CyH 20 33 33 QO O}QowS xtWJ \|xR=Ov= (mm) \|rw] CyH 2 2 2 (mm) Ct=N[ PolyVinyl Chloride "\|m=N Kr=Yt \|m}R}i C=}YwYN "2 pwOH RAP RCA NA xvwtv 0=33 0=8 0=51 D10(mm) 1=7 2=4 1=98 D30(mm) 3=77 4=15 4=08 D50(mm) 4=28 5=18 5=26 D60(mm) 14=39 6=45 10=21 Cu 1=8 1=0 1=43 CC 2=44 2=6 2=68 GS SW SW SW USCS l=N \|Ov@xk@] 0=8 0=7 0=8 QDR}Q C=QP OYQO 200 lr= R= 1=1 4=1 3=2 ?; ?PH 7=82 10=7 7=36 pH RAP RCA NA xvwtv 0=33 0=8 0=51 D10(mm) 1=7 2=4 1=98 D30(mm) 3=77 4=15 4=08 D50(mm) 4=28 5=18 5=26 D60(mm) 14=39 6=45 10=21 Cu 1=8 1=0 1=43 CC 2=44 2=6 2=68 GS SW SW SW USCS l=N \|Ov@xk@] 0=8 0=7 0=8 QDR}Q C=QP OYQO 200 lr= R= 1=1 4=1 3=2 ?; ?PH 7=82 10=7 7=36 pH "\|iQYt O}QowS Q}wYD "3 pmW O}QowS "2"2 \|Q=HD \|=ys=v =@ PET x}wUlD O}QowS `wv xU 'Q[=L VywSB QO KrUt \|=Q@GPGRID60=20 w GPGRIR60=30 'GPGRID110=30 s}kDUt VQ@ V}=tR; s=Hv= \|=Q@ u}vJty w \|a}@] \|Di=}R=@ Kr=Yt uOQm \|Qt}rB VmwQ l} \UwD q=@ \|WWm Ctw=kt =@ QDU=\|rB \|=yMv R= xm T=}ktnQR@ \|WWm \|=yCtw=kt 'QwmPt O}QowS `wv xU "CU= xOW xO=iDU= 'xOW xOv=WwB x=}U Qy QO q=@ Ctw=kt =@ =yO}QowS "Ovvm\|t O=H}= lJwm \|=yVvQm QO =Q \|yHwDp@=k =Q u;,qwtat xm x=oDUO ZQa O=ODt= w MD x=oDUO CyH pwQ pw] CyH wO CmQW R= \|iQYt \|=yO}QowS "OvwW\|t xDi=@ Ovt=v\|t CD \|[Qa CyH s=v =@ VywSB QO xOW xO=iDU= \|=yO}QowS Q}wYD '3 pmW QO "Ov=xOW x}yD u=}UQ=BwS w =yxtWJ w u=Um} \|UOvy Q=DN=U '\|iQYt \|=yO}QowS "OwW\|t xOy=Wt Q[=L \|m}v=mt w \|m}R}i C=}YwYN '3 pwOH QO "OvQ=O Cw=iDt \|WWm \|=yCtw=kt CrwyU\|=Q@"CU=xOWx=Q='CU=xOWx}yDu=}UQ=BwSCmQW\UwDxm=yO}QowS 'GPGRID110=30 O}QowS \|=Q@ ?}DQD x@ GP3 w 'GP2 'GP1 \|=ys=v R= "CU= xOW xO=iDU= GPGRID60=30 w GPGRID60=20 "\|iQYt O}QowS Q}wYD "3 pmW "R}Qm=N \|Ov@xv=O \|xOwOLt w Kr=Yt \|Ov@xv=O \|vLvt "4 pmW "\|iQYt O}QowS Q}wYD "3 pmW "\|iQYt O}QowS Q}wYD "3 pmW O}QowS "2"2 \|Q=HD \|=ys=v =@ PET x}wUlD O}QowS `wv xU 'Q[=L VywSB QO KrUt \|=Q@GPGRID60=20 w GPGRIR60=30 'GPGRID110=30 s}kDUt VQ@ V}=tR; s=Hv= \|=Q@ u}vJty w \|a}@] \|Di=}R=@ Kr=Yt uOQm \|Qt}rB VmwQ l} \UwD q=@ \|WWm Ctw=kt =@ QDU=\|rB \|=yMv R= xm T=}ktnQR@ \|WWm \|=yCtw=kt 'QwmPt O}QowS `wv xU "CU= xOW xO=iDU= 'xOW xOv=WwB x=}U Qy QO q=@ Ctw=kt =@ =yO}QowS "Ovvm\|t O=H}= lJwm \|=yVvQm QO =Q \|yHwDp@=k =Q u;,qwtat xm x=oDUO ZQa O=ODt= w MD x=oDUO CyH pwQ pw] CyH wO CmQW R= \|iQYt \|=yO}QowS "OvwW\|t xDi=@ Ovt=v\|t CD \|[Qa CyH s=v =@ VywSB QO xOW xO=iDU= \|=yO}QowS Q}wYD '3 pmW QO "Ov=xOW x}yD u=}UQ=BwS w =yxtWJ w u=Um} \|UOvy Q=DN=U '\|iQYt \|=yO}QowS "OwW\|t xOy=Wt Q[=L \|m}v=mt w \|m}R}i C=}YwYN '3 pwOH QO "OvQ=O Cw=iDt \|WWm \|=yCtw=kt CrwyU\|=Q@"CU=xOWx=Q='CU=xOWx}yDu=}UQ=BwSCmQW\UwDxm=yO}QowS 'GPGRID110=30 O}QowS \|=Q@ ?}DQD x@ GP3 w 'GP2 'GP1 \|=ys=v R= "CU= xOW xO=iDU= GPGRID60=30 w GPGRID60=20 "R}Qm=N \|Ov@xv=O \|xOwOLt w Kr=Yt \|Ov@xv=O \|vLvt "4 pmW QO xOW xO=iDU= R}Qm=N [21]'101 \|x}QWv w [20]'FHWA OQ=Ov=DU= j@] KrUt l=N \|=y?}W w MSEW xOW C}@FD \|m}v=mt \|m=N \|=yQ=w}O OQ=Ov=DU= j@] \|Ov@xv=O V}=tR; "CU= \|Ov@xv=O \|xOwOLt l} \|=Q=O 21RSS xOW xO=iDU= l=N \|Ov@xv=O \|vLvt "CU= xOW s=Hv= [22] ASTM42287 USCSOQ=Ov=DU=T=U=Q@"OwW\|txOy=Wt4pmWQO\|Ov@xv=OV}=tR;R=pY=L j@=]t"OW\|Q=Pos=v SWu=wvax@xOWxO=iDU=l=N'O}=i}vw}\|Ov@xk@]sDU}U xt=vu}}; Q_vOQwt \|xOwOLt QO 'xOW xO=iDU= Kr=Yt xU Qy \|Ov@xv=O 'Q}N= pmW KrUt l=N \|=yQ=w}O \|=Q@ FHWA \|Ov@xv=O \|xOwOLt \|=Q@ =t= 'CU= xOw@ j@] "CU= xOw@ GQ=N xOwOLt R= \|RH \|Q=Okt Kr=Yt \|Ov@xv=O 'MSE V}=Ri= Ov=wD\|t QDt\|Dv=U 10 =D R}Qm=N \|xv=O u}QDnQR@ \|xR=Ov= '101 \|x}QWv Kr=Yt R= xO=iDU= QF= QO =yl}DDvUwS \|oO}O?}U; sOa O}=@ 'Cr=L u}= QO "O@=} 38 R= QDnQR@ \|=yxv=OovU CU= QDy@ "OwW \|UQQ@ \|W}=tR; C=a]k QO xv=OCWQO \|xt=vu}}; "OvyO p}mWD =Q \|R}Qm=N Kr=Yt 20 R= QDtm 'Iv}= 1=5 QDt\|r}t 30 "Kr=Yt sm=QD Q=Owtv "5 pmW "xv}y@ C@w]Q w xv}W}@ lWN XwYNt uRw Q}O=kt "4 pwOH xv}y@ C@w]Q lWN XwYNt uRw xvwtv (kN=m3) xv}W}@ 6=2 18=8 NA 9=35 17=88 RCA 5=75 18=6 RAP 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|UOvyt "x=oW}=tR; QO OwHwt T=}ktnQR@ s}kDUt VQ@ x=oDUO "6 pmW xOW C@F DATA LOGGER \|}xv=}=Q sDU}U l} \UwD G}=Dv w \|Q}oxR=Ov= "CU= 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|UOvyt "Kr=Yt sm=QD Q=Owtv "5 pmW "xv}y@ C@w]Q w xv}W}@ lWN XwYNt uRw Q}O=kt "4 pwOH xv}y@ C@w]Q lWN XwYNt uRw xvwtv (kN=m3) xv}W}@ 6=2 18=8 NA 9=35 17=88 RCA 5=75 18=6 RAP 2/1 \| "x=oW}=tR; QO OwHwt T=}ktnQR@ s}kDUt VQ@ x=oDUO "6 pmW xOW C@F DATA LOGGER \|}xv=}=Q sDU}U l} \UwD G}=Dv w \|Q}oxR=Ov= "CU= xvwtv \|x}yD VwQ "4 %CU= xOw@ KQW u}= x@ xvwtv \|R=UxO=t; pL=Qt V}=tR;R=xOt;CUOx@\|xv}W}@lWNXwYNtuRwR==yxvwtv\|x}yDQO hr= R=pY=L\|xv}W}@XwYNtuRwQ=OktxmCU=xOWxO=iDU=xOWKqY=sm=QD \|@Uv sm=QD =@ \|}=yxvwtv =D CU= xOW ?Q[ 95 QO xOW KqY= sm=QD V}=tR; s}kDUtVQ@x=oDUO\|x@aHsHLQOxOt;CUOx@Q=Okt"O};CUOx@ 22RC CUOx@ V}=tR; \|QU Qy \|=Q@ R=}vOQwt \|xvwtv pm uRw Q=Okt w xOW ?Q[ u=R}t \|xv}tm '308 w 101 \|=yx}QWv w FHWA \|xt=vu}}; j@] CU= xOt; 95 RSS w MSE KrUt l=N ?}W w KrUt l=N \|=yQ=w}O QO \|oO}@wm "CU= 19 =D =Q l}DDvUwS =@ KrUt l=N R}Qm=N \|xv=O u}QDnQR@ \|xR=Ov= 'FHWA O}=@ 'QDnQR@ \|xR=Ov= =@ =yxv=O R= xO=iDU= \|=Q@ "Ovm\|t x}YwD Iv}= 3=4 QDt\|r}t u}}aD \|=Q@ "OwW \|UQQ@ =yl}DDvUwS \|oO}O?}U; sOa x@ \w@Qt \|=yV}=tR; j@=]t xOW KqY= sm=QD V}=tR; R= xv}y@ C@w]Q w xv}W}@ lWN XwYNt uRw QO w 5 pmW QO "CU= xOW xO=iDU= [23] ASTM D1557 OQ=Ov=DU= \@=w[ "CU= xOW x=Q= xv}y@ C@w]Q w lWN \|xv}W}@ XwYNt uRw Q}O=kt '4 pwOH ASTM D[24]85487 OQ=Ov=DU= R= l=N Ot=H C=QP \|xS}w \|r=oJ u}}aD \|=Q@ lr= R= xm OQ=O OQ@Q=m \|Ot=H C=QP \|=Q@ Q}N= OQ=Ov=DU= "CU= xOW xO=iDU= Q=Okt 'QDtwvm}B l} R= xO=iDU= =@ xm OvW=@ xOQm Qw@a QDt\|r}t 4=75 4 \|xQ=tW 4=75 lr= R= QDnQR@ C=QP \|w=L l=N xm \|t=ovy "CU= xOW u}}aD xS}w \|r=oJ \|wQ Q@ xOv=t =H C=QP \|=Q@ ASTM C127[25] uwtR; VwQ R= O}=@ 'OW=@ QDt\|r}t xS}w \|r=oJ Q=Okt u=wvax@ =Q u; u}ov=}t w OQm xO=iDU= QDt\|r}t 4=75 4 lr= [25] ASTM C127 \|=yOQ=Ov=DU= R= ?; ?PH \|x@U=Lt \|=Q@ "CiQo Q_v QO \UwD Q_vOQwt Kr=Yt PH xHQO "CU= xOW xO=iDU= [26] ASTM C128 w j@]R}Qm=NKr=Yt PHu=R}t"CU=xOWu}}aD [27] ASTM D4972OQ=Ov=DU= =yl}DDvUwS \|=Q@ FHWA \|xt=vu}}; j@] w 510 u}@ 308 w 101 \|=yx}QWv Kr=Yt w PH > 3 w 3 < PH < 9 ?}DQD x@ PP/HDPE w PET `wv R= "CU= xOw@ 7=82 w 10=7 '7=36 ?}DQD x@RAP w RCA 'NA xOW \|Ov@xv=O Kr=Yt R= R=}vOQwt sm=QD x@ xHwD =@ xvwtv Qy uRw u}}aD R= Oa@ ? Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt ,qt=m \|DUO CQwY x@ =D 'CU= xOW xDN}Q \|v}U l} QO xOW x@U=Lt u=R}t x@ x@ w xi=[= \wrNt x@ xv}y@ C@w]Q OYQO u=R}t TBU 'OwW uoty w \wrNt "OUQ@ =yxv=O \|xty x@ C@w]Q =D Ov=xOW \wrNt Kr=Yt xk}kO 10 COt CtUk Qy xm CU= xOW s}UkD \|w=Ut VN@ 5 x@ VQ@ \|x@aH 'xt=O= QO G x@aH CmQL R= =D OvOW smLt x@aH \|=yI}B "CU= xOw@ QDt\|Dv=U 3 `=iDQ= x@ 5 x@ \wrNt '\wrNt uOW xO=t; R= TB "OwW \|Q}owrH xvwtv CN=U s=ovy \|x@wm=@x}qQywxDN}QVQ@\|x@aHQOCtUkQywOWs}UkD\|w=UtCtUk \|=Q@ C=@Q[ O=OaD "OW sm=QDt Q_vOQwt sm=QD \|xHQO x@ uO}UQ =D XwYNt \|aU CQwY x@ w \|D=tOkt \|=yV}=tR; \|QU l} \|] Q_vOQwt sm=QD x@ uO}UQ \|=yx}q 'ASTM D5321 OQ=Ov=DU= j@] "28 \|r= 20 OW u}}aD =]N w "OvQ}ov Q=Qk VQ@ \|xLiY QO x}q wO RQt QO xm OvwW xDN}Q \|}xvwox@ O}=@ l=N xm \|Qw]x@ 'CiQo Q=Qk xvwtv \|wQ O}QowS 'u; uO}@wm w swO \|x}q uDN}Q R= TB OQ=Ov=DU= T=U=Q@ "Ow@ O=R; Qo}O hQ] R= w smLt xQ}o l} \UwD hQ]l} R= uwO@ w h=Y O}=@ 'x=oDUO QO O}QowS uO=O Q=Qk s=ovy 'ASTS D5321 \|=yx}q "OQ}o Q=Qk VQ@ \|xLiY R= QDu}}=B \|QDt\|r}t 2 \|r= 1 QO \|oOW`tH CW=O xHwD xDmv u}= x@ O}=@ "OvOW sm=QDt w xDN}Q ?}DQD x@ sHvB w sQ=yJ 'swU Q=OQ=}W u; K]U O}=@ '\|Oa@ \|x}q x@ uDiQ R= p@k 'OwW\|t xO}@wm xm x}q Qy K]U 'pm \|xt=vu}}; \|x}YwD j@] "OwW =}yt =yx}q \|DN=wvm} w \|Q}oQO =D OwW s}kDUt VQ@ x=oDUO "3 \|=yxOvvm KrUt w \|m=N Kr=Yt u}@ T=tD K]U \|WQ@ Ctw=kt \|@=}RQ= \|=Q@ "CU= xOW xO=iDU= 6 pmW j@=]t T=}ktnQR@ s}kDUt VQ@ x=oDUO R= O}QowS O=a@= w C=t=Rr= s=tD w xOw@ QDt\|Dv=U 30 30 15 O=a@= \|=Q=O x=oDUO \|x@aH xO=iDU=x=oDUO"CU=xOWxOQw;Q@ [28] ASTM D5321\|xt=vu}};j@]\|}xv}tm C}iQ_ =@ \|m}rwQO}y lH l} \UwD \|ki= \|wQ}v w xOw@ l}D=twvB CQwY x@ xOW LVDT GvU\|}=Hx@=H \UwD s=k w \|ki= \|=y\|}=Hx@=H w xOW OQ=w uD 2 31 \|ki= \|}=Hx@=H \|vLvt ? Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt '\|ki= \|}=Hx@=H \|WQ@ Ctw=kt \|vLvt hr= "8 pmW "KrUtQ}e \|a}@] w \|Di=}R=@ Kr=Yt \|Owta \|}=Hx@=H p=tQv VvD QO "OwW\|t xOy=Wt =yxvwtv s=tD QO ,=@}QkD \|WvQm \|oOvwW sQv Q=DiQ w QO xm \|Qw]x@ 'OvQ=O Qo}Om} x@ x@=Wt Q=DiQ RCA w NA Kr=Yt 'p=mU=Bwr}m 50 \|}=Hx@=H =D w xO}UQ xv}W}@ \|WQ@ Ctw=kt x@ QDt\|r}t 6 ,=OwOL \|ki= \|}=Hx@=H OvwQ'QDt\|r}t45 \|}=Hx@=H=DC}=yvQOw xDi=}Vy=mCtw=kt'QDt\|r}t25,=OwOL xm \|Qw]x@ 'CU= xOw@ Cw=iDt \|tm RAP Kr=Yt Q=DiQ "CU= xO=O xt=O= =Q \|D@=F TBU w xO}UQ xv}W}@ Ctw=kt x@ \|QDnQR@ \|ki= \|}=Hx@=H QO 'Q=@QU V}=Ri= =@ "CU= xDi=} Vy=m Ctw=kt 'RCA w NA Kr=Yt x@ C@Uv \|QDs}qt ?}W =@ K]U \|Q@R p=L u}a QO "OW=@ QDQ@R \|Di=}R=@ uD@ \|=yxv=O K]U OwQ\|t Q=_Dv= Ctw=kt V}=Ri= Ea=@ Qt= u}= xm OW Oy=wN =yxv=O QDW}@ \|Q}oQO Ea=@ =yxv=O CU= QDh=Y w sQv,=Qy=_ '\|Dr=iU; \|=yxv=O K]U 'p@=kt QO "OwW\|t xv}W}@ \|WQ@ \|Dr=iU; \|=yxv=OnvU K]U Q@ Q}k OwHw,q=tDL= "Ow@ Oy=wN QDtm =yu; \|Q}oQO w Vy=m Ea=@ C}=yvQO w xOW Cr=iU; \|=yxv=O u}@ l=m]Y= w \|Q}oQO R= `v=t \|vD@ \|Dr=iU; Kr=Yt SEM \|vwQDmr= Q}w=YD "CU= xOW \|WQ@ Ctw=kt \|=OD@= QO "OwW\|t xOy=Wt 10 w 9 \|=ypmW QO ?}DQD x@ Cw=iDt \|=yxR=Ov= =@ xDN=U 95 sm=QD =@ Kr=Yt xmv}= =@ "CU= xO=O MQ \|RH \|x}rw= Z=@kv= 'V}=tR; =yxv=O CU= xOW Ea=@ xm OQ=O OwHw =yxv=O u}@ \|r=N \|=[i \|Q=Okt \|rw 'Ov=xOW "OvyO sHL Vy=m TBU w OvwW l}ORv sy x@ \|Q=Okt =OD@= V}=tR; s=Hv= "5 \|=yG}o s}_vD x@ C@wv 'VQ@ \|x@aH QO u; uOQm sm=QDt w xvwtv CN=U R= TB xOW KrUt Cr=L Qy \|=Q@ "OUQ\|t V}=tR; \|=Q@ \|ki= w s=k Q=@ p=ta= w \|Q}oxR=Ov= =@ p=mU=Bwr}m 150 w '100 '50 s=k \|=yVvD CLD V}=tR; xU 'xOWv KrUtw C}OwOLt 'xt=vu}}; "CU= xOW s=Hv= xk}kO Q@ QDt\|r}t l} \|ki= \|Q=PoQ=@ CaQU x@ "CU= xDiQo Q_v QO xk}kO QO QDt\|r}t 6=35 0=025 u}@ =Q VQ@ CaQU CQwY x@ V}=tR; \}=QW w xOw@ \|}xv=O `wv R= 'xOW xO=iDU= Kr=Yt xmv}= p}rO QO \|v=OvJ Q}F-=D '\|WQ@ \|}=Hx@=H uOQm OQ=w CaQU u}=Q@=v@ 'CU= xOw@v `=@W= \|xU}=kt w \|ovy=ty Qw_vt x@ w [29]&OQ=Ov =yV}=tR; R= xOt; CUOx@ \|=yxDi=} Q@ QDt\|r}t 1 VvQm MQv R= =yV}=tR; \|t=tD QO 'xOW s=Hv= C=ar=]t Qo}O =@ G}=Dv ?rY \|xLiY \|wQ s=k \|Q=PoQ=@ sQy= 'O=@ Q=Wi \UwD "CU= xOW xO=iDU= xk}kO VvD "OvOW ?Yv Q_vOQwt u=mt QO s=k w \|ki= \|=yG}o w CiQo Q=Qk \|Q=PoQ=@ CQwY x@ xvwtv CUWv =D 'CU= xOW OQ=w xvwtv x@ xk}kO 15 COt x@ Q_vOQwt s=k OQ=Ov=DU= "OW `wQW V}=tR; w R=@ VQ@ \|x@aH \|=yI}B TBU "OQ}o s=Hv= pt=m QO xm Ovm\|t x}YwD =Q QDt\|r}t 75 =D 25 \|WQ@ \|}=Hx@=H ASTM D5321 Q_v QO 'CU= xvwtv pw] 15 pO=at xm QDt\|r}t 45 \|}=Hx@=H 'Q[=L VywSB "CU= xOW hkwDt x=oDUO u; R= TB xm\|Qw]x@ 'CU= xOW xDiQo G}=Dv Q}UiD "6 s=Hv= KrUtQ}e Cr=L QO s}kDUt VQ@ V}=tR; 9 =OD@= QO 'OW xDio xm Qw]u=ty \|oOv@UJ w \|rN=O l=m]Y= \|x}w=R Kr=Yt \|WQ@ Ctw=kt \|=yQDt=Q=B =D 'OW \|}=Hx@=Hp@=ktQO\|Owta\|}=Hx@=Hw\|WQ@VvD\|vLvt'8pmWQO"O};CUOx@ "OwW\|t xOy=Wt KrUtQ}e RAP w NA 'RCA Kr=Yt \|ki= CU= xDi=} V}=Ri= l}B \|WQ@ VvD 'p=tQv VvD V}=Ri= =@ 'Q}N= pmW j@=]t 32 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|UOvyt "RAP w RCA 'NA Kr=Yt \|=Q@ \|oDN}Uo VwB Q=Owtv "11 pmW \|}=Hx@=H \|vLvt ? Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt '\|ki= \|}=Hx@=H \|WQ@ Ctw=kt \|vLvt hr= "12 pmW "KrUtQ}e w KrUt NA Kr=Yt \|=Q@ \|ki= \|}=Hx@=H s=k 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|UOvyt "RAP w RCA 'NA Kr=Yt \|=Q@ \|oDN}Uo VwB Q=Owtv "11 pmW \|}=Hx@=H \|vLvt ? Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt '\|ki= \|}=Hx@=H \|WQ@ Ctw=kt \|vLvt hr= "13 pmW "xOvvmKrUt uwO@ w =@ RCA Kr=Yt \|=Q@ \|ki= \|}=Hx@=H s=k p=mU=Bwr}m 150 w 100 '50 \|=yQ=@QU \|=Q@ KrUtQ}e RCA \|xv}W}@ \|WQ@ x@ l}ORv ,=@}QkD xm 'CU= xO=O MQ QDt\|r}t 9 w 7 '7 \|=y\|}=Hx@=H QO ?}DQD x@ 7 \|=y\|}=Hx@=H QO xv}W}@ \|WQ@ Ctw=kt 'xOvvmKrUt =@ xm \|r=L QO "OvDUy sy Vy=m xm CU= u; Q}N= Kr=Yt QO Qo}O \|xDmv "CU= xO=O MQ QDt\|r}t 13 \|r= CQwY \|QDtm ?}W =@ NA \|a}@] Kr=Yt x@ C@Uv KrUtQ}e Cr=L QO Ctw=kt "CU= xOw@ KrUtQ}e Cr=L x@ x}@W ,=@}QkD 'KrUt Cr=L QO Kr=Yt Q=DiQ w xDiQo KrUtQ}e w KrUt Cr=L QO xOv=t\|k=@ w xv}W}@ \|WQ@ Ctw=kt hqDN= u}vJty R= u=Wv R}v \|Owta \|}=Hx@=H Q@=Q@ QO \|ki= \|}=Hx@=H u}@ \|x]@=Q "CU= xOw@ sm O}QowS w Kr=Yt lQDWt T=tD K]U QO w KrUt \}=QW QO w CU= \|a=UD= Q=DiQ "CU= xOW O}OWD xrUt '\|a}@] Kr=Yt Ovv=ty Kr=Yt \|=Q@ \|ki= \|}=Hx@=H Q@=Q@ QO \|WQ@ Ctw=kt Q=Owtv 'hr=14 pmW QO C@Uv RAP Kr=Yt Q=DiQ "OwW\|t xOy=Wt KrUtQ}e w KrUt Cr=L QO RAP KrUtQ}e RAP Kr=Yt \|WQ@ Ctw=kt "CU= Cw=iDt NA w RCA Kr=Yt x@ \|=y\|}=Hx@=H QO u; R= Oa@ w xOw@ \|W}=Ri= QDt\|r}t 19 \|r= 11 \|ki= \|}=Hx@=H =D = = m r= = = = m = = Ctw=kt 'p=tQv VvD V}=Ri= =@ xm OwW\|t xOy=Wt KrUtQ}e w KrUt Cr=L QO "CU= xDi=} V}=Ri= NA+GP3 'NA+GP2 'NA+GP1 'NA \|=Q@ \|WQ@ R= TB w xDi=} V}=Ri= \|WQ@ Ctw=kt '\|}=Hx@=H p=ta= =@ 'KrUtQ}e \|xvwtv QO V}=Ri= =@ xt=O= QO 'QDt\|r}t 5 ,=@}QkD \|}=Hx@=H QO xv}W}@ \|WQ@ VvD x@ uO}UQ OvwQ V}=tR; u=}=B =D TBU w xDi=} Vy=m Ctw=kt QDt\|r}t 23 =D,=@}QkD \|}=Hx@=H pmW "CU= xOvwWsQv Q=DiQ \|xOvyOu=Wv 'Q}N= Q=DiQ "CU= xDiQo OwN x@ \|D@=F Ctw=kt V}=Ri= Ea=@ =yxvwtv uDN=U KrUt xm OyO\|t u=Wv Kw[w x@ QwmPt NA Kr=Yt uOQm KrUt R= TB xm CU= u}= xHwD p@=k \|xDmv "CU= xOW \|WQ@ xOWv KrUt Cr=L R= Cw=iDt xvwtv Q=DiQ 'GP3 w GP2 'GP1 \|=yO}QowS =@ QO \|WQ@ Ctw=kt 'p=mU=Bwr}m 100 w 50 p=tQv \|=yVvD QO xm \|Qw] x@ 'CU= 150 VvD QO \|rw 'CU= xO}UQ OwN \|xv}W}@ x@ QDt\|r}t 11 ,=@}QkD \|}=Hx@=H Q=DiQ u}vJty "CU= xO}UQ xv}W}@ Q=Okt x@ QDt\|r}t 17 \|}=Hx@=H QO p=mU=Bwr}m Ctw=kt "CU= xOW p}O@D xOvwWCNU Cr=L x@ ,=@}QkD xOvwWsQv Cr=L R= Kr=Yt O}QowS QO p=mU=Bwr}m 150 w 50 p=tQv \|=yVvD QO T=tD K]U \|xv}W}@ \|WQ@ VvD QO xm \|r=L QO &CU= xOw@ QDW}@ GP3 w GP2 \|=yO}QowS x@ C@Uv GP1 Qw]u=ty "Ov=xOw@ sy x@ l}ORv ,=@}QkD \|WQ@ \|=yCtw=kt 'p=mU=Bwr}m 100 p=tQv sm=QDt \|=yxv=O \|Q}oQO 'O}QowS K]U =@ =yxv=O l=m]Y= %pt=a xU 'OW u=}@ xm Vkv Ctw=kt V}=Ri= u=R}t QO Ovv=wD\|t \|[Qa \|=yQ=wv w =yxtWJ pN=O QO \|tm 'OQ=O \|QDW}@ Vkv \|Qo}O x@ C@Uv pt=a s=Om xmv}= uDUv=O =t= &OvW=@ xDW=O lQDWt K]U QO \|WQ@ Ctw=kt V}=Ri= p}rO u=wD\|t \|rm Qw]x@ "CU= xO}J}B \|=yxv=O CU@ w pik u}vJty w \|[Qa \|=yQ=wv =@ sm=QDt \|=yxv=O \|Q}oQO x@ =Q xm syt w Q=PoQ}F-=D pt=wa R= \|m} "CUv=O \@DQt O}QowS \|=yxtWJ pN=O l=N K]U w =yxv=OnvU u}@ l=m]Y= 'OQ=O Ctw=kt V}=Ri= QO =Q \|rY= Vkv,q=tDL= 'GP1 \|=yO}QowS \|=Q@ O}QowS pm K]U x@ \|r=N \|=[i C@Uv "CU= O}QowS xm Qw]u=ty "CU= xOw@ 0=72 w 0=74 '0=64 =@ Q@=Q@ ?}DQD x@ GP3 w GP2 u=R}t \|va} 'CU= xOw@ QDtm GP1 O}QowS \|=Q@ QwmPt C@Uv 'OwW\|t x_Lqt QO \|QDW}@ QF= \|m=m]Y= Ctw=kt xOW Ea=@ xm CU= xDW=O \|QDtm \|r=N \|=[i p@=kt QO \|ki= \|}=Hx@=H Q=Owtv "OW=@ xDW=O GP1 O}QowS QO Ctw=kt V}=Ri= \|Owta Z=@kv= l} 'V}=tR; \|=OD@= QO xm OyO\|t u=Wv =yxvwtv \|Owta \|}=Hx@=H "CU= xOQm `=UD= sHL V}=Ri= x@ `wQW xvwtv TBU w xO=O MQ \|RH \|x}rw= T=tD K]U QO u; Q=Okt R= QDtm 'xOvvmKrUt uwO@ NA Kr=Yt QO `=UD= Q=Okt xOW QDW}@ \|a=UD= Q=DiQ Ea=@ O}QowS =@ =yxv=O uOW CU@ w pik "CU= xOw@ \|xvwtv QO '`k=w QO "CU= xDi=} Vy=m `=UD= u=R}t 'p=tQv VvD V}=Ri= =@ "CU= 'OQ}o\|t CQwY QDCL=Q VQ@ K]U QO =yxv=O \|}=Hx@=H w VRer u=mt= 'KrUtQ}e =yxtWJ pN=O QO xOQm Q}o \|=yxv=O VRer u=mt= 'KrUt \|=yCr=L QO xm \|r=L QO CmQL q=@ CtU x@ xtWJ pN=O R= O}=@ =yxv=O Q=J=v x@ w OQ=Ov u=mt= \|DL=Q x@ Qy "OwW\|t xvwtv `=UD= x@ QHvt pta u}= xm Ovvm =O}B VNQJ w VRer u=mt= w p}rO x@ hrDNt \|=yO}QowS QO w O@=}\|t Vy=m Q=@QU V}=Ri= =@ `=UD= Q=Okt OvJ pt=a u}ty '`k=w QO "CU= Cw=iDt sy=@ \|[Qa \|=ys}U Cw=iDt \|=yCtw=kt "OwW\|t sy Ctw=kt V}=Ri= Ea=@ r e r r= RCA v v = V= ; = v 13 pmW \|}=Hx@=H \|vLvt ? Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt '\|ki= \|}=Hx@=H \|WQ@ Ctw=kt \|vLvt hr= "12 pmW "KrUtQ}e w KrUt NA Kr=Yt \|=Q@ \|ki= \|}=Hx@=H s=k "\|Dr=iU; \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "9 pmW "\|vD@ \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "10 pmW V}=Ri= O=H}= Ea=@ sR}v=mt u}= w Ov=xO}Rer w xO}Dre sy \|wQ =yxv=O 'xt=O= QO "O@=}\|t Vy=m `=UD= x@ p}=tD p=tQv VvD V}=Ri= =@ "CU= xOW `=UD= sHL Kr=Ytxm\|DQwYQO'OvQ=O\|QDW}@`=UD=x@p}=tD\|Di=}R=@uD@Ovv=tQDQ@RKr=Yt Kr=Yt "OvQ=O \|QDtm `=UD= x@ p}=tD '\|Di=}R=@ Cr=iU; Ovv=t 'QDh=Y \|=yxv=O =@ QHvt =yxv=O \|wQ u=t}U \|=yQwr@ OWQ '\|DQ=@a x@ "OvQ=O \|v}@=v}@ \|Q=DiQ R}v \|a}@] K]U uOW QDh=Y x@ QHvt =yxv=O \|wQ Q}k \|x}q w `=UD= V}=Ri= w uOW QDQ@R x@ Qo}O OQwt wO R= QDtm \|oOvwWsQv Q=Okt '\|rm Qw]x@ "OwW\|t `=UD= Vy=m w =yxv=O xOv=WwB =Q =yxv=O K]U xm CU= \|Q}k VWwB u=ty u; p}rO,q=tDL= xm CU= xOw@ \|rw "CU= xO=O Vy=m =Q =yxv=O \|Q}oQO w xOQm O=H}= =Q \|QDh=Y ,=D@Uv \|=yxv=O w \|O=}R Cw=iD xvwtv xU Qy \|=Q@ V}=tR; \|=yDv= QO xOv=t}k=@ VvD Q=Okt '\|rmQw] x@ "OvDW=O \|y@=Wt Q=DiQ \|Di=}R=@ uD@ w \|a}@] \|xvwtv wO Qw]u}ty "OvDW=Ov sy =@ xOy=Wt11pmWQOxOv=t\|k=@wxv}W}@\|WQ@Ctw=kt\|oDN}UoVwBQ=Owtv ?}DQD x@ RAP w NA 'RCA Kr=Yt \|=Q@ xv}W}@ l=m]Y= \|x}w=R "OwW\|t x@ Q}N= Q=Okt 'xOv=t}k=@ Cr=L QO "CU= xOw@ xHQO 40=6 w 42=5 '45 =@ Q@=Q@ Kr=Yt \|=Q@ \|oOv@UJ u=R}t "CU= xOw@ xHQO 35=7 w 36=5 '36 =@ Q@=Q@ ?}DQD ?}DQD x@ RAP w RCA \|Di=}R=@ Kr=Yt \|=Q@ w p=mU=Bwr}m 24 =@ Q@=Q@ NA OwHw x@ u=wD\|t =Q \|oOv@UJ V}=Ri= p}rO "CU= xOw@ p=mU=Bwr}m 22 w 31=2 uD@ Kr=Yt \|wQ u=t}U Cqt u}vJty w RAP \|=yxv=OnvU \|wQ Q}k VWwB \|oOv@UJ \|Q=Okt 'C@w]Q uOW xi=[= =@ xm 'CUv=O \@DQt RCA \|xOWOQN ?}QkD p}rOx@ \|Qy=_ \|oOv@UJ Ov=wD\|t \|oOv@UJ u}= OvJ Qy "OwW\|t Qy=_ "OW=@ sy \|]NQ}e \|oDN}Uo VwB \N NA Kr=Yt \|wQ Q@ s}kDUt VQ@ V}=tR; G}=Dv '12 pmW QO u}vJty / \| Q \| Qw u @ }Q uQ \| y 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|UOvyt 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|UOvyt "\|Dr=iU; \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "9 pmW "\|vD@ \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "10 pmW "\|Dr=iU; \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "9 pmW 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|U "\|Dr=iU; \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "9 pmW "\|Dr=iU; \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "9 pmW "\|vD@ \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "10 pmW "RAP w RCA 'NA Kr=Yt \|=Q@ \|oDN}Uo VwB Q=Owtv "11 pmW "\|vD@ \|=yxv=OovU SEM \|vwQDmr= Q}w=YD "10 pmW V}=Ri= O=H}= Ea=@ sR}v=mt u}= w Ov=xO}Rer w xO}Dre sy \|wQ =yxv=O 'xt=O= QO "O@=}\|t Vy=m `=UD= x@ p}=tD p=tQv VvD V}=Ri= =@ "CU= xOW `=UD= sHL Kr=Ytxm\|DQwYQO'OvQ=O\|QDW}@`=UD=x@p}=tD\|Di=}R=@uD@Ovv=tQDQ@RKr=Yt Kr=Yt "OvQ=O \|QDtm `=UD= x@ p}=tD '\|Di=}R=@ Cr=iU; Ovv=t 'QDh=Y \|=yxv=O =@ QHvt =yxv=O \|wQ u=t}U \|=yQwr@ OWQ '\|DQ=@a x@ "OvQ=O \|v}@=v}@ \|Q=DiQ R}v \|a}@] K]U uOW QDh=Y x@ QHvt =yxv=O \|wQ Q}k \|x}q w `=UD= V}=Ri= w uOW QDQ@R x@ Qo}O OQwt wO R= QDtm \|oOvwWsQv Q=Okt '\|rm Qw]x@ "OwW\|t `=UD= Vy=m w =yxv=O xOv=WwB =Q =yxv=O K]U xm CU= \|Q}k VWwB u=ty u; p}rO,q=tDL= xm CU= xOw@ \|rw "CU= xO=O Vy=m =Q =yxv=O \|Q}oQO w xOQm O=H}= =Q \|QDh=Y ,=D@Uv \|=yxv=O w \|O=}R Cw=iD xvwtv xU Qy \|=Q@ V}=tR; \|=yDv= QO xOv=t}k=@ VvD Q=Okt '\|rmQw] x@ "OvDW=O \|y@=Wt Q=DiQ \|Di=}R=@ uD@ w \|a}@] \|xvwtv wO Qw]u}ty "OvDW=Ov sy =@ xOy=Wt11pmWQOxOv=t\|k=@wxv}W}@\|WQ@Ctw=kt\|oDN}UoVwBQ=Owtv ?}DQD x@ RAP w NA 'RCA Kr=Yt \|=Q@ xv}W}@ l=m]Y= \|x}w=R "OwW\|t x@ Q}N= Q=Okt 'xOv=t}k=@ Cr=L QO "CU= xOw@ xHQO 40=6 w 42=5 '45 =@ Q@=Q@ Kr=Yt \|=Q@ \|oOv@UJ u=R}t "CU= xOw@ xHQO 35=7 w 36=5 '36 =@ Q@=Q@ ?}DQD ?}DQD x@ RAP w RCA \|Di=}R=@ Kr=Yt \|=Q@ w p=mU=Bwr}m 24 =@ Q@=Q@ NA OwHw x@ u=wD\|t =Q \|oOv@UJ V}=Ri= p}rO "CU= xOw@ p=mU=Bwr}m 22 w 31=2 uD@ Kr=Yt \|wQ u=t}U Cqt u}vJty w RAP \|=yxv=OnvU \|wQ Q}k VWwB \|oOv@UJ \|Q=Okt 'C@w]Q uOW xi=[= =@ xm 'CUv=O \@DQt RCA \|xOWOQN ?}QkD p}rOx@ \|Qy=_ \|oOv@UJ Ov=wD\|t \|oOv@UJ u}= OvJ Qy "OwW\|t Qy=_ "OW=@ sy \|]NQ}e \|oDN}Uo VwB \N NA Kr=Yt \|wQ Q@ s}kDUt VQ@ V}=tR; G}=Dv '12 pmW QO u}vJty 33 \|}=Hx@=H \|vLvt ? Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt '\|ki= \|}=Hx@=H \|WQ@ Ctw=kt \|vLvt hr= "13 pmW m r = RCA r= = ki= = = =k Kr=Yt \|=Q@ \|ki= \|}=Hx@=H Q@=Q@ QO \|WQ@ Ctw=kt Q=Owtv 'hr=14 pmW QO C@Uv RAP Kr=Yt Q=DiQ "OwW\|t xOy=Wt KrUtQ}e w KrUt Cr=L QO RAP KrUtQ}e RAP Kr=Yt \|WQ@ Ctw=kt "CU= Cw=iDt NA w RCA Kr=Yt x@ \|=y\|}=Hx@=H QO u; R= Oa@ w xOw@ \|W}=Ri= QDt\|r}t 19 \|r= 11 \|ki= \|}=Hx@=H =D C@Uv Ctw=kt Ci= xm \|r=L QO "CU= xOW Ci= Q=JO \|tm Q=Okt 'QDW}@ \|ki= KrUtQ}e w KrUt Cr=L QO RCA \|xvwtv \|=Q@ V}=tR; G}=Dv '13 pmW QO u}=xHwDp@=k\|xDmv\|rw"CU=\|a}@]Kr=Ytx@x}@W,=@}QkDG}=Dv"OwW\|txOy=Wt \|QDW}@Ctw=kt GP3O}QowS'p=mU=Bwr}m100w50p=tQv\|=yVvDQOxmCU= GP1 O}QowS p=mU=Bwr}m 150 VvD QO w GP2 w GP1 \|=yO}QowS x@ C@Uv Ctw=kt "CU= xO=O u=Wv OwN R= \|QDW}@ \|WQ@ Ctw=kt Qo}O O}QowS wO x@ C@Uv 34 34 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|UOvyt O=H}= CU@ w pik u}QDy@ 'O}QowS \|xtWJ uOw@ QDlJwm p}rO x@ xm OUQ\|t u}ty w xOW Qo}Om} \|wQ Q@ =yxv=O uO}Rer R= `v=t O}QowS u}=Q@=v@ "CU= xOW Kr=Yt u}vJty "CU= xOW =yxtWJ pN=O \|=yxv=O \|Q}oQO V}=Ri= Ea=@ Qt= \|}=Hx@=H Q=Owtv "Ov=xDW=O GP3 O}QowS =@ =Q 1=25 VvmQOv= u}QDq=@ QwmPt wO TmaQ@ xm OyO\|t u=Wv ?14 pmW QO \|ki= \|}=Hx@=H Q@=Q@ QO \|Owta OwN x@ C@Uv \|QD\|[=@kv= Q=DiQ '\|Di=}R=@ Cr=iU; lQDWt K]U '\|r@k Kr=Yt QO \|Q}k \|x}q R= \|WWwB OwHw p}rO x@ ,q=tDL= j=iD= u}= "CU= xDW=O Kr=Yt QO "CU= \|r@k Kr=Yt wO x@ C@Uv =yu; K]U uOw@ QDh=Y w =yxv=OovU \|wQ Qw]u=ty "CU= xOW x=Q= O}QowS uwO@ w =@ =yV}=tR; G}=Dv \|xYqN '5pwOH "CU= xDi=} V}=Ri= \|WQ@ Ctw=kt u=R}t 'O}QowS uOwRi= =@ 'OwW\|t xOy=Wt xm C@Uv GP1 O}QowS =@ NA \|a}@] Kr=Yt QO Ctw=kt OYQO V}=Ri= u=R}t 150 p=tQv VvD QO xm \|Qw] x@ 'CU= xOw@ QDW}@ GP3 w GP2 \|=yO}QowS x@ \|=yVvD x@ C@Uv xm 'CU= xOw@ 20=35 'Ctw=kt V}=Ri= u=R}t p=mU=Bwr}m Ctw=kt OYQO V}=Ri= xm \|r=L QO "CU= QDtm p=mU=Bwr}m 100 w 50 p=tQv x@ C@Uv GP3 O}QowS =@ RAP Cr=iU; w RCA uD@ \|Di=}R=@ Kr=Yt QO \|Dr=iU; w \|vD@ Kr=Yt QO xm \|Qw] x@ 'CU= QDW}@ GP3 w GP2 \|=yO}QowS OYQO V}=Ri= u}QDW}@ 'p=mU=Bwr}m 100 w 150 p=tQv \|=yVvD QO ?}DQD x@ u=OvJ QwmPt Kr=Yt `wv xU Q=DiQ \|xU}=kt O}=W u}=Q@=v@ "CU= xDW=O =Q Ctw=kt w \|Q@R '=yxv=OovU K]U VWwB w =yxv=O C=QP pmW xm =QJ 'OW=@v CUQO \|=yxv=O K]U OwQ\|t Q=_Dv= "CU= Cw=iDt Qo}Om} =@ =yu; K]U VWwB TvH QDxOvvmW =yxv=O K]U VWwB p=Lu}aQO '10 pmW OW=@ QDQ@R \|Di=}R=@ uD@ "OW=@ \|}=Hx@=H \|vLvt ? Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt '\|ki= \|}=Hx@=H \|WQ@ Ctw=kt \|vLvt hr= "14 pmW "xOvvmKrUt uwO@ w =@ RAP Kr=Yt \|=Q@ \|ki= \|}=Hx@=H s=k 2/1 \|xQ=tW '382 \|xQwO '1401 u=DU@=D h}QW u=Qta \|UOv "OW Oy=wN O}QowS w Qo}Om} =@ =yu; QDW}@ \|Q}oQO Ea=@ =yxv=O K]U \|Q@R \|Q}oQO xm 9 pmW CU= Q}k \|=Q=O w h=Y Cr=iU; \|=yxv=O K]U u}vJty =} CU= QDW}@ =yxv=O u}@ l=m]Y= xmv}= 'u}=Q@=v@ "Ow@ Oy=wN QDtm =yxv=O u}@ \|}=yv \|xH}Dv x@ uO}UQ =Pr &CU}v \|DL=Q p=-wU 'O}QowS =yxv=O u}@ l=m]Y= \|=yV}=tR;OvtR=}v'=yxtWJ\|xR=Ov==}wO}QowSpmx@R=@K]UC@UvQF=OQwtQO =@ R}v Kr=Yt `wv xU \|=yxv=O xm CU= sRq xDmv u}= QmP "CU= \|QDj}kO w QDW}@ \|Di=}R=@ Cr=iU; Kr=Yt,q=tDL= "Ov=xDW=Ov \|v=Um} -=Wvt w xOw@ Cw=iDt Qo}Om} \|=ypmWQ}}eD xm \|OQ=wt QO =Pr 'OvyO\|t u=Wv OwN R= \|WRN Q=DiQ 'COtR=QO QO OvJ Qy &CW=O xHwD `w[wt u}= x@ O}=@ 'OW=@ xDW=O C}OwOLt \|m=N \|xR=U w xS}w \|=yVywSB x@ R=}v u; Q=Okt w OvwQ w \|Di=}R=@ Kr=Yt QO VRN EL@ C=ar=]t "CU= xOW xO}WwB Q}k R= \|}x}q =@ =yxv=O K]U \ki xm =QJ 'OQ=O \|QDW}@ =@ \|Di=}R=@ Kr=Yt VvmQOv= w \|WQ@ Ctw=kt \|=yQDt=Q=B \|xv}tR QO xOW s=Hv= \|tm \|r}N O=OaD w xOW s=Hv= \|R=UwQ w =yxO=H T=U=Q}R EL@ QO QDW}@ O}QowS \|=yQ=w}O R}Qm=N QO \|Di=}R=@ Kr=Yt R= OOHt \|xO=iDU= \|wQ Q@ u=QoWywSB R= \UwD xOW s=Hv= \|=yVywSB =@ Q[=L VywSB Cw=iD "Ov=xDN=OQB KrUt l=N xm CU= u}= 2021 u=Q=mty w 24\|=JtwOw} w 2020 u=Q=mty w 23nvBOwU s}rk= w \}=QW x@ xHwD =@ u}vJty w OvDUy \|tw@ 'xOW ?=NDv= \|Di=}R=@ Kr=Yt xDmv u}= QmP "OW=@ Cw=iDt Ov=wD\|t \|v=tDN=U \|=yxr=Nv O}rwD `@vt `wv '\|}=}i=QeH Kr=Yt R= \|m} \|wQ \ki 'xOW xQ=W= u=QoWywSB R= l} Qy xm CU= \|wQwQ[ \|Di=}R=@ Kr=Yt `wv wO Qy Q[=L VywSB QO xm \|r=L QO 'Ov=xOQm xar=]t \|Di=}R=@ u}vJty "Ov=xOW xO=iDU= xU}=kt \|=Q@ \|a}@] Kr=Yt w \|Di=}R=@ uD@ w Cr=iU; OwHw xOW QmP \|=yQ=DWwv =@ O}QowS `wv w Kr=Yt \|Ov@xv=O Q_v R= \|}=yCw=iD "OQ=O \|}=Hx@=H \|vLvt ? Original Article K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= O}QowS w \|Di=}R=@ Cr=iU; w uD@ lQDWt s}kDUt VQ@ V}=tR; R= xO=iDU= =@ OWQ= \|U=vWQ \|wHWv=O \|DQ}L u}UL Q=}Wv=O O=QwrvUL OwtLt Q=}Wv=O \|vOQ= =[Q}ra u}wRk 'xQ \|v}tN s=t= \|rrtr=u}@ x=oWv=O '\|UOvyt \|vi \|xOmWv=O '\|Di=}R=@ Kr=Yt lQDWt T=tD K]U \|WQ@ Ctw=kt C=}YwYN 'Q[=L VywSB QO l=N \|=yxR=U QO u}Ro}=H R}Qm=N Kr=Yt u=wvax@ O}QowS =@ 'Cr=iU; w uD@ pt=W Kr=Yt Q=vm QO "CU= xOW \|UQQ@ T=}ktnQR@ s}kDUt VQ@ V}=tR; R= xO=iDU= =@ KrUt ,=@}QkD \|Ov@xv=O w \|m}R}i C=YNWt =@ \|a}@] Kr=Yt l} R= 'Cr=iU; w uD@ \|Di=}R=@ 'x}wUlD O}QowS `wv xU "CU= xOW xO=iDU= xU}=kt CyH SW \|Ov@xk@] =@ u=Um} \|x}w=R =@ QwmPt \|Di=}R=@ Kr=Yt xm O=O u=Wv G}=Dv w OvOW V}=tR; xOvvmKrUt u=wvax@ l=N \|=yxR=U QO xO=iDU= \|=Q@ \|@wN p}Uv=DB 'xHQO 40 R= QDW}@ \|rN=O l=m]Y= OvJ Qy 'OQm xO=iDU= \|a}@] Kr=Yt \|=H x@ \|D=_Lqt =@ =Q =yu; u=wD\|t w OvQ=O KrUt 'O}QowS \|WWm Ctw=kt xm OvO=O u=Wv =yV}=tR; "OvQ=O \|WQ@ Q=DiQ QO \|}=yCw=iD xm V}=Ri= Ea=@ O}QowS =@ =yxv=O \|Q}oQO "OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D Ctw=kt ?}Q[ xm \|Qw]x@ 'CU= xOW VvmQOv= ?}Q[ w T=tD K]U \|WQ@ Ctw=kt u}@ \|wk VvmQOv= \|xOvyOu=Wv xm Ot; CUO x@ 1 R= QDnQR@ 'QwmPt Kr=Yt \|=Q@ \|WQ@ "CU= \|Di=}R=@ Kr=Yt w O}QowS 'T=}ktnQR@ s}kDUt VQ@ 'KrUt l=N 'VvmQOv= 'O}QowS %\|O}rm u=oS=w "\|Di=}R=@ Kr=Yt '\|ki= \|}=Hx@=H \|WQ@ Ctw=kt \|vLvt hr= "14 pmW "xOvvmKrUt uwO@ w =@ RAP Kr=Yt \|=Q@ \|ki= \|}=Hx@=H s=k Kr=Yt \|Dkw "CU= xOQm =O}B Vy=m \|t}qt \|r}N ?}W =@ Qo}O Kr=Yt wO x@ w xO=O MQ \|QDnQR@ \|=y\|}=Hx@=H QO Ctw=kt V}=Ri= 'Ov=xOW KrUt 'O}QowS =@ 'Qo}O \|DQ=@a x@ "CU= xDi=} xt=O= QDt\|r}t 45 \|}=Hx@=H =D C@=F ,=D@Uv OvwQ =@ RAP Kr=Yt "CU= xOw@ xOvwWCNU 'xOW QmP Kr=Yt QO lQDWt K]U Q=DiQ \|=Q@ BGG/D50 C@Uv "Ov=xDW=O GP3 O}QowS =@ \|QDq=@ \|WQ@ Ctw=kt xOw@ 5=3 w 6=63 '6=63 =@ Q@=Q@ ?}DQD x@ GP3 w GP2 'GP1 \|=yO}QowS \|Di=}R=@ Cr=iU; \|=yxv=OovU R= SEM \|vwQDmr= Q}w=YD '9 pmW QO "CU= Ov=wD\|t =yxv=O K]U QO Q}k OwHw 'QwmPt Q}w=YD x@ xHwD =@ "OwW\|t xOy=Wt x@ xHwD =@ "OyO u=Wv OwN R= \|a}@] w \|vD@ \|=yxv=OovU C@Uv \|Dw=iDt G}=Dv uOWQDsQv Ea=@ =yxv=OovU K]U QO Q}k OwHw,q=tDL= 'KrUtQ}e Cr=L QO G}=Dv x@ w xOW xv=O x@ xv=O l=m]Y= Vy=m Ea=@ Qt= u}ty w xOW =yxv=OovU =@ uOQm KrUt s=ovy xm \|DQwY QO &CU= xDi=} Vy=m \|WQ@ Ctw=kt u; p=@vO Kr=Yt "CU= xO=O MQ \|yHwDp@=k V}=Ri= 'T=tD K]U \|WQ@ Ctw=kt QO 'O}QowS w Ov=xOQm O=H}= =Q \|QDW}@ T=tD K]U \|WQ@ Ctw=kt 'GP3 O}QowS =@ \|Dr=iU; Q_vx@ Qw]u}= "CU= xOw@ 0=72 'GP3 O}QowS \|=Q@ \|r=N \|=[i Q=Okt u}vJty 35 35 "\|WQ@ Ctw=kt \|=y QDt=Q=B Q}O=kt "5 pwOH kP a (c) \|oOv@UJ (') l=m]Y= x}w=R Kr=Yt xHQO xOv=t}k=@ xv}W}@ xOv=t}k=@ xv}W}@ 3=7 24 36 45 NA 10 29 46=9 47=3 NA + geo60=20 15 24 47=6 48=8 NA + geo110=30 23 22 43=5 49=7 NA + geo60=30 1 31=2 36=6 42=7 RCA 24=7 37=7 41=6 49=6 RCA + geo60=20 16 15 46 52 RCA + geo110=30 24=7 37=7 44=7 51=1 RCA + geo60=30 4 22 36=7 40=6 RAP 11 19 48 47 RAP + geo60=20 5=5 19 48=6 47=6 RAP + geo110=30 6=7 20 50 49=2 RAP + geo60=30 "\|iQYt \|=yO}QowS =@ Kr=Yt T=tD K]U VvmQOv= ?}Q[ Q}O=kt "6 pwOH (kP a) p=tQv VvD xvwtv u}ov=}t 150 100 50 1=1 1=1 1=11 1=12 NA + geo60=20 1=21 1=2 1=19 1=16 NA + geo110=30 1=11 1=14 1=12 1=09 NA + geo60=30 1=25 1=26 1=25 1=25 RCA + geo60=20 1=13 1=22 1=15 1=02 RCA + geo110=30 1=29 1=32 1=29 1=28 RCA + geo60=30 1=18 1=22 1=17 1=15 RAP + geo60=20 1=18 1=22 1=17 1=15 RAP + geo110=30 1=25 1=29 1=26 1=21 RAP + geo60=30 \|WQ@ Ctw=kt \|=yQDt=Q=B \|xv}tm '\|Di=}R=@ Kr=Yt 'CU= XNWt xm Qw]u=ty u}@ VvmQOv= ?}Q[ "Ovvm\|t u}t-=D 'OvwW\|t KrUt O}QowS =@ xm \|v=tR =Q KrUt l=N \|=yxR=U \|L=Q] QO \|O}rm \|=yQDt=Q=B R= \|m} 'xOvvmKrUt w R}Qm=N ?}Q[ "OwW u=}@ KrUt l=N l=m]Y= ?}Q[ u=wva x@ Ov=wD\|t w CU= x@ KrUt Cr=L QO \|WQ@ Ctw=kt \|xv}W}@ C@Uv u=wvax@ u=wD\|t =Q VvmQOv= Q=Wi u=ty CLD 1 \|x]@=Q j@] KrUtQ}e Cr=L QO \|WQ@ Ctw=kt \|xv}W}@ 3 CLD w Q=@ 3 's}kDUt VQ@ V}=tR; Qy xm =Hv; R= "OQm h}QaD p=tQv u}ov=}t R= VvmQOv= ?}Q[ 'CU= xOW s=Hv= x@=Wt \}=QW w hrDNt Q=@QU u}@ VvmQOv= "CU= xOt; CUOx@ p=mU=Bwr}m 150 w 100 '50 Q=@QU xU 'KrUt l=N \|=yQ=w}O xm OwW\|t Ea=@ KrUt l=N R}Qm=N w xOvvmKrUt pta Q}PBh=]av= OL=w l} Ovv=t w Ovvm ptLD =Q \|yHwDp@=k pmWQ}}eD w \|Q=PoQ=@ ?}Q[ w T=tD K]U \|WQ@ Ctw=kt \|=yQDt=Q=B "7 VvmQOv= "Ov=xOW x=Q= 6 pwOH QO KrUtQ}e w KrUt Cr=L QO '\|WQ@ Ctw=kt \|=yQDt=Q=B x@ s}kDUt VQ@ V}=tR; G}=Dv T=U=Q@ \|oDN}Uo VwB sUQ =@ QwmPt \|=yQDt=Q=B QO \|a}@] Kr=Yt 'OwW\|t xOy=Wt Q}N= pwOH QO xm Qw]u=ty "Ov=xOt; CUO K]U \|Q@R w \|oWwoR}D p}rO x@ \|Di=}R=@ Kr=Yt x@ C@Uv KrUtQ}e Cr=L xOvvmKrUt xm \|v=tR w xDW=O =Q \|rN=O l=m]Y= \|x}w=R u}QDW}@ 'u; \|=yxv=O \|=yl=N "CU= xDi=} V}=Ri= T=tD K]U l=m]Y= \|x}w=R 'CU= xOW xi=[= \|xv}W}@ l=m]Y= \|x}w=R Q}O=kt \|=Q=O,qwtat 'sm=QDt \|xU=t w uW Ovv=t \|}xv=O [30]"OvDUy xHQO 36 \|r= 32 xOv=t}k=@ l=m]Y= \|x}w=R Q}O=kt w xHQO 48 =D 40 36 "xOvvmKrUt uwO@ w =@ s}kDUt VQ@ V}=tR; G}=Dv \|xYqN "7 pwOH Ctw=kt V}=Ri= Ctw=kt =@ Q_=vDt \|}=Hx@=H xv}W}@ \|WQ@ Ctw=kt xOvvmKrUt xvwtv (mm) xv}W}@ \|WQ@ (kP a) 1500 100 50 150 100 50 150 100 50 (kP a) (kP a) (kP a) (kP a) (kP a) (kP a) (kP a) (kP a) (kP a) 6 4 5 172=7 121 75=5 NA Kr=Yt 20=35 19 16 17 9 10 207 145 87 NA+GP1 \|a}@] 11=62 14 10 11 12 9 192 138 83 NA+GP2 18 11=5 13=3 13 11 14 203 135 85 NA+GP3 9 7 6 168 126=7 76 RCA Kr=Yt 25 18=25 5=3 7 10 7 210 149 79 RCA+GP1 \|vD@ 28=6 19 32 7 8 9 216 150 99 RCA+GP2 \|Di=}R=@ 32 29=4 29=3 10 13 17 222 163 97 RCA+GP3 19 12 11 150 108 64 RAP Kr=Yt 20 17=5 11 29 16 26 180 127 71 RAP+GP1 \|Dr=iU; 22 16=6 15=6 32 33 15 183 126 74 RAP+GP2 \|Di=}R=@ 29=3 26=85 21=8 32 33 15 194 137 78 RAP+GP3 60 w 110 =@ Q@=Q@ ?}DQD x@ GP60=20 w GP110=30 `wv O}QowS \|WQ@ \|xtWJ ZQa =@ QDt\|r}t 33 25 =yxtWJ \|xR=Ov= "CU= QDt QO uDw}vwr}m Ctw=kt'OvyO\|tu=WvG}=DvxmQw]u=ty"CU=xOw@O}QowS`wvwOQy\|=Q@u=Um} O}QowS \|oDN}Uo =Q}R 'OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D \|WWm =yQ=Owtvu}@\|y=oxm\|QYDNt\|=yu=Uwv"CU=xOWvxOy=WtVQ@V}=tR;R=Oa@ u=Q=mty w nvBOwU "CU= V}=tR; \|=y=]N p}rO x@ ,q=tDL= 'OwW\|t xOy=Wt w OvOQm \|UQQ@ RCA Kr=Yt QO =Q O}QowS \|WWm Ctw=kt Q}F-=D R}v '2018 T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D O}QowS \|WWm Ctw=kt xm OvDi=}QO \|WWm Ctw=kt 'OyOv MQ O}QowS QO \|oQ=B xm \|v=tR =D Cio u=wD\|t =Pr [6]"OQ=Ov "OQ=Ov l=N =@ u; VvmQOv= QO \|yHwD p@=k Q}F-=D 'O}QowS \|Q}oxH}Dv "9 \|WQ@ Q=DiQ =OD@= 'T=}ktnQR@ s}kDUt VQ@ V}=tR; ltm x@ 'Q[=L Q=DWwv QO Cr=iU; w uD@ %pt=W '\|a}@] Kr=Yt u}Ro}=H u=wva x@ \|Di=}R=@ Kr=Yt `wv wO ?wN \|xU=t \|Ov@xk@] =@ w x@=Wt \|Ov@xv=O =@ \|a}@] Kr=Yt l} Q=vm QO \|Di=}R=@ xU =@ QwmPt Kr=Yt lQDWt K]U Q=DiQ TBU "CU= xOW \|UQQ@ xOW \|Ov@xv=O KrUt l=N p=L \|=yQ=w}O QO \|Di=}R=@ Kr=Yt R= xO=iDU= hOy =@ O}QowS `wv [31]"Ovvm = rienforce=unreinforce (1) w xOW KrUt rienfoce 'T=tD K]U \|WQ@ Ctw=kt ?}Q[ 'u; QO xm u}@ T=tD K]U \|xv}W}@ \|WQ@ Ctw=kt ?}DQD x@ xOWv KrUt unreinforc VvmQOv=?}Q[xm\|DQwYQO"CU=\|r=Nl=N\|xvwtvwO}QowSw\|m=N\|xvwtv Qo= w h}a[ 'l}DDvUwS w l=N u}@ Ovw}B xm CU= \|vat u}O@ 'OW=@ 0=5 R= QDtm '7 pwOH QO [31]"CU= Q=QkQ@ l}DDvUwS w l=N u}@ \|wk p=YD= 'OW=@ 1 R= QDW}@ xOy=Wt xm Qw]u=ty "CU= xOW x=Q= T=tD K]U \|WQ@ Ctw=kt ?}Q[ Q}O=kt VvmQOv= Qov=}@ xm CU= 1 R= QDnQR@ Kr=Yt xU Qy \|=Q@ QwmPt ?}Q[ 'OwW\|t "CU=yO}QowS w Kr=Yt u}@ \|wk \|WQ@ Ctw=kt QO O}QowS \|WWm Ctw=kt Q}F-=D "8 VvmQOv= G}=Dv \|xU}=kt =@ T=tD K]U \|WQ@ Ctw=kt QO O}QowS \|WWm Ctw=kt Q}F-=D 15pmWQO GPGRID60=20w GPGRID110=30\|=yO}QowS=@V}=tR; CyH QO \|WWm Ctw=kt "OwW\|t xOy=Wt RAP w RCA 'NA Kr=Yt \|=Q@ 60 w 110 =@ Q@=Q@ ?}DQD x@ GP60=20 w GP110=30 `wv O}QowS \|WQ@ \|xtWJ ZQa =@ QDt\|r}t 33 25 =yxtWJ \|xR=Ov= "CU= QDt QO uDw}vwr}m Ctw=kt'OvyO\|tu=WvG}=DvxmQw]u=ty"CU=xOw@O}QowS`wvwOQy\|=Q@u=Um} O}QowS \|oDN}Uo =Q}R 'OQ=Ov T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D \|WWm =yQ=Owtvu}@\|y=oxm\|QYDNt\|=yu=Uwv"CU=xOWvxOy=WtVQ@V}=tR;R=Oa@ u=Q=mty w nvBOwU "CU= V}=tR; \|=y=]N p}rO x@ ,q=tDL= 'OwW\|t xOy=Wt w OvOQm \|UQQ@ RCA Kr=Yt QO =Q O}QowS \|WWm Ctw=kt Q}F-=D R}v '2018 T=tD K]U \|WQ@ Ctw=kt QO \|v=OvJ Q}F-=D O}QowS \|WWm Ctw=kt xm OvDi=}QO \|WWm Ctw=kt 'OyOv MQ O}QowS QO \|oQ=B xm \|v=tR =D Cio u=wD\|t =Pr [6]"OQ=Ov "OQ=Ov l=N =@ u; VvmQOv= QO \|yHwD p@=k Q}F-=D 'O}QowS (1) (1) \|Q}oxH}Dv "9 \|WQ@ Q=DiQ =OD@= 'T=}ktnQR@ s}kDUt VQ@ V}=tR; ltm x@ 'Q[=L Q=DWwv QO Cr=iU; w uD@ %pt=W '\|a}@] Kr=Yt u}Ro}=H u=wva x@ \|Di=}R=@ Kr=Yt `wv wO ?wN \|xU=t \|Ov@xk@] =@ w x@=Wt \|Ov@xv=O =@ \|a}@] Kr=Yt l} Q=vm QO \|Di=}R=@ xU =@ QwmPt Kr=Yt lQDWt K]U Q=DiQ TBU "CU= xOW \|UQQ@ xOW \|Ov@xv=O KrUt l=N p=L \|=yQ=w}O QO \|Di=}R=@ Kr=Yt R= xO=iDU= hOy =@ O}QowS `wv \|Q}oxH}Dv "9 37 %CU= KQW u}= x@ pY=L G}=Dv sy= "CU= xOW \|UQQ@ \|@wN \|WQ@ Ctw=kt \|=Q=O xOW OQN Cr=iU; w uD@ pt=W \|Di=}R=@ Kr=Yt "1 \|=yQ=w}O QO \|a}@] Kr=Yt u}Ro}=H u=wva x@ \|D=_Lqt =@ Ovv=wD \|t w OvDUy "CU= Cw=iDt \|tm =yu; \|WQ@ Q=DiQ OvJ Qy 'OvwW xO=iDU= KrUt l=N p=L \|WQ@Ctw=kt'\|Di=}R=@uD@w\|a}@]Kr=Ytx@C@Uv\|Di=}R=@Cr=iU;Kr=Yt \|=yxv=OovU \|wQ Q@ Q}k VWwB OwHw x@ u=wD\|t =Q Qt= u}= p}rO "OvQ=O \|QDtm xmv}= p}rO x@ "OwW\|t l=m]Y= Vy=m Ea=@ xm O=O C@Uv \|Di=}R=@ Cr=iU; R= QD\|[=@kv= u; \|tHL Q=DiQ =Pr 'CU= QDsQv '\|Dr=iU; Kr=Yt \|L]U \|x}q "CU= \|Di=}R=@ uD@ w \|a}@] Kr=Yt V}=Ri= x@ QHvt 'O}QowS =@ \|Di=}R=@ \|}xv=O Kr=Yt ?U=vt \|Q}oQO \|rm Qw]x@ "2 Kr=Yt OwN x@ C@Uv O}QowS w QwmPt Kr=Yt lQDWt K]U \|WQ@ Ctw=kt Cr=L x@ \|tm xOvwWsQv Cr=L R= uD@ w \|a}@] Kr=Yt \|WQ@ Q=DiQ "CU= xOW Cr=iU; OQwt QO OvwQ u}= "CU= xOW p}O@D lQDWt K]U QO xOvwWCNU w \|a}@] Kr=Yt lQDWt K]U \|tHL Q=DiQ u}vJty "CU= xOw@ QDO}OW QO `w[wt u}= 'CU= xOw@ Kr=Yt OwN R= QD\|a=UD= 'O}QowS =@ \|Di=}R=@ uD@ uOW QDh=Y Ea=@ xm =yxv=O K]U Q@ Q}k VWwB OwHw p}rO x@ Cr=iU; OQwt K]U \|WQ@ Ctw=kt 'O}QowS uOwRi= =@ "CU= TmaQ@ 'OwW\|t =yxv=OnvU w O}QowS \|=yxtWJ QO =yxv=O QDW}@ \|Q}oQO p}rO x@ \|Dr=iU; Kr=Yt T=tD "CU= xDi=} V}=Ri= Qo}Om} \|wQ Q@ =yxv=O uO}Rer R= \|Q}owrH \|WQ@ Ctw=kt \|=yQDt=Q=B \|xv}tm 'Cr=iU; w uD@ \|Di=}R=@ Kr=Yt '\|rm Qw]x@ "3 VvmQOv=?}Q[u}vJty"Ovvm\|tu}t-=D'OvwW\|tKrUtO}QowS=@xm\|v=tR=Q xOw@ 1 =@ Q@=Q@ QDW}@ O}QowS =@ Cr=iU; w uD@ \|Di=}R=@ Kr=Yt lQDWt K]U "OQ=O =yu; ?U=vt \|Q}oQO w l=m]Y= R= u=Wv xm CU= lQDWt T=tD K]U \|WQ@ Ctw=kt QO \|O=}R Q}F-=D =yO}QowS \|WWm Ctw=kt "4 VQ@ V}=tR; u=}=B QO O}QowS \|oDN}Uo =Q}R &OQ=Ov O}QowS w Kr=Yt u}@ "CU= xOWv xOy=Wt s}kDUt Cr=iU; ,=YwYNt 'QwmPt \|Di=}R=@ Kr=Yt R= xO=iDU= QO OW Qw;O=} O}=@ =yDv= QO "OQm xHwD =yu; \|WRN Q=DiQ x@ O}=@ '\|Di=}R=@ QmWD w Q}OkD xO=iDU=\|=yO}QowSuDW=PoQ=}DN=QOwO}rwDp}rOx@u=}UQ=BwSCmQWR='C}=yvQO "OwW\|t \|v=OQOk w QmWD 'Q[=L VywSB QO xOW "T=tD K]U \|WQ@ Ctw=kt QO O}QowS \|WWm Ctw=kt Q}F-=D "15 pmW """ K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= "T=tD K]U \|WQ@ Ctw=kt QO O}QowS \|WWm Ctw=kt Q}F-=D "15 pmW %CU= KQW u}= x@ pY=L G}=Dv sy= "CU= xOW \|UQQ@ \|@wN \|WQ@ Ctw=kt \|=Q=O xOW OQN Cr=iU; w uD@ pt=W \|Di=}R=@ Kr=Yt "1 \|=yQ=w}O QO \|a}@] Kr=Yt u}Ro}=H u=wva x@ \|D=_Lqt =@ Ovv=wD \|t w OvDUy "CU= Cw=iDt \|tm =yu; \|WQ@ Q=DiQ OvJ Qy 'OvwW xO=iDU= KrUt l=N p=L \|WQ@Ctw=kt'\|Di=}R=@uD@w\|a}@]Kr=Ytx@C@Uv\|Di=}R=@Cr=iU;Kr=Yt \|=yxv=OovU \|wQ Q@ Q}k VWwB OwHw x@ u=wD\|t =Q Qt= u}= p}rO "OvQ=O \|QDtm xmv}= p}rO x@ "OwW\|t l=m]Y= Vy=m Ea=@ xm O=O C@Uv \|Di=}R=@ Cr=iU; R= QD\|[=@kv= u; \|tHL Q=DiQ =Pr 'CU= QDsQv '\|Dr=iU; Kr=Yt \|L]U \|x}q "CU= \|Di=}R=@ uD@ w \|a}@] Kr=Yt """ K]U \|WQ@ Ctw=kt \|=yQDt=Q=B \|@=}RQ= \|=yxv=OovU \|wQ Q@ Q}k VWwB OwHw x@ u=wD\|t =Q Qt= u}= p}rO "OvQ=O \|QDtm xmv}= p}rO x@ "OwW\|t l=m]Y= Vy=m Ea=@ xm O=O C@Uv \|Di=}R=@ Cr=iU; R= QD\|[=@kv= u; \|tHL Q=DiQ =Pr 'CU= QDsQv '\|Dr=iU; Kr=Yt \|L]U \|x}q "CU= \|Di=}R=@ uD@ w \|a}@] Kr=Yt V}=Ri= x@ QHvt 'O}QowS =@ \|Di=}R=@ \|}xv=O Kr=Yt ?U=vt \|Q}oQO \|rm Qw]x@ "2 Kr=Yt OwN x@ C@Uv O}QowS w QwmPt Kr=Yt lQDWt K]U \|WQ@ Ctw=kt Cr=L x@ \|tm xOvwWsQv Cr=L R= uD@ w \|a}@] Kr=Yt \|WQ@ Q=DiQ "CU= xOW Cr=iU; OQwt QO OvwQ u}= "CU= xOW p}O@D lQDWt K]U QO xOvwWCNU w \|a}@] Kr=Yt lQDWt K]U \|tHL Q=DiQ u}vJty "CU= xOw@ QDO}OW QO `w[wt u}= 'CU= xOw@ Kr=Yt OwN R= QD\|a=UD= 'O}QowS =@ \|Di=}R=@ uD@ uOW QDh=Y Ea=@ xm =yxv=O K]U Q@ Q}k VWwB OwHw p}rO x@ Cr=iU; OQwt K]U \|WQ@ Ctw=kt 'O}QowS uOwRi= =@ "CU= TmaQ@ 'OwW\|t =yxv=OnvU w O}QowS \|=yxtWJ QO =yxv=O QDW}@ \|Q}oQO p}rO x@ \|Dr=iU; Kr=Yt T=tD "CU= xDi=} V}=Ri= Qo}Om} \|wQ Q@ =yxv=O uO}Rer R= \|Q}owrH \|WQ@ Ctw=kt \|=yQDt=Q=B \|xv}tm 'Cr=iU; w uD@ \|Di=}R=@ Kr=Yt '\|rm Qw]x@ "3 VvmQOv=?}Q[u}vJty"Ovvm\|tu}t-=D'OvwW\|tKrUtO}QowS=@xm\|v=tR=Q xOw@ 1 =@ Q@=Q@ QDW}@ O}QowS =@ Cr=iU; w uD@ \|Di=}R=@ Kr=Yt lQDWt K]U "OQ=O =yu; ?U=vt \|Q}oQO w l=m]Y= R= u=Wv xm CU= lQDWt T=tD K]U \|WQ@ Ctw=kt QO \|O=}R Q}F-=D =yO}QowS \|WWm Ctw=kt "4 VQ@ V}=tR; u=}=B QO O}QowS \|oDN}Uo =Q}R &OQ=Ov O}QowS w Kr=Yt u}@ "CU= xOWv xOy=Wt s}kDUt Cr=iU; ,=YwYNt 'QwmPt \|Di=}R=@ Kr=Yt R= xO=iDU= QO OW Qw;O=} O}=@ =yDv= QO "OQm xHwD =yu; \|WRN Q=DiQ x@ O}=@ '\|Di=}R=@ QmWD w Q}OkD xO=iDU=\|=yO}QowSuDW=PoQ=}DN=QOwO}rwDp}rOx@u=}UQ=BwSCmQWR='C}=yvQO "OwW\|t \|v=OQOk w QmWD 'Q[=L VywSB QO xOW "T=tD K]U \|WQ@ Ctw=kt QO O}QowS \|WWm Ctw=kt Q}F-=D "15 pmW =yCWwv=B 1. 1. land ll 2. recycled concrete aggregate (RCA) 3. reclaimed asphalt pavement (RAP) 4. large direct shear test References `@=vt 1. 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Liu 38 V}=Ri= x@ QHvt 'O}QowS =@ \|Di=}R=@ \|}xv=O Kr=Yt ?U=vt \|Q}oQO \|rm Qw]x@ "2 Kr=Yt OwN x@ C@Uv O}QowS w QwmPt Kr=Yt lQDWt K]U \|WQ@ Ctw=kt Cr=L x@ \|tm xOvwWsQv Cr=L R= uD@ w \|a}@] Kr=Yt \|WQ@ Q=DiQ "CU= xOW Cr=iU; OQwt QO OvwQ u}= "CU= xOW p}O@D lQDWt K]U QO xOvwWCNU w \|a}@] Kr=Yt lQDWt K]U \|tHL Q=DiQ u}vJty "CU= xOw@ QDO}OW QO `w[wt u}= 'CU= xOw@ Kr=Yt OwN R= QD\|a=UD= 'O}QowS =@ \|Di=}R=@ uD@ uOW QDh=Y Ea=@ xm =yxv=O K]U Q@ Q}k VWwB OwHw p}rO x@ Cr=iU; OQwt K]U \|WQ@ Ctw=kt 'O}QowS uOwRi= =@ "CU= TmaQ@ 'OwW\|t =yxv=OnvU w O}QowS \|=yxtWJ QO =yxv=O QDW}@ \|Q}oQO p}rO x@ \|Dr=iU; Kr=Yt T=tD "CU= xDi=} V}=Ri= Qo}Om} \|wQ Q@ =yxv=O uO}Rer R= \|Q}owrH "T=tD K]U \|WQ@ Ctw=kt QO O}QowS \|WWm Ctw=kt Q}F-=D "15 pmW \|WQ@ Ctw=kt \|=yQDt=Q=B \|xv}tm 'Cr=iU; w uD@ \|Di=}R=@ Kr=Yt '\|rm Qw]x@ "3 VvmQOv=?}Q[u}vJty"Ovvm\|tu}t-=D'OvwW\|tKrUtO}QowS=@xm\|v=tR=Q xOw@ 1 =@ Q@=Q@ QDW}@ O}QowS =@ Cr=iU; w uD@ \|Di=}R=@ Kr=Yt lQDWt K]U "OQ=O =yu; ?U=vt \|Q}oQO w l=m]Y= R= u=Wv xm CU= lQDWt T=tD K]U \|WQ@ Ctw=kt QO \|O=}R Q}F-=D =yO}QowS \|WWm Ctw=kt "4 VQ@ V}=tR; u=}=B QO O}QowS \|oDN}Uo =Q}R &OQ=Ov O}QowS w Kr=Yt u}@ "CU= xOWv xOy=Wt s}kDUt Cr=iU; ,=YwYNt 'QwmPt \|Di=}R=@ Kr=Yt R= xO=iDU= QO OW Qw;O=} O}=@ =yDv= QO "OQm xHwD =yu; \|WRN Q=DiQ x@ O}=@ '\|Di=}R=@ =yCWwv=B 1. land ll 2. recycled concrete aggregate (RCA) 3. reclaimed asphalt pavement (RAP) 4. large direct shear test 5. 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https://openalex.org/W4361278871	https://repository.ubn.ru.nl//bitstream/handle/2066/293033/293033.pdf	English	null	Sex and gender differences in primary care help-seeking for common somatic symptoms: a longitudinal study	Scandinavian journal of primary health care	2,023	cc-by	6,417	Sex and gender differences in primary care help-seeking for common somatic symptoms: a longitudinal study. Ballering, A.V.; Olde Hartman, T.C.; Verheij, R.; Rosmalen, J.G.M. Sex and gender differences in primary care help-seeking for common somatic symptoms: a longitudinal study. 2023, Article / Letter to editor (Scandinavian Journal of Primary Health Care, 41, 2, (2023), pp. 132- 139) Doi link to publisher: https://doi.org/10.1080/02813432.2023.2191653 Doi link to publisher: https://doi.org/10.1080/02813432.2023.2191653 g y Ballering, A.V.; Olde Hartman, T.C.; Verheij, R.; Rosmalen, J.G.M. Scandinavian Journal of Primary Health Care Scandinavian Journal of Primary Health Care ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ipri20 ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ipri20 Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Sex and gender differences in primary care help-seeking for common somatic symptoms: a longitudinal study Aranka V. Ballering, Tim C. Olde Hartman, Robert Verheij & Judith G. M. Rosmalen To cite this article: Aranka V. Ballering, Tim C. Olde Hartman, Robert Verheij & Judith G. M. Rosmalen (2023) Sex and gender differences in primary care help-seeking for common somatic symptoms: a longitudinal study, Scandinavian Journal of Primary Health Care, 41:2, 132-139, DOI: 10.1080/02813432.2023.2191653 To link to this article: https://doi.org/10.1080/02813432.2023.2191653 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. View supplementary material Published online: 30 Mar 2023. Submit your article to this journal Article views: 492 View related articles View Crossmark data p g © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. View supplementary material Published online: 30 Mar 2023. Submit your article to this journal Article views: 492 View related articles View Crossmark data ABSTRACT Objective: Women are reported to consult general practitioners (GPs) more frequently than men. However, previous studies on sex differences in help-seeking behavior for somatic symp- toms do not distinguish between sex and gender, do not account for sex differences in pre- sented symptoms, and are frequently conducted in clinical settings, automatically excluding non-help seekers. Therefore, we aim to assess the independent associations of sex and gender with primary care help-seeking for somatic symptoms in the general population. p y p g y p g p p Design and setting: Records from the longitudinal population-based Lifelines Cohort Study were linked to routine electronic health records from GPs. Subjects: Participants reporting new-onset common somatic symptoms. j g y Main outcome measures: Associations between sex and gender, operationalized via a novel gender-index, with primary care help-seeking for somatic symptoms and differences in the strength of the association between gender and help-seeking for somatic symptoms between women and men. Results: Of 20,187 individuals with linked data, 8325 participants (67.5% female; mean age ¼ 44.5 years [SD ¼ 12.9]) reported at least one new-onset somatic symptom. Hereof, 255 (3.1%) consulted the GP within 6 weeks of symptom onset. Female sex was positively associated with consulting the GP (OR ¼ 1.78; 95%CI ¼ 1.13–2.80), whereas feminine gender was not (OR ¼ 0.67; 95%CI ¼ 0.39–1.16). The latter association did not differ in strength between men and women. More paid working days are negatively associated with help-seeking (OR ¼ 0.95; 95%CI ¼ 0.91–0.98). ) Conclusions: The results suggest that female sex rather than feminine gender is associated with primary care help-seeking behavior for somatic symptoms. Nevertheless, clinicians should be aware that gender-related variables, such as mean paid working days, may be associated with help-seeking behavior. Conclusions: The results suggest that female sex rather than feminine gender is associated with primary care help-seeking behavior for somatic symptoms. Nevertheless, clinicians should be aware that gender-related variables, such as mean paid working days, may be associated with help-seeking behavior. rates in women. To understand how help-seeking for somatic symptoms is affected by sex and gender, it is important to clearly distinguish between these latter two concepts [9]. Sex refers to biological characteris- tics, including but not limited to chromosomes, hor- mones and anatomy, of female and male bodies. ABSTRACT In contrast, gender is a socioculturally-constructed, multi- dimensional concept that entails the embodiment of different roles, behaviors, identities and relationships of women and men prescribed by social norms in a given time and society [10]. Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ipri20 SCANDINAVIAN JOURNAL OF PRIMARY HEALTH CARE 2023, VOL. 41, NO. 2, 132–139 https://doi.org/10.1080/02813432.2023.2191653 Aranka V. Balleringa , Tim C. Olde Hartmanb , Robert Verheijc,d and Judith G. M. Rosmalena ringa , Tim C. Olde Hartmanb , Robert Verheijc,d and Judith G. M. Rosmalena Aranka V. Balleringa , Tim C. Olde Hartmanb , Robert Verheijc,d and Judith G. M. R aDepartment of Psychiatry, University of Groningen, University Medical Center of Groningen, Groningen, The Netherlands; bDepartment of Primary and Community Care, Radboud University Medical Center, Nijmegen, The Netherlands; cNivel, Netherlands Institute of Health Services Research, Utrecht, The Netherlands; dTilburg School of Social and Behavioral Sciences, Tranzo Scientific Center for Care and Welfare, Tilburg University, Tilburg, The Netherlands Supplemental data for this article can be accessed online at https://doi.org/10.1080/02813432.2023.2191653. Supplemental data for this article can be accessed online at https://doi.org/10.1080/02813432.2023.2191653. 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. CONTACT Aranka V. Ballering a.v.ballering@umcg.nl Department of Psychiatry, P.O. Box 30.001, Internal Netherlands CONTACT Aranka V. Ballering a.v.ballering@umcg.nl Department of Psychiatry, P.O. Box 30.001, Internal Code CC72, 9700 RB Groningen, The Netherlands Supplemental data for this article can be accessed online at https://doi.org/10.1080/02813432.2023.2191653. 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. Department of Psychiatry, P.O. Box 30.001, Internal Code CC72, 9700 RB Groningen, The Introduction Previous studies have suggested that women more frequently consult their general practitioner (GP) for common somatic symptoms than men [1–5]. This may partly be explained by sex and gender differences in the occurrence of such symptoms: female participants reported more frequent, severe and persistent somatic symptoms, whereas femininity (i.e. feminine gender) was protective of the persistence of symptoms [6–8]. However, also help-seeking behavior for these symp- toms might contribute to the increased consultation SCANDINAVIAN JOURNAL OF PRIMARY HEALTH CARE 133 and complex genetics. The Lifelines Cohort Study is approved by the Medical Ethical Committee of University Medical Center Groningen (2007/152) and participants provided written consent. Extensive infor- mation on the cohort, design considerations and recruitment procedures is provided elsewhere [17,18]. In this study, we included Lifelines data collected from 2008 up until 2018. The idea of increased female primary care help- seeking for common somaticsymptoms, however, is under debate. A recent systematic review that directly compared the consultation patterns in women and men experiencing headaches and lower back pain concluded that the evidence for a female preponder- ance in help-seeking was surprisingly weak and incon- sistent [11]. Previous studies on sex and gender differences in primary care help-seeking for somatic symptoms are characterized by several methodological limitations. The NPCD encompasses electronic health records data from general practices located across the Netherlands, with diagnoses, contacts and prescription medicine coded via the International Classification of Primary Care (ICPC-2) system. Virtually all people resid- ing in the Netherlands are listed as a patient in a GP practice. The NPCD population is a representative sam- ple of the Dutch population. Data on GP consultations for common somatic symptoms as described in the Symptom CheckList-90 Somatization subscale (SCL-90 SOM; Table S1) were retrieved from electronic health records for all participants that were listed in one of the 63 NPCD GP practices located in the North of the Netherlands. This study has been approved according to the governance code of NPCD under number NZR- 00319.049. First, potentially due to the absence of adequate gender measures in epidemiological studies, these could not quantify independent sex and gender differ- ences in the frequency of help-seeking behavior for somatic symptoms [6,9,12–14]. Introduction Second, most studies focusing on help-seeking behavior in relation to som- atic symptoms are conducted in clinical populations or patient registries, which is problematic as it automatic- ally excludes people who do not seek help for their somatic symptoms [11]. Third, previous studies are largely based on self-reported measures of help-seek- ing, making these studies prone to recall bias. Fourth, most study designs do not correct for the type of somatic symptom and sex differences in the presenta- tion of these [15]. Variables We present the first large epidemiological cohort study on the association between sex, gender and help-seeking for common somatic symptoms that overcomes these problems. We linked the Lifelines general population cohort to the Nivel Primary Care Database (NPCD) [16], allowing us to assess independ- ent sex and gender differences in symptom-specific help-seeking behavior in the general population. We examined whether sex and gender have a unique association with help-seeking behavior for common somatic symptoms and whether the association between gender and help-seeking behavior for com- mon somatic symptoms varies between sexes. The presence of self-reported common somatic symp- toms was assessed in Lifelines surveys by the 12-item ordinal SCL-90 SOM, which has been recommended for large-scale studies and has sufficient measurement invariance over time [19,20]. Table S2 shows the defin- ition of new-onset symptoms. The symptoms (ICPC) of the SCL-90 SOM comprise headache (N01), dizziness (N17), heart pain (K01), (lower) back pain (L02 and L03), nausea (D09), muscle pain (L18), shortness of breath (R02), chills (A02), tingling of fingers, feet and/ or toes (N05), swallowing/throat problems (D21 and R21), general tiredness (A04) and heavy arms and/or legs (L09 and L14). The SCL-90 SOM subscale assesses the extent of distress or bother participants experi- enced during the past seven days due to these symp- toms. Presence of the new-onset symptoms was assessed with Lifelines’ survey data, whereas GP con- sults for these symptoms were listed in the NPCD. Lifelines and the Nivel Primary Care Database Lifelines is a multi-disciplinary prospective population- based cohort study examining in a unique three-gen- eration design the health and health-related behaviors of 167,729 persons living in the North of The Netherlands. It employs a broad range of investigative procedures in assessing the biomedical, socio-demo- graphic, behavioral, physical and psychological factors which contribute to health and disease of the general population, with a special focus on multi-morbidity Participants’ sex (female or male) and age in years were derived from the municipal population registry. Participants’ self-reported highest attained educational level was categorized into ‘high’, ‘medium’ and ‘low’ [21]. Participants’ burden of somatic symptoms was measured by the SCL-90 SOM sumscore at the moment of reporting a new-onset symptom [22]. A. V. BALLERING ET AL. 134 Participants’ feminine and masculine gender roles were operationalized via a recently developed, data- driven gender index, which accounts for the place-, time- and society-bound nature of gender roles [6]. In a subsample of baseline Lifelines adult participants that had no suspected intersex variation or gender-diverse gender identity, we performed LASSO logistic regression analyses that used 153 psychosocial characteristics, including dietary preferences, hobbies, time spend on household tasks or odd jobs, type of profession and personality traits, to predict partici- pants’ municipally-registered sex. The majority of the included psychosocial variables referred to gender roles. Using the obtained estimates of the regression coefficients of the LASSO logistic regression model (AUC ¼ 92%), an individual score regarding feminine and masculine gender roles (i.e. the gender index) was calculated for each adult participant. In other words, participants’ individual adherence to prototypical fem- inine and masculine psychosocial characteristics was calculated. The gender index ranges from 0% (fully masculine) to 100% (fully feminine). An index of 50% indicates androgyny, with equal levels of feminine and masculine characteristics present. Extensive informa- tion on the development and applicability of the gen- der index is provided elsewhere [6,7]. allows for accounting for the dependency of residual errors due to the hierarchical structure of the data; these models also handle missing data efficiently. Our data were clustered on three levels: (1) observations of help-seeking (2) were nested within patients, (3) who in turn were nested in GP practices. Our initial model included only the intercept as independent variable and allowed intercepts to vary across individ- uals and GP practices (i.e. random intercepts for the second and third level). One-by-one we included inde- pendent variables as fixed effects. Lifelines and the Nivel Primary Care Database Thereafter, we also allowed the effects of sex and gender to vary across patients and GP practices (i.e. random intercepts and slopes for the second and third level). Model fit was assessed using the Akaike information criterion (AIC), with a lower AIC indicating better model fit. One-way ANOVAs were applied to assess the significance of dif- ferences in model fit. The random effects’ covariance matrix was unstructured and no mean centering of continuous independent variables was performed, as these had meaningful zero points. Included independent variables were participants’ sex, age, educational level, score on the gender index, reported new-onset symptom, burden of somatic symp- tom experience at the moment of symptom reporting, and self-reported lifetime presence of chronic somatic and psychiatric diseases at the moment of symptom reporting. We also included a sex-by-gender interaction term to assess whether the association between gender and help-seeking differed for female and male partici- pants. We repeated these analyses with help-seeking within 3 months as dependent variable. To define help-seeking behavior of participants, we linked data derived from Lifelines with the NPCD (for detailed procedure: Figure S1). A Dutch trusted third party (Statistics Netherlands) used Record Identification Number pseudonymization to temporarily link health records on an individual level to facilitate analyses [23]. The presence of help-seeking was defined as the pres- ence of a GP contact (either face-to-face, by phone or digital) for the reported symptom provided 6 weeks before, or 6 weeks after reporting a new-onset common somatic symptom during a Lifelines assessment. Presence of help-seeking within 3 months of symptom reported was defined using a similar strategy. Since we only included contacts with ICPC < 30 codes, we restricted our analyses to help-seeking for common som- atic symptoms that the GP did not diagnose with an underlying disease (i.e. disease diagnosis; ICPC 70), but with a symptom diagnosis (i.e. symptoms that remain symptoms over time) [24]. In post hoc analyses, we assessed whether gender- related factors are associated with help-seeking. We repeated the abovementioned analyses, but replaced the gender index with gender-related factors, namely being a healthcare professional (yes/no), mean days per week one performs paid labor (1–7 days) and whether one considers oneself a homemaker (yes/no) as these were indicated by previous qualitative research to be of importance for help-seeking [9,13,14]. Lifelines and the Nivel Primary Care Database We assessed the presence of multicollinearity among independent variables, but found no indication of problems with multicollinearity as the variance inflation factor was 5 in all analyses [25]. We adhered to a two-sided a-level of 0.003, corrected for multiple comparisons (0.05/20; 19 independent varia- bles and one interaction term within the family of tests). Data on independent variables were imputed as described earlier [6]. All analyses were conducted using the lme4 package in R version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria). Patient population and GP consults Patient population and GP consults The Lifelines baseline population consisted of 152,728 adult participants, whereas the NPCD population com- prised 277,881 patients. In total, we linked 20,187 individu- als of whom 2709 (32.5%) male and 5616 (67.5%) female participants reported new-onset symptoms (Table 1). Ultimately, 255 individual participants (3.1%) sought help 360 times for 255 different symptoms within 6 weeks of reporting a new-onset symptom. Within 3 months of reporting a new-onset symptom, 387 individual participants (4.6%) sought help 596 times for 389 different new-onset symptoms (Table 2). The most frequently reported new-onset symptoms are muscle pains and (lower) back pain in 1037 (38.3%) and 903 (33.3%) of the male and in 1979 (35.2%) and 1626 (29.0%) of the female participants with new- onset symptoms, respectively (Table S3). The associations between the independent varia- bles and seeking help within 6 weeks of symptom reporting are similar in effect size to those of help- seeking within 3 months of symptom reporting. Female patients seek help more often within 6 weeks of new-onset symptom reporting than male patients Statistical analyses To assess whether sex and gender were independently associated with primary care help-seeking behavior, we applied generalized linear mixed-effect models with maximum-likelihood estimation. This approach SCANDINAVIAN JOURNAL OF PRIMARY HEALTH CARE 135 135 Table 1. Characteristics of the study population at baseline (N ¼ 8325). Table 1. Characteristics of the study population at baseline (N ¼ 8325). Male participants (N ¼ 2709; 32.5%) Female participants (N ¼ 5616; 67.5%) Age (years), mean (SD) 44.8 (12.7) 42.9 (13.0) Gender index, median (IQR) 0.08 (0.02–0.30) 0.95 (0.82–0.99) Educational level, N (%) Low 905 (33.4%) 1724 (30.7%) Medium 968 (35.7%) 2229 (39.7%) High 836 (30.9%) 1663 (29.6%) Symptom burden, mean SCL-90 SOM score (SD) 1.38 (0.37) 1.49 (0.41) Chronic disease, N (%) Present 1241 (45.8%) 3035 (54.0%) Absent 1468 (54.2%) 2581 (46.0%) Self-rated health, N (%) Good to excellent 2326 (87.1%) 4728 (85.4%) Poor to mediocre 345 (12.9%) 807 (14.6%) New-onset symptom 5439 12,416 Help-seeking 6 weeks 70 185 Help-seeking 3 months 111 278 Table 2. Reported new-onset symptoms in Lifelines and concomitant GP contacts in 8325 participants. New-onset common somatic symptom (ICPC) Symptoms reported, N (%) GP contacts within 6 weeks, N (%) GP contacts within 3 months, N (%) Headache (N01) 1868 (10.5%) 19 (7.5%) 26 (6.7%) Dizziness (N17) 761 (4.3%) 17 (6.7%) 24 (6.2%) Heart pain (K01) 483 (2.7%) <10 (<3.9%) <10 (2.6%) (Lower) back pain (L02/L03) 2529 (14.2%) 88 (34.5%) 138 (35.5%) Nausea (D09) 1406 (7.9%) <10 (<3.9%) <10 (<2.6%) Muscle pain (L18) 3016 (16.9%) 20 (7.8%) 39 (10.0%) Shortness of breath (R02) 633 (3.5%) 13 (5.1%) 17 (4.4%) Hot-and-cold spells (A02) 1627 (9.1%) <10 (<3.9%) <10 (<2.6%) Tingling extremities (N05) 1566 (8.7%) 10 (3.9%) 12 (3.1%) Throat/swallowing problems (D21/R21) 798 (4.5%) 17 (6.7%) 25 (6.4%) General tiredness (A04) 1698 (9.5%) 43 (16.9%) 66 (17.0%) Arm/leg symptoms (L09/L14) 1470 (8.2%) 19 (7.5%) 28 (7.2%) Total 17,855 (100.0%) 255 (100.0%) 389 (100.0%) between sex and gender, and help-seeking. For help- seeking within 6 weeks of new-onset symptom report- ing, the model that allowed the effect of only sex (AIC ¼ 2495.1), or of both sex and gender to vary across GP practices (AIC ¼ 2501.1; i.e. models including ran- dom slopes) did not fit the data significantly better than the model that included only random intercepts for patients and GP practices (AIC ¼ 2491.6; v2 (DF¼2) ¼ 0.58, p ¼ .75 and v2 (DF¼5) ¼ 0.58, p ¼ .99). Statistical analyses Similarly, for the 3 month timeframe, the model that included random intercepts for patients and GP practices (AIC ¼ 3454.4) fitted the data better than the models that allowed sex to vary across GP practices (AIC ¼ 3458.1), or sex and gender to vary across GP practices (AIC ¼ 3464.0). However, the differences in model fit were not statistically significant (v2 (DF¼2)¼ 0.37, p¼ .83 and v2 (DF¼5)¼ 0.41, p¼ .99, respectively). Therefore, we present the models including random intercepts, as these had the lowest AIC (Table 3). between sex and gender, and help-seeking. For help- seeking within 6 weeks of new-onset symptom report- ing, the model that allowed the effect of only sex (AIC ¼ 2495.1), or of both sex and gender to vary across GP practices (AIC ¼ 2501.1; i.e. models including ran- dom slopes) did not fit the data significantly better than the model that included only random intercepts for patients and GP practices (AIC ¼ 2491.6; v2 (DF¼2) ¼ 0.58, p ¼ .75 and v2 (DF¼5) ¼ 0.58, p ¼ .99). Similarly, for the 3 month timeframe, the model that included random intercepts for patients and GP practices (AIC ¼ 3454.4) fitted the data better than the models that allowed sex to vary across GP practices (AIC ¼ 3458.1), or sex and gender to vary across GP practices (AIC ¼ 3464.0). However, the differences in model fit were not statistically significant (v2 (DF¼2)¼ 0.37, p¼ .83 and v2 (DF¼5)¼ 0.41, p¼ .99, respectively). Therefore, we present the models including random intercepts, as these had the lowest AIC (Table 3). Sex and gender in association with help-seeking behavior for somatic symptoms We defined generalized linear mixed-effect models with increasing complexity to assess the associations 136 A. V. BALLERING ET AL. A. V. BALLERING ET AL. Table 3. Generalized linear mixed-effects models: estimated associations between independent variables and help-seeking within different time frames. Help-seeking (N ¼ 17,855) Within 6 weeks Within 3 months Fixed effects OR (95%CI) Female sex 1.78 (1.13–2.80) 1.53 (1.05–2.21) Femininity 0.67 (0.39–1.16) 0.76 (0.49–1.20) Age 1.03 (1.02–1.04) 1.02 (1.01–1.03) Presence of chronic disease 1.00 (0.76-1.31) 1.04 (0.84–1.30) Educational level Low Ref. Ref. Medium 1.22 (0.89–1.67) 0.95 (0.74–1.23) High 1.18 (0.84–1.66) 0.94 (0.71–1.24) Symptom burden 0.98 (0.96–1.01) 0.99 (0.97–1.01) Presence of symptom Headache 0.78 (0.41–1.48) 0.74 (0.43–1.28) Dizziness 1.77 (0.92–3.41) 1.67 (0.97–2.90) Heart pain 0.47 (0.14–1.58) 0.54 (0.21–1.38) (Lower) back pain 2.67 (1.62–4.40) 3.00 (1.99–4.53) Nausea 0.29 (0.11–0.76) 0.31 (0.14–0.68) Muscle pain 0.48 (0.26–0.90) 0.65 (0.40–1.06) Shortness of breath 1.57 (0.77–3.19) 1.40 (0.76–2.56) Hot-and-cold spells 0.05 (0.01–0.32) 0.03 (0.01–0.22) Tingling extremities 0.47 (0.28–0.99) 0.38 (0.19–0.75) Throat problems 1.67 (0.87–3.21) 1.68 (0.98–2.90) General tiredness 2.10 (1.22–3.59) 2.24 (1.43–3.49) Heavy arms/legs 1.27 (0.68–2.41) 1.33 (0.78–2.28) Table 3. Generalized linear mixed-effects models: estimated associations between independent variables and help-seeking within different time frames. Table 3. Generalized linear mixed-effects models: estimated associations between independent variables and help-seeking within different time frames. help-seeking within 6 weeks and 3 months of symp- tom reporting, whereas femininity was not. We found increased mean working days per week to be nega- tively associated with help-seeking behavior. (OR ¼ 1.78, 95%CI ¼ 1.13–2.80), but femininity was not associated with help-seeking (OR ¼ 0.67, 95%CI ¼ 0.39– 1.16). The sex-by-gender interaction terms were not statistically significant (OR ¼ 0.54, 95%CI ¼ 0.18–1.65), indicating that the association between gender and help-seeking within 6 weeks does not differ between female and male patients. Strengths and limitations This study had several strengths. First, we directly compared help-seeking in women and men, adjusted for the reported symptoms. Previous studies did not adjust for the reported symptoms and sex differences in presentation hereof. Second, previous studies assessing differences in primary care help-seeking behavior did not distinguish between sex and gender [11,26]. Here, we used a novel gender measure to dis- entangle associations of sex and gender with help- seeking behavior. Third, this study included consult- ation patterns based on primary care registries as opposed to self-reported measures, resulting in min- imal risk of recall bias. Lastly, this study assessed help- seeking in the general population instead of clinical populations, which allowed for participants who have not sought help to be included in the analyses. Post hoc analyses Femininity operationalized by the gender index showed no association with help-seeking for new- onset common somatic symptoms. We assessed whether specific gender-related factors that were iden- tified by previous qualitative research were associated with help-seeking. We found that working as a health- care professional or considering oneself as a home- maker showed no association with help-seeking within 6 weeks (OR ¼ 0.93; 95%CI ¼ 0.65–1.32 and OR ¼ 0.68; 95%CI ¼ 0.42–1.14, respectively). Weekly mean days of paid work did associate with help-seeking within 6 weeks (OR ¼ 0.95; 95%CI ¼ 0.91–0.98). No associations were found with help-seeking within 3 months. However, this study also had limitations. First, the NPCD is physician-centered and diagnosis-based, and the reason for encounter as reported by patients was not recorded. The use of recorded final diagnoses in this study implies that the frequency of help-seeking behavior was underestimated: GP consults in which a disease was recorded (i.e. ICPC 70) were excluded from analyses while those for which a symptom SCANDINAVIAN JOURNAL OF PRIMARY HEALTH CARE SCANDINAVIAN JOURNAL OF PRIMARY HEALTH CARE 137 diagnosis was recorded were included (i.e. ICPC 30). This may explain the negative association between nausea, muscle pain, hot-and-cold spells and tingling extremities with GP contacts, as these are hardly diag- nosed with symptom diagnoses. A recent study showed that men had a 6%-increase in odds of being provided with a disease diagnosis for their somatic symptoms compared to women [27]. This may imply that male help-seeking is underestimated in this study, and that the reported sex difference is overestimated. Often, sex differences in help-seeking are, at least partly, attributed to gender differences between women and men [15]. However, the results of this study suggest that factors associated with sex differen- ces in help-seeking should be sought in either the biological realm or in factors that go beyond the com- posite gender index. Multiple reasons grounded in biology have been explored for the female preponder- ance in help-seeking behavior. First, women experi- ence, describe and report their symptoms in a different manner, and more readily attribute these to somatic causes than men [8,32]. Second, depressive and anxiety-related symptoms are more prevalent in women than in men, and these depressive and anx- iety-related symptoms strongly associate with help- seeking [5,8]. Analyses in this study were adjusted for diagnosed psychiatric disorders, but not for depressive or anxiety-related symptoms. Last, female patients may have a lower threshold to seek help from their GP, as they are more familiar with primary care. This familiarity may arise, for example, because of more frequent visits to the GP related to gynecological interventions (i.e. the recurring smear test or preg- nancy-related visits). Second, the exact moment of symptom onset is unknown and symptoms could have been present for a substantial amount of time before individuals decide to consult their GP. Possibly, patients already sought help for symptoms in an earlier stage which would erroneously classify these participants as non-help seekers. This may also lead to an underestimation of help-seeking behavior, potentially explaining why a mere 3.1% of the patients with new-onset symptoms sought help, which is less than reported by previous studies [28]. Moreover, many previous studies assess- ing primary care help-seeking behavior include symp- toms beyond the 12 common somatic symptoms analyzed in this study, including sex-related symptoms and symptoms that require acute help-seeking, such as traumatic injuries, and do not distinguish between first and follow-up visits [2–4]. Comparison with previous studies We found that female patients sought help more fre- quently from their GP for common somatic symptoms than male patients. This is in line with previous studies [1,2,8,31], including those that adjusted for sex-specific symptoms [5,15]. Other studies stated that the evi- dence for a sex difference in help-seeking for common somatic symptoms is weak [11,26]. However, these studies did not solely focus on primary care, included other symptoms than the current study, defined symp- tom experience very broadly, or relied on self-reported data about help-seeking behavior. These differences in methodology may result in differing outcomes between the studies. SCANDINAVIAN JOURNAL OF PRIMARY HEALTH CARE Although no studies about sex differences in the amount of time between the moment of first symptoms until the moment of first contact in primary care for common somatic symptoms are known to the authors, studies on symp- toms associated with cancer and stroke among 10,297 and 162,856 adult patients, respectively, report no or inconclusive sex differences in time to help-seeking [29,30]. This suggests that no sex bias was introduced due to delays in help-seeking in this study. Gender-related factors that are not incorporated or are diluted in the gender index may also affect help- seeking. The gender index is based on psychosocial variables that predominantly reflect gender roles. Although studies show that more practical gender- related factors, such as household responsibilities [33,34], influence help-seeking behavior, others argue that mainly gender stereotypes influence people’s help-seeking behavior [35,36]. Gender stereotypes are not incorporated in the gender index, yet these do pose a social framework on people resulting in gen- dered behaviors and ideas. For example, traditional western gender stereotypes prescribe that it is more culturally and socially accepted for women to openly express their symptoms compared to men [6]. In con- trast, these gender stereotypes state that men should be stoical about bodily experiences and should con- ceal their symptoms, even from care providers [36,37]. Although the gender index does not associate with help-seeking behavior, gender as an influencing factor on help-seeking should not be discarded completely. Discussion To our knowledge, this is the first large-scale study that assesses separate associations of sex and gender with primary care help-seeking behavior for common somatic symptoms. Female sex was associated with References [1] Rodriguez Gonzalez AM, Ramırez Aranda JM, de los Santos Resendiz H, et al. Health care utilization of Mexican patients with medically unexplained physical symptoms. Colomb Med. 2016;47:155–159. Consent form [9] Doyal L. Sex and gender: the challenges for epidemi- ologists. Int J Health Serv. 2003;33(3):569–579. Informed consent was obtained from all individual partici- pants included in the study. [10] Johnson JL, Greaves L, Repta R. Better science with sex and gender: facilitating the use of a sex and gender-based analysis in health research. Int J Equity Health. 2009;8(1):14. Data availability statement Given the privacy of participants included in Lifelines and NPCD, the authors cannot publicly share the databases used in this study. Access to data can be requested by contacting Lifelines and NIVEL. Ethical approval [4] Bertakis KD, Azari R, Helms LJ, et al. Gender differen- ces in the utilization of health care services. J Fam Pract. 2000;49:147. The Lifelines Cohort Study is performed according to the principles of the Declaration of Helsinki and in accordance with UMCG’s research code. It is approved by the Medical Ethical Committee of the University Medical Center Groningen, The Netherlands (number: 2007/152) and all par- ticipants have provided written consent. The use of elec- tronic health records, including those provided by the NPCD, for research purposes is allowed under certain conditions. When these conditions are fulfilled, neither obtaining informed consent from patients nor approval by a medical ethics committee is obligatory for this type of observational studies containing no directly identifiable data (art. 24 GDPR Implementation Act jo art. 9.2. sub j GDPR). [5] Wang Y, Hunt K, Nazareth I, et al. Do men consult less than women? An analysis of routinely collected UK general practice data. BMJ Open. 2013;3(8): e003320. [6] Ballering AV, Bonvanie IJ, Olde Hartman TC, et al. Gender and sex independently associate with com- mon somatic symptoms and lifetime prevalence of chronic disease. Soc Sci Med. 2020;253:112968. [7] Ballering AV, Wardenaar KJ, Olde Hartman TC, et al. Female sex and femininity independently associate with common somatic symptom trajectories. Psychol Med. 2020;52:1–11. [8] Barsky AJ, Peekna HM, Borus JF. Somatic symptom reporting in women and men. J Gen Intern Med. 2001;16(4):266–275. Acknowledgements The Lifelines initiative has been made possible by subsidy from the Dutch Ministry of Health, Welfare and Sport, the Dutch Ministry of Economic Affairs, the University Medical Center Groningen (UMCG), Groningen University and the Provinces in the North of the Netherlands (Drenthe, Friesland, Groningen). The authors wish to acknowledge the services of the Lifelines Cohort Study, the contributing research centers delivering data to Lifelines, and all the study participants. [2] de Waal M, Arnold IA, Eekhof JAH, et al. Follow-up study on health care use of patients with somato- form, anxiety and depressive disorders in primary care. BMC Fam Pract. 2008;9(1):5. [3] Redondo-Sendino A, Guallar-Castillon P, Banegas JR, et al. Gender differences in the utilization of health- care services among the older adult population of Spain. BMC Public Health. 2006;6(1):155. ORCID Aranka V. Ballering http://orcid.org/0000-0002-3491-8990 Tim C. Olde Hartman http://orcid.org/0000-0003-2078- 1206 Robert Verheij http://orcid.org/0000-0003-2234-4819 Judith G. M. Rosmalen http://orcid.org/0000-0002-6393- 0032 Further research should consider whether the sex difference in help-seeking behavior also results in a sex difference in conducted diagnostic tests and diagnosed diseases [27,39]. Men are often typed as more reluctant seekers of healthcare and health information, whereas women are portrayed as frequent help-seekers. Such ideas may inadvertently prompt the GP to consider men’s symptoms as more serious than women’s symp- toms, resulting in less watchful waiting in men. Clinical implications This study found that female sex and the amount of days performing paid labor both associate with help- seeking behavior. For clinicians, the patient’s charac- teristics, such as sex, frequency of help-seeking and A. V. BALLERING ET AL. 138 occupational factors, are pivotal in the clinical deci- sion-making processes. 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W4293421566.txt	https://journals.wlb-stuttgart.de/ojs/index.php/sh/article/download/3601/3594	de		Kirchliches Wirken und wirtschaftliches Handeln	Schwäbische Heimat	2,022	cc-by	3,632	Ansicht Korntals von Süden über die Teichwiesen auf Betsaal, Gemeindegasthaus und Wirtschaftsgebäude des früheren Allodialguts. Kolorierte Lithografie von H. Pons um 1820 Kirchliches Wirken und wirtschaftliches Handeln Die Korntaler Güterkaufsgesellschaft Albrecht Rittmann Die Darstellung eines Unternehmens mit über 200-jähriger Geschichte heißt die Brücke zu schlagen zwischen dessen betriebswirtschaftlichem Handeln und dem korres pondierenden politischen und sozialen Umfeld. Versetzen wir uns also in die Zeit von 1817 bis 1819, als Gottlieb Wilhelm Hoffmann, Notar aus Leonberg, und das 15-köpfige Brüder-Collegium mit dem Oberkonsistorium und dem Geheimen Rat der Regierung von Württemberg die Verhandlungen über eine eigenständige politisch-religiöse Gemeinde führten. Eine Vielzahl der Pietisten im Kulturerbe Land, die im Streit mit der Amtskirche waren, strebte dies an, während zur selben Zeit um die zehntausend Menschen aus wirtschaftlicher Not das Königreich verließen, um nach Südrussland auszuwandern; dort hatte ihnen der russische Zar Land zu günstigen Bedingungen versprochen. Im Vordergrund der Verhandlungen mit der württembergischen Regierung standen zunächst Fragen des Religions- und Glaubensbekenntnisses sowie der Kirchenordnung und der Liturgien. Zu dem Punkt, wie denn eine in 77 Das Gemeindegasthaus war das ehemalige Herrschaftshaus der Freiherrn von Münchingen und der eingeheirateten Grafen von Görlitz. Mit dem Erwerb des Hofguts Korntal wurde es zum Verwaltungssitz der Brüdergemeinde und Gasthaus umgebaut und 1921 durch einen großen Anbau mit Festsaal ergänzt. Foto vom Saalplatz aus um 1955 sich geschlossene und abgeschottete Gesellschaft überhaupt wirtschaftlich funktionieren kann, machte man sich auf beiden Seiten bei den ersten Verhandlungen noch keine großen Gedanken. Es war vielmehr ein zähes Ringen um religiöse Fragen. Auf Seiten der Regierung und der Landeskirche entstand die Sorge, dass die geplante Gemeinschaft sich zu sehr in Gegnerschaft zur Amtskirche begeben und zu einer Sekte abgleiten könnte. Denn es gab nicht nur die Pietisten, die einen Glaubensabfall der Amtskirche beklagten, sondern deutlich radikalere Gruppen, beispielsweise in der Gegend um Maulbronn die Sekte der Separatisten, auch Harmonisten oder Harmoniten genannt, die aber staatlicherseits verfolgt wurden. Auch die württembergischen Pietisten waren keine glaubensmäßig völlig übereinstimmende Gruppe, sondern es gab unter ihnen sehr verschiedene Ausrichtungen. Mit der Vorlage eines Religions- und Glaubensbekenntnisses der Gemeinde und einer Abhandlung über Kirchenordnung und Zeremonien der Gemeinde konnte Hoffmann bei den Verhandlungen mit dem Oberkonsistorium offensichtlich überzeugend darlegen, dass die zu gründende Gemeinde im Gegensatz zu anderen religiösen Bewegungen das Fundament der Amtskirche nicht grundlegend verlassen und treu zur Krone stehen würde. Suche nach einem geeigneten Gelände Rund 18 Monate nach Hoffmanns erster Eingabe bei König Wilhelm I. bekam er am 1. Oktober 1818 den Bescheid, dass die Zusicherung zu einer politisch religiösen Gemeinde gegeben werde, sobald das erforderliche Gelände erworben sei. Rund 700 Familien hatten sich zuvor dazu bekannt, Mitglied einer neuen Gemeinde werden zu wollen. Von ihnen wurde nun eine Gruppe, das sog. BrüderCollegium, beauftragt, Hoffmann beim Kauf eines geeigneten Geländes zu unterstützen. 78 Daraufhin wurde mit den Sondierungen über ein passendes Areal begonnen. Schon früher hatten die Brüder ihr Augenmerk auf Hohenheim gerichtet, es aber nicht erhalten. Hoffmann schwebte der Kauf einer Staatsdomäne vor, so kamen das Hofgut Einsiedel bei Tübingen, dann der Ihringer Hof bei Weil der Stadt und der Lauterbacher Hof bei Heilbronn ins Gespräch. Alle diese Verhandlungen führten nicht zum erhofften Erfolg. Vermutlich wurde es Hoffmann und dem Collegium dann schon etwas mulmig: Das Erreichen des Ziels war greifbar nahe, aber es fand sich nicht die notwendige Liegenschaft. In dieser Phase wurde dem Collegium das Rittergut Korntal angetragen, das den Freiherren von Münchingen und den eingeheirateten Grafen von Görlitz gehörte und offensichtlich keinen großen Ertrag abwarf. Der Kauf kam am 12. Januar 1819 zustande und damit lagen die Voraussetzungen vor, dass das endgültige Privilegium, also die Erlaubnis zur Gründung und die Festlegung der Korntaler Grundordnung seitens Wilhelm I. erteilt werden konnte. Zuvor war natürlich noch die Frage zu klären, wer der Käufer des Ritterguts sein würde. Hoffmann allein konnte es nicht sein: um den Kaufpreis von 113.700 Gulden aufzubringen, mussten viele Geldgeber bereitstehen. Am 17. Februar 1819 wurde deshalb zunächst mit 42 Gründerfamilien provisorisch die Güterkaufsgesellschaft gegründet, die aus allen bäuerlichen Siedlern bestand, die sich ernsthaft in Korntal niederlassen wollten. Dass die G.K.G. zunächst nur als Interimslösung gedacht war, geht daraus hervor, dass die Gesellschaft nach ihren Statuten die Aufgabe hatte, das Hofgut zu erwerben, es in Bonitätsklassen einzustufen und dann nach einem bestimmten Modus unter den Siedlern zu verteilen. Schwäbische Heimat 2022\|2 Eigentumsverhältnisse Grundsätzlich sollten die Grundstücke, die mit den Häusern der Siedler bebaut wurden, in deren freies Eigentum übergehen. Auch wurden die zum Rittergut gehörenden Weinberge und der Wald den Käufern zum Eigentum übertragen und schieden somit aus dem Gesellschaftsvermögen aus. Die Feldgrundstücke blieben dagegen im Eigentum der G.K.G. Die einzelnen Bewirtschafter hatten an den durch Los zugeteilten unbebauten Grundstücken nur ein erbpachtähnliches Besitz- und Nutzungsrecht. Es konnte vererbt und veräußert werden, aber nur an ein Mitglied der Güterkaufsgesellschaft. Auswärtige Personen konnten keine Mitglieder sein. Im Gesellschaftsvertrag war verankert, dass keiner seine durch Los erhaltene Liegenschaft als eigen zu betrachten habe. Jeder sei schuldig, bei Abgabe der Grundstücke diese wieder der Gesellschaft zurückzugeben. Nur der Mehrwert durch die Bewirtschaftung, wie der Bau von Zäunen und die Anpflanzung von Bäumen, konnte bei Rückgabe erstattet werden. Die G.K.G. war dann in einem solchen Falle gehalten, die eingenommenen Grundstücke wieder durch Los an ihre Mitglieder zu verteilen. Wollte ein G.K.G.-Mitglied eines seiner Grundstücke als Bauplatz verkaufen, so musste dies die G.K.G. als formale Eigentümerin für ihn tun. Grundsätzlich erhielt das G.K.G.-Mitglied den Kaufpreis. Bei einem Verkauf an Nichtmitglieder, was grundsätzlich erst ab dem Freizügigkeitsgesetz von 1867 möglich war, mussten bis zu 20 Prozent des Kaufpreises als sog. »Überlos« an die G.K.G. gezahlt werden. An eine vollständige Vergemeinschaftung der landwirtschaftlichen Flächen war nicht gedacht. Da waren die Korntaler weitsichtig und erfahren genug. Es schwebte ihnen zwar als bibeltreue Gläubige entsprechend Apg. 4, 32 »Keiner sage von seinen Gütern, dass sie sein wären« ein gemeinschaftliches Wirtschaften vor, und nach dem 3. Buch Mose 25, 23 »Ihr sollt das Land nicht verkaufen für immer, denn das Land ist mein und ihr seid Fremdlinge und Gäste von mir« waren sie der Auffassung, dass Gott der eigentliche Eigentümer des Bodens sei. Aber ihre bisherige Lebenserfahrung sagte ihnen, dass nur Arbeit und Fleiß auf eigener Scholle zu Wohlstand führt. Die Korntaler Neusiedler gehörten allesamt zu den eher begüterten Bürgern des Landes, jedenfalls nicht zu den Habenichtsen. Es war ja gerade der Trumpf in den Händen Hoffmanns gewesen, dass er Wilhelm I. vortragen konnte, ohne religiöse Freiheit würden viele wichtige Steuerzahler auswandern. dort eine wichtige Rolle spielte. Auch äußere Umstände, nämlich die Notwendigkeit, das Wilhelmsdorfer Moorgebiet gemeinsam trockenzulegen, um landwirtschaftlich nutzbare Flächen zu gewinnen, führten dort zunächst zu einer kollektiven Wirtschaftsform. Die Vergesellschaftung des Eigentums führte aber beinahe zum Bankrott der Wilhelmsdorfer Brüdergemeinde, in den auch die G.K.G. hineinzugeraten drohte. Diese Wirtschaftsform wurde deshalb wieder aufgegeben. Es waren aber nicht die Wilhelmsdorfer allein, die nach dem angeblichen Vorbild der apostolischen Kirche eine kirchliche und bürgerliche Verfassung mit Gütergemeinschaft herstellen wollten. Auch die schon erwähnten Separatisten strebten eine Vergesellschaftung aller ihrer Güter an. Radikalpietistische Gruppen um den Iptinger Leinenweber Johann Georg Rapp gründeten in Indiana/USA die Siedlung »Harmony«, in der jegliches Privateigentum abgeschafft war. Nachdem Korntal als sog. königlich-privilegierte Gemeinde am 22. August 1819 durch die sog. Fundationsurkunde seine staatliche Anerkennung gefunden hatte und die Gemeindeorgane – weltlicher Vorsteher und Gemeinderat – gewählt waren, wurden diese Organe kurzerhand auch zu Organen der G.K.G. Ein bürgerliches Leitungsorgan gab es nicht. So kam es zu einer engen Verquickung von Gemeinde und G.K.G., ja man darf wohl Gemeinde und G.K.G als religiös und wirtschaftlich einheitliches Gebilde ansehen. Den weltlichen Part der Brüdergemeinde hatte also die G.K.G übernommen. Diese Entscheidung war aber nicht gänzlich unstrittig, wie sich später zeigen sollte. Immer wieder kam es zu Streit und Anfeindungen, weil einzelne Mitglieder die G.K.G. beschuldigten, nur im Interesse der Gemeinde zu handeln. Beispielsweise kam es immer wieder vor, dass die G.K.G. einzelne Grundstücke, die wieder zurückgegeben wurden, nicht durch Los an ihre Mitglieder weitergab, sondern mit dem Verweis auf Eigenbedarf Sparkassenbuch der Güterkaufsgesellschaft. Die »Leihkasse« der Güterkaufsgesellschaft, die bis 1921 existierte, gilt neben der Württembergischen Landessparkasse als erste dieser Art in Württemberg und ist damit eine Vorläuferin des Raiffeisen- und Sparkassenwesens. Eine andere Ideologie in Wilhelmsdorf Interessant ist es, Wilhelmsdorf als Ausgründung von Korntal im Vergleich zur Korntaler Entwicklung zu betrachten. Wilhelmsdorf schlug einen anderen Weg ein. Wie Theodor Steimle in seinem Buch Korntal und Wilhelmsdorf schrieb, orientierten sich die Wilhelmsdorfer eng an der Urchristengemeinde, sodass ein von ihnen unterstellter urchristlicher Kommunismus in den Anfangsjahren Kulturerbe 79 selbst behielt. Das wurde damit begründet, dass das allgemeine Wohl das oberste Gesetz der Gesellschaft sei. Schon immer wurde den Korntalern eingeprägt, sich nach der Gemeinde und nicht nach dem Eigenwohl zu richten. Mit Streitereien zwischen G.K.G und einzelnen Mitgliedern musste die G.K.G. bis in die 1980er-Jahre leben. Haftungsregelungen und Bodenrechte Eine Besonderheit der G.K.G. war die Haftungsregelung. Jedes Mitglied haftete unbegrenzt für alle Verbindlichkeiten der G.K.G. Diese Solidarhaftung war sinnvoll, weil die G.K.G. dadurch gegenüber allen Geschäftspartnern in hohem Maße kreditwürdig war, insbesondere für die beiden Verkäufer des Korntaler Hofguts. Die einzelnen Mitglieder begaben sich aber in eine existentielle Abhängigkeit zur Gesellschaft, die nicht dauerhaft sein konnte. Gemeindevorsteher Johannes Daur schrieb in der Festschrift zur Jahrhundertfeier der G.K.G., dass durch das äußere Band einer teilweisen Vermögensgemeinschaft der Siedler durch die Soldidarhaftung »alle für einen, einer für alle« im Inneren wie gegen außen die Ziele und Bestrebungen der ersten apostolischen Gemeinde sichtbar wurden. Bei einem kritischen Rückblick auf die Geschichte der G.K.G. muss allerdings die Frage erlaubt sein, ob nicht einzelne Bestimmungen der G.K.G. eher einen wirtschaftlichen als einen religiösen Hintergrund hatten. Spekulativ ist auch die Annahme, dass die Regelung, nach welcher die Gebäude und Baugrundstücke in das Privateigentum ihrer Besitzer übergehen, ihre Wurzel in 3. Buch Mose 25,29 hatte. Nach dem israelitischen Bodenrecht konnten Häuser innerhalb ummauerter Städte für ewig verkauft werden. Es war wohl ein ständiges Ringen von notwendigem wirtschaftlichem Pragmatismus mit alttestamentlichen Idealvorstellungen, das die G.K.G. durchlebte. Darauf weist auch eine Äußerung Hoffmanns aus dem Jahre 1825 hin: »Die Frage, ob die Gemeinde Korntal nach Art der Gemeinde nach Apostelgeschichte 4 eingerichtet sei, kann ich mit Das 1851 erbaute Krankenhaus der Güterkaufsgesellschaft diente der Krankenpflege der Korntaler. Das Gebäude existiert nicht mehr. mehr Gewißheit beantworten. Es war der sehnlichste und herzlichste Wunsch meiner Brüder, sowohl als auch mein eigener, eine solche Gütergemeinschaft zu haben; aber es haben sich unüberwindliche Hindernisse in den Weg gestellt. Wir glauben, dass eine solche Gemeinschaft erst zustande kommt, wenn die Weissagung aus Joel 3,1 erfüllt werden wird.« Zweifellos haben die bodenrechtlichen Bestimmungen der G.K.G. segensreich bewirkt, dass es in Korntal keine Bodenspekulation gab. Ob allerdings, wie Steimle in seiner schon erwähnten Schrift ausführt, Korntal seine Blüte in erster Linie seinen eigenartigen, von der G.K.G. getragenen Bodenrechtsverhältnissen verdankt, kann aufgrund der Wilhelmsdorfer Entwicklung bezweifelt werden. Ich schreibe die Erfolgsgeschichte von Korntal eher dem Fleiß, den Fähigkeiten, der Weitsicht der handelnden Personen und den bestens geführten wirtschaftlichen Unter- 1921 gründete Hoffmann die »Töchteranstalt« zunächst in einer bestehenden Kelter nördlich des Saalplatzes. Sie musste dann einem Neubau weichen, der ein Schülerinnenwohnheim sowie eine höhere Mädchenschule beherbergte. Wegen des schlechten Baugrunds aus Gipskeuper wurde das Gebäude in den 50er-Jahren abgebrochen. Das Foto zeigt das Töchterinstitut um 1896. 80 Schwäbische Heimat 2022\|2 nehmungen der G.K.G. zu und gehe damit konform mit dem in Korntal verstorbenen und begrabenen Missionar Johannes Hesse, dem Vater von Hermann Hesse, der meinte: »Der Wohlstand der Gemeinde geht auf die vortrefflichen Einrichtungen der G.K.G. zurück.« Sein Grabstein wurde auf Initiative des Schwäbischen Heimatbundes im vergangenen Jahr mit Mitteln der G.K.G. restauriert. Eine der ältesten Sparkassen Württembergs Als die G.K.G. ein Jahr nach ihrer Gründung auch als Leihkasse firmierte, war faktisch der Grundstein für ihr Fortbestehen gelegt. Da die Korntaler Siedler nach den Statuten keine Kredite außerhalb Korntals aufnehmen durften, gab es nur die Möglichkeit, über die G.K.G. an notwendige Investitionsmittel zu kommen. Die Leihkasse nahm von den Gemeindegliedern Geld auf, das zu 4 Prozent verzinst und zu 4,5 Prozent ausgeliehen wurde. Schuldenmacherei war verpönt und so hatte man auch die Kontrolle über die finanziellen Verhältnisse der Mitbürger. Gleichwohl war diese Einrichtung der G.K.G. damals sehr fortschrittlich, wie überhaupt alle ihre Einrichtungen. Die Leih- und Sparkasse der G.K.G. gilt neben der Württembergischen Landessparkasse als älteste Sparkasse in Württemberg. Bis zum Jahre 1869, also ein halbes Jahrhundert lang, hatte die G.K.G. in ihrer ursprünglichen Rechtsform fortbestanden. Es ist schon bemerkenswert und außergewöhnlich, dass durch einen privatrechtlichen Vertrag jeder mit jedem verbunden war. Auf den Namen aller Mitglieder wurden die Korntaler Erziehungsanstalten, z. B. 1821 das Töchterinstitut, das Pfarrhaus, das Gasthaus und vieles mehr übernommen, ein Krankenhaus und ein Witwenhaus gebaut, den Grund zum Besoldungs- und Pensionsfonds für Pfarrer, Vorsteher und Schullehrer gelegt und schließlich im Jahre 1863 die Gemeindehandlung von Kaufmann Schrade übernommen. Wäre nur eine dieser Unternehmungen schiefgelaufen, hätte das die Existenz von manchem Korntaler vernichtet. Man muss sich einmal vorstellen, wie hoch das Haftungsrisiko jedes Mitglieds war. Kein Mensch würde heute ein vergleichbares Risiko eingehen. Es musste also doch eine tiefe, glaubensmäßig verwurzelte Verbindung unter den Korntalern gegeben haben. Anders ist das ursprüngliche Konstrukt der G.K.G. nicht zu erklären. Von Anfang an verband die Gemeindeglieder ein Gemeinsinn, beruhend auf dem Gefühl der gegenseitigen Verpflichtung für einander und für die Ordnung der Gemeinde. Dieses enge Band der Verbundenheit innerhalb der G.K.G. führte auch zur Einrichtung einer »Vieh-Assekuranz« für die G.K.G.-Mitglieder. Wo Licht ist, ist aber auch Schatten: 1853 wurde beschlossen, »daß derjenige, welchem künftig ein Stück Vieh verunglückt, das er von einem Juden erkauft hat, keinen Anspruch auf Vergütung an die Versicherungsgesellschaft zu machen haben soll, weil mehrfache Erfahrung lehrt, dass die meisten bei dem Handel mit Juden Schaden leiden«. Kulturerbe Königliches Dekret zur Verleihung des Status einer juristischen Person für die Güterkaufsgesellschaft Die weitere Entwicklung der Gesellschaft In der zweiten Hälfte des 19. Jahrhunderts standen Reformen im Vordergrund, die durch den Generationenwechsel, die zunehmende Zahl an Mitgliedern und der fehlenden Rechtsfähigkeit notwendig wurden. Sie begannen damit, dass ab 1863 die Mitglieder der G.K.G aus der Einzelhaftung entlassen wurden. Darüber hinaus durften sie auch außerhalb der Gemeinde Kredite aufnehmen. Auf einen entsprechenden Antrag wurde am 9. September 1867 die G.K.G in eine juristische Person umgewandelt, die nur noch mit ihrem eigenen Vermögen haftete mit der Folge, dass sie nunmehr auch freier agieren konnte. Immerhin gehörten in dieser Zeit von den 270 Hektar Fläche der Gemeinde Korntal 190 Hektar der G.K.G. Heute sind nur noch 27 Hektar im Besitz der Gesellschaftsmitglieder und ca. 5,5 Hektar im Besitz der Gesellschaft. Aber auch das Bestreben, Korntal seinen eigenen, ursprünglichen Charakter zu bewahren, stand auf der Agenda der G.K.G. Den im Zuge der Reichsgesetzgebung möglich gewordenen unbeschränkten Zuzug nach Korntal versuchte die G.K.G. mit ihren Mitteln, nämlich der Herrschaft über den Grundbesitz, zu steuern. Hierzu gab sie sich 1906 weise Grundsätze, deren erster Abschnitt meines Erachtens auch noch heute Richtschnur sein könnte. »Eine Ausdehnung der Gemeinde wird nicht angestrebt, aber die natürliche Entwicklung derselben auch nicht hintangehal- 81 1863 übernahm die Güterkaufsgesellschaft die zunächst im Privatbesitz befindliche »Gemeindehandlung« und baute sie zu einem Kaufhaus mit sechs Abteilungen aus. Das Foto zeigt die Gemeindehandlung um 1950. ten; doch soll weise Mäßigung und Vorsicht innerhalb der gesetzlichen Schranken bei Behandlung künftiger Baugesuche und Ansiedlungen walten. Im Baustil und in der äußeren Ausführung ist auf edle Einfachheit und Harmonie des Einzelnen mit der Umgebung Gewicht zu legen.« In einem weiteren Abschnitt der Baugrundsätze der G.K.G. steht: »Durch ausreichende Grund- und Gartenfläche der einzelnen Neubauten ist auf Erhaltung des ländlichen Bildes der Gemeinde Bedacht zu nehmen.« Wir haben es also den internen Bestimmungen der G.K.G. zu verdanken, dass Korntal den Charakter einer Gartenstadt bekam und daher ihren Charme hat. Dennoch besaß Korntal im Gegensatz zu den umliegenden Nachbargemeinden immer ein eher städtisches Gepräge. Bei den landwirtschaftlichen Gebäuden waren zum Beispiel die Düngerstätten und Jauchegruben zumeist hinter dem Haus angelegt. Störende Gewerbebetriebe sind auszuschließen In den Regeln zur Überbauung von Gesellschaftsgrundstücken findet sich eine weitere interessante Bestimmung: »Bauspekulationen, Fabriken und Wirtschaften sind tunlichst auszuschließen; ebenso störende Gewerbebetriebe.« Hier stoßen wir auf den grundsätzlichen Vorbehalt der Korntaler Brüder gegenüber einer industriellen Wirtschaftsform. Schon zu Lebzeiten Hoffmanns betrachtete es die Gemeinde als ihre Aufgabe, Gewerbe und Industriebetriebe, die den Geist ihrer Einrichtung stören, fernzuhalten. Das zeigt sich am Beispiel der Korntaler Manchesterfabrik, also einer Wollspinnerei. 1835 von der Familie Schüle begründet, lief das Geschäft von Anfang an sehr gut. 1839 arbeiteten dort bereits 35 Webstühle, die alle gar nicht von Korntalern besetzt werden konnten, sondern von auswärtigen Arbeitern betrieben wurden. Als dann wegen des weiteren Wachstums die Fabrik um ein drittes Stockwerk erweitert werden sollte, lehnte der Gemeinderat dies ab mit der Begründung, dass die Fremdarbeiter der Fabrik Unruhe in die Gemeinde gebracht hätten. Die langfristige 82 Folge war, dass die Manchesterfabrik geschlossen werden musste. Nur ein Tuchhandelsgeschäft verblieb in Korntal. Nur notgedrungen beschloss 1899 der Gemeinderat die Übernahme des bis dahin verpachteten Korntaler Gipswerkes, um »schädlichen Einflüssen auf die Gemeinde einen Damm zu setzen«. Noch 1912 wurde eine Ortsbausatzung zum Zwecke der Fernhaltung »störender Gewerbebetriebe« erlassen. Die damalige Grundhaltung prägt bis heute Korntal als Wohnstadt. Bei seiner günstigen Verkehrslage nach der Eröffnung der württembergischen Schwarzwaldbahn von Stuttgart nach Calw im Jahre 1872 und der Nähe zu den Stuttgarter Industriebetrieben wäre Korntal mit großer Wahrscheinlichkeit zu einer Industrie- oder Arbeiterwohngemeinde geworden. So jedoch wurde das dann neu erschlossene Gebiet der Neuen Halde – siehe die Straßenbezeichnung Neuhaldenstraße – zum Villengebiet. An anderen Orten gab es jedoch durchaus aus christlicher Gesinnung heraus gegründete erfolgreiche gewerbliche oder industrielle Unternehmungen, wie beispielsweise die Gustav Werner’schen Anstalten in Reutlingen. Auch die Geschichte des Gipswerkes zeigt die industrieskeptische Haltung der Korntaler. Schon ein Jahr nach dessen Errichtung kam man zu der Einsicht, dass der nach sozialen und christlichen Grundsätzen durchgeführte Regiebetrieb nicht rentierte. Er wurde deshalb verpachtet, nach Ablauf des Pachtvertrags wieder mit zwei eigenen Geschäftsführern weiterbetrieben, dann weitere Gesellschafter aufgenommen, um es schließlich an die Schwäbische Gipsverkaufstelle GmbH zu verpachten, bis sich der Betrieb wegen mangelnder Rentabilität auflöste. Von ihrer Struktur und den handelnden Persönlichkeiten war die G.K.G. durchaus in der Lage, gewinnbringend zu agieren, wenn ihr auch die Tatsache zu Gute kam, dass nach dem Privilegium und der Gemeindeordnung Gewerbe, Handel und Krämerei zunächst auf je einen Vertreter ihrer Gattung beschränkt waren. So gab es in Korntal ein Schwäbische Heimat 2022\|2 Spezereifachgeschäft, das zunächst in privater Hand war. 1856 erwarb die G.K.G. dieses Geschäft und führte es als Gemeindehandlung fort. Schon im ersten Jahr wurde ein Gewinn von 838 Gulden verzeichnet. 1873 war die Gemeindehandlung schuldenfrei. Der Gewinn steigerte sich dann drei Jahre später um ein Mehrfaches. Bis ins 20. Jahrhundert hinein war die G.K.G der allumfassende Wirtschaftsbetrieb der Gemeinde. Ab 1912 kaufte man den elektrischen Strom für Korntal ein und errichtete die dafür notwendige Infrastruktur. 1915 legte man eine Gasleitung von Feuerbach nach Korntal und legte das eigene Gaswerk still. Umbruchszeit und Unfrieden In der Zeit der Weimarer Republik kam dann der große Umbruch nicht nur für die kirchliche Gemeinde, sondern auch für die G.K.G, da das königliche Privilegium keine Gültigkeit mehr besaß. Zwar verblieb zunächst der gesamte Grundbesitz der ehemals privilegierten Gemeinde bei der G.K.G. und auch deren Leitungsorgan blieb identisch mit der Kirchenleitung. Schon 1916 hatte man beschlossen, dass für den Fall einer Trennung von bürgerlicher und kirchlicher Gemeinde die G.K.G. bei der kirchlichen Gemeinde verbleiben soll. Aber im Lauf der Zeit unterschieden sich die Mitglieder der G.K.G. und die der Brüdergemeinde dann durch Erbgänge und Verkäufe an Nichtmitglieder erheblich. So war es nicht mehr zu rechtfertigen, dass der nur von den Mitgliedern der Brüdergemeinde gewählte Kirchengemeinderat auch die Geschicke der G.K.G lenkte. 1934 unternahm man den ersten Versuch zu einer sinnvollen Aufteilung des Vermögens der G.K.G. zwischen der bürgerlichen Gemeinde, der Brüdergemeinde und der Gesellschaft als solcher. Das brachte, wie man sich denken kann, viel Unfrieden und Unruhe mit sich. Von vielen Mitgliedern der G.K.G., die der Brüdergemeinde fernstanden, konnte man auch nicht erwarten, dass sie sich an kirchlich-sozialen Aufgaben beteiligten. Erst im Jahre 1952 fand der Streit nach einer langen und schmerzlichen Phase der Auseinandersetzungen ein gerichtliches Ende. Zuvor hatte der Brüdergemeinderat auf seine Vertretungsbefugnis zugunsten Über den Autor Prof. Dr. Albrecht Rittmann ist stellvertretender Vorsitzender des Schwäbischen Heimatbundes und Autor zahlreicher Publikationen. Bis zu seiner Pensionierung war er Ministerialdirektor und Amtschef des Ministeriums für ländlichen Raum, Ernährung und Verbraucherschutz. 2018 wurde er zum Honorarprofessor an der Hochschule für öffentliche Verwaltung und Finanzen Ludwigsburg ernannt. In der SH 2019/1 veröffentlichte er: »Vor 200 Jahren: Die Gründung der Brüdergemeinde Korntal«. Kulturerbe eines von der G.K.G. selbst zu wählenden Organs verzichtet. Pfarrer Grünzweig schrieb dazu, »die Not um die G.K.G. war ein unübersehbares Zeichen, eine offene Wunde am Leib der Brüdergemeinde«. Heute ist die Güterkaufsgesellschaft ein eigenständiger Rechtskörper mit eigener Leitung. Sie hat in den vergangenen Jahren viele Projekte der kirchlichen wie kommunalen Gemeinde finanziell großzügig unterstützt. Was können wir nun aus der Geschichte der G.K.G erkennen? Ein begrenzter, homogener Kreis religiös gleichgesinnter Personen kann erfolgreich sozial und wirtschaftlich Großes bewirken und kirchliches Wirken und wirtschaftliches Handeln miteinander verbinden. Sobald aber durch äußerliche Einflüsse diese Einheit gestört wird, tiefgreifende Änderungen der äußeren Verhältnisse eintreten, kommt das Modell einer religiösen und wirtschaftlichen Partnerschaft an seine Grenzen, es lässt sich nicht mehr aufrechterhalten. In einem langen und schwierigen Abnabelungsprozess hat die G.K.G. aber heute ihre neue Aufgabe in der Unterstützung von sozialen Projekten wie dem Bau eines Mehrgenerationenhauses oder der Einrichtung eines Schulbauernhofs auf ökologischer Grundlage gefunden. Das Aquarell zeigt das 1892 von der Güterkaufsgesellschaft in Betrieb genommene Gipswerk. Literaturverzeichnis Dieter Feucht, Kleine Geschichte Korntals. Rede anlässlich der 60. Abiturfeier. Metzingen 2015 Fritz Grünzweig, Die evangelische Brüdergemeinde Korntal. Weg – Wesen – Werk. Metzingen 1957 Johannes Hesse, Korntal einst und jetzt. Stuttgart 1910 Paul Link, Die Geschichte Korntals von der Gründung bis zur Gegenwart. Korntal 2017 Sixt Karl Kapff, Korntal und Wilhelmsdorf, ihre Geschichte, Einrichtung und Erziehungsanstalten. Stuttgart 1839 Theodor Steimle, Die wirtschaftliche und soziale Entwicklung der württembergischen Brüdergemeinde Korntal und Wilhelmsdorf. Korntal 1929 83
https://openalex.org/W4220855531	https://zenodo.org/records/6356734/files/193-202.pdf	Swahili	null	MAKTAB JIHOZLARNING O'QUVCHILAR SALOMATLIK HOLATIGA TA'SIRINI GIGIENIK TAHLILI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,129	Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vebsayt: https://involta.uz/ MAKTAB JIHOZLARNING O‘QUVCHILAR SALOMATLIK HOLATIGA TA’SIRINI GIGIENIK TAHLILI Samarqand Davlat Tibbiyot Instituti To’xtarov Baxrom Eshnazarovich Umumiy gigiena va ekologiya kafedrasi mudiri E-mail: baxromtuxtarov@gmail.com Soatov Mirmuhsin Maxmatmurot o’g’li Umumiy gigiena va ekologiya kafedrasi 1-kurs magistratura rezidenti E-mail: mirmuhsinsoatov@gmail.com Maxmanazarov Gafur Axnazarovich Umumiy gigiena va ekologiya kafedrasi assistenti E-mail: gafuraxnazarov@gmail.com Annotatsiya. Maqolada O„zbekiston Respublikasi aholi Bolalar va o„smirlar jamoasi salomatlik holati jamoaning mehnat sharoitlari, maktablarda xonalarning jihozlanishi profilaktik chora tadbirlari taminlanganlik “Involta” Ilmiy Jurnali Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vebsayt: https://involta.uz/ MAKTAB JIHOZLARNING O‘QUVCHILAR SALOMATLIK HOLATIGA TA’SIRINI GIGIENIK TAHLILI Samarqand Davlat Tibbiyot Instituti To’xtarov Baxrom Eshnazarovich Umumiy gigiena va ekologiya kafedrasi mudiri E-mail: baxromtuxtarov@gmail.com Soatov Mirmuhsin Maxmatmurot o’g’li Umumiy gigiena va ekologiya kafedrasi 1-kurs magistratura rezidenti E-mail: mirmuhsinsoatov@gmail.com Maxmanazarov Gafur Axnazarovich Umumiy gigiena va ekologiya kafedrasi assistenti E-mail: gafuraxnazarov@gmail.com Annotatsiya. Maqolada O„zbekiston Respublikasi aholi Bolalar va o„smirlar jamoasi salomatlik holati jamoaning mehnat sharoitlari, maktablarda xonalarning jihozlanishi profilaktik chora tadbirlari taminlanganlik “Involta” Ilmiy Jurnali Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vebsayt: https://involta.uz/ “Involta” Ilmiy Jurnali Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vebsayt: https://involta.uz/ “Involta” Ilmiy Jurnali Involta Ilmiy Jurnali Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vebsayt: https://involta.uz/ “Involta” Ilmiy Jurnali Involta Ilmiy Jurnali Vol. 1 No.4 (2022) школьного оборудования на здоровье школьников Аннотация: в статье описаниы состояние здоровья детей и подростков Республиков Узбекистан, условия труда общества, оснащение учебных помещений в школах, проведение профилактических мероприятий Ключевые слова: школьное оборудование, дети, подростки, освещенность, учебные помещения Hygienic analysis of the impact of school equipment school equipment on the health of schoolchildren MAKTAB JIHOZLARNING O‘QUVCHILAR SALOMATLIK HOLATIGA TA’SIRINI GIGIENIK TAHLILI Samarqand Davlat Tibbiyot Instituti To’xtarov Baxrom Eshnazarovich Umumiy gigiena va ekologiya kafedrasi mudiri E-mail: baxromtuxtarov@gmail.com Soatov Mirmuhsin Maxmatmurot o’g’li Umumiy gigiena va ekologiya kafedrasi 1-kurs magistratura rezidenti E-mail: mirmuhsinsoatov@gmail.com Maxmanazarov Gafur Axnazarovich Umumiy gigiena va ekologiya kafedrasi assistenti E-mail: gafuraxnazarov@gmail.com Annotatsiya. Maqolada O„zbekiston Respublikasi aholi Bolalar va o„smirlar jamoasi salomatlik holati jamoaning mehnat sharoitlari, maktablarda xonalarning jihozlanishi, profilaktik chora-tadbirlari taminlanganlik. Tayanch so‘zlar: Maktab jihozlari, bolalar va o„smirlar jamoasi, yoritilganlik, o„quv xonalari. Гигиенический анализ влияния школьного оборудовани 193 Hygienic analysis of the impact of school equipment school equipment on the health of schoolchildren Annotation: the article describes the state of health of children and adolescents of the Republic of Uzbekistan, the working conditions of society, the equipping of classrooms in schools, and the implementation of preventive measures Key words: school equipment, children, teenagers, lighting, classrooms Muammoning dolzarbligi. Bolalar va o„smirlar jamoasi salomatlik holati jamoaning mehnat va intelektual potensialni tashkil qilganligi uchun bugungi kunda jahondagi eng asosiy dolzarb muamolardan biri hisoblanadi. Mamlakatimiz umumiy aholisining 14 million 170 minggini 41% ortig„ini 18 yoshgacha bolalar va o„smirlar jamoasi tashkil qiladi. Bu ko„rsatkichi yaqin turgan qo„shni mamlakatlar aholisining har birini alohida hisoblaganda umumiy sonidan ortiqdir. Ushbu jamoani tarbiyalash mamlakatimizda qonuniylashtirilgan bo„lib, 1996 yilda ta‟limning zamonaviy modelini amalga oshirish maqsadida Ta‟lim to„g„risdagi qonun qabul qilingan. Qonunning 10- moddasida Respublikamizdagi ta‟lim tizimining asosiy bosqichlari keltirilgan. Zamonaviy ta‟lim muassasalarning asosiy vazifalari komil insonni tarbiyalab etishtirish bilan birgalikda bolalar va o„smirlar ongiga demokratik jamiyat tamoyillarini singdirish, kattalarga hurmat, kichiklarga izzat, kasbga o„rgatishning boshlang„ich siralarni o„rgatish, o„zi tug„ilib o„sgan Vatanga 194 Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vol. 1 No.4 (2022) Vol. 1 No.4 (2022) sadoqat ruhida tarbiyalashdan iborat . Akademik YU.P.Lisitsin (1992) ma‟lumotlariga ko„ra salomalikka ta‟sir qiluvchi omillardan 50% turmush tarziga bog„liq bo„lsa, 25% ekologik vaziyatga bog„liqdir. Salomatlik holatiga ta‟sir ko„rsatadigan maktab ichi omillari material- texnik, atrof-muhit, o„quv yuklamalar va salomatlikni oldini olish va mustahkamlagshga qaratilgan guruhlarga ajratib tahlil qilinadi . Tayanch maktablarida ta‟lim olayotgan o„quvchilar orasida sog„lom muhit yaratish bilan birgalikda jahon andozalari talablariga javob beradigan etuk farzandlarni tarbiyalash, barkamol avlodni kamol toptirish va shakllantirish, ularning salomatlik holatini himoyalash va mustaxkamlash, gormonik normal tarzda rivojlangan etuk mutaxassislar etishtirib chiqarish, bugungi kunda nafaqat SEO va JSXning Bolalar va o„smirlar gigienasi bo„limi xodimlari, balki barcha jamoatchilik oldida turgan asosiy vazifalardan biri hisoblanadi. Oqilona tarzda tashkillashtirilgan kun tartibi bolalar va o„smirlar jamoasida sog„lom muhitni yaratish va ta‟minlashning asosiy manbalaridan biridir (SHayxova G.I., 2004,). Milliy kadrlar tayyorlash tizimini samaradorligini oshirish va yaxshilashga qaratilgan qator qonun va dasturlar ishlab chiqarilishiga qaramasdan O„zbekiston sharoitida ta‟lim olayotgan o„quvchilar orasida sog„lom maktab zamonaviy va qulay jixozlardan foydalanishni tashkillashtirish va ularning davriy ravishda gigienik baholash bugungi kunda hal qilinishi lozim bo„lgan dolzarb muammolardan biri hisoblanadi. Ishning maqsadi. Maktab jihozlarini va to„g„ri ovqatlanishning o„quvchilar salomatlik holatiga ta‟sirini o„rganish va uni gigienik tahlili. Natijalar muxokamasi. Inson organizmining o„sib rivojlanishida 3 ta bosqich amalga oshirildi. Bularga bolalik davri, etilish, karilik davri. Maktabgacha tarbiya yoshidagi bolalar oranizmining o„sib va rivojlanishi filogentik jarayonida bola organizmining o„sib rivojlanishida 3 ta qonuniyatga asosan shakllanish amalga oshiriladi. 195 Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Ularning asosiy o„lchamlari partalarning o„quvchilarga qaragan yuzasining kengligi va sinfxonalarning erdan balandligi santimetrli lenta yordamida aniqlanib baholanadi. Maktab jihozlarning funksional o„lchamlari, ya‟ni distanitsiya va differensiya esa aniq belgilangan GOST 0159-2003 «GOST shkolnыx mebeli» bo„yicha baxolandi va taxlil qilindi. Xonalarning xavosining almashtirilishi tabbiy yo„l aeratsiya yordamida fortochka va framuglar yordamida olib boriladi. Mexanik tortuvchi ventilyasiya oshxona va xojatxonalarga o„rnatilgan biroq ishlamaydi. Buning oqibatida sinfxonalarda changning miqdori – 0,25 mg/m3 ni tashkil qildi. Tabbiy yoritilganlik xonalarga chap tomomdan tushishiga qarmasdan TYOK qishda 0,5%, yozda – 1% (normada – 1,5%), sport zali va mexnat ta‟lim xonalarida esa 0,6-0,7%ni tashkil qildi. Barcha o„quv xonalarida su‟niy chiroqlar cho„g„lanma chiroqlar asosida yoritiladi va gigienik talablarga mos kelmaydi. ( 1-jadval). Maktab xonalarning yoritilganlik darajasi Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vol. 1 No.4 (2022) Maktabgacha bo„lgan davrda o„sib rivojlanishning 1- qonuniga bola qancha yosh bo„lsa, shuncha tez o„sib rivojlanish tushunchasiga to„g„ri keladi. Bu davrda xayotining birinchi yilida 47%, 2chi yilida 21%, 3 yilida 13%, keyinchalik 3-8%gacha usib rivojlanish amalga oshriladi (8,10,22,60). Bu davrda ona suti bilan birgalikda yoshiga va jinsiga mos ravishda ovkatlanish katta ahamiyatga egadir. Organizmning me‟yoriy faoliyat ko„rsatishi, uning barcha tizimlari o„zaro mutanosiblikda ishlashi, organizmning oziq-ovqatlardan oladigan quvvatining, uning extiyojlarini to„la qoplashi, ovqatlar tarkibidagi ko„p sonli biologik faol bo„lgan va muxim fiziologik vazifalarni bajaruvchi moddalar oqsillar, karbonsuvlar, yog„lar, vitaminlar va mikroelementlarning etarli bo„lishi bilan uzviy bog„langan. Optimal ovqatlanish tushunchasiga, organizmning fiziologik ritmlari talabiga mos, sifatli qilib tayyorlangan, to„yimli va shirin ta‟mli, tarkibi esa tengi yo„q biologik faol moddalar bilan to„ldirilgan, hamda organizmning me‟yoriy xayot kechirishi uchun zarur bulgan oqsillar, karbonsuvlar, yog„lar, vitaminlar va mikroelementlarni o„z ichiga olgan ovqat mahsulotlari bilan ovqatlanish kiradi. Fiziologik extiyojlarga mos ovkatlanish orkali organizm ichki muxiti (gomeostazining) doimiyligi ta‟minlanadi. Optimal ovqatlanish alohida aholi qatlamlari, guruxlar va ayrim odamlar talablariga mos kelishi, o„lkalarning geografik-iqlim sharoitlarini, milliy an‟analarini hisobga olgan holda tashkil qilinishi lozim. Maktab jihozlarni gigienik baholash usullari. Maktab o„quv xonalarining o„lchamlari SanMvaQ 01-02-2000 «Gigienicheskie trebovaniya k usloviyam obucheniya shkolnikov v obщeobrazovatelnыx shkolax» qonuniy hujjatlari bilan solishtirildi va gigienik baholandi. Maktablardagi sinfxonalarida va boshqa o„quv xonalarida jixozlarning joylashtirilish tartibi GOST 0159-2003 «GOST shkolnыx mebeli» asosida tahlili qilinib baholanadi. 196 Maktab xonalarning yoritilganlik darajasi Maktab xonalarning yoritilganlik darajasi Xonalar O„lchov soni YOritilganlik, lk, M±m Gigienik daraja Lyumin. chiroq CHo„g„lanma chiroq Lyumin. CHiroq CHo„g„lanma chiroq Sinfxonalar 5 220 170 300 200 O„qituvchilar xonasi 5 175 220 200 200 Rasm xonasi 5 400 200 500 300 Kompyuter sinflari 5 200 180 300 200 Ustaxonalar 5 200 180 500 300 Tikuvchilik 5 300 190 400 300 197 Involta Ilmiy Jurnali Vol. 1 No.4 (2022) xonasi Sport zal 5 150 100 200 150 Involta Ilmiy Jurnali Yoritilganlikning etishmovchilik darajasi asosan chiroqlar o„rni va sonining etishmasligi asosida kelib chiqqanligini bildiradi. Nazorat ostiga olingan maktabning binosi va undagi xonalar soni, ularning o„lchamlari va belganlingan ko„rsatkichlaridan kam ekanligi aniqlandi. SHuni ta‟kidlashimiz lozimki, nazorat ostiga olingan maktabning barcha ko„rsatkichlari sanitariya me‟yor ko„rsatkichlariga mos kelmasligi aniqlandi. Maktabning asosiy ko„rsatkichlari 2-jadvalda keltirilgan. Maktabning ta’lim va tarbiya sharoitining solishtirma tavsifnomasi Maktabning ta’lim va tarbiya sharoitining solishtirma tavsifnomasi Maktabning ta’lim va tarbiya sharoitining solishtirma tavsifnomasi Maktabning ta’lim va tarbiya sharoitining solishtirma tavsifnomasi Ko‘rsatkichlari SanMvaQ 0102-00 №29 maktab 1 2 3 joylashtirilishi kvartallararo Kvartallararo er maydoni 1 o„quvchiga 35-40 m2 40 m2 er maydonining mintaqalari: Qurilish foizi 12% gacha 25% Ko„kalamzorlash-tirilgan 45-50% 40% Jismoniy tarbiya sport (JTS) 35-40% 30% Sport maydoni Sport zali tomon 25 m Sport zali tomon 25 m Sport maydoni tarkibi: Stadion, maydonlar, yugurush yo„lakchasi, sakrash maydoni, o„yin uchun mos keladigan jixozlar To„liq to„plam, jixozlar bilan ta‟minlanmagan Sport maydoning Futbol maydoni-chim, hamma maydonlarga 198 Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Involta Ilmiy Jurnali Vol. 1 No.4 (2022) qoplamlari maydonlar –qattiq q. grunt qoplangan JTS saqlash tartibi Kunlik suv sepish Suv sepish xaftada 1 marotaba Dam olish mintaqasi 1-4 sinf – o„yin maydonlari 5- 9 sinf – jim dam olish mintaqasi Jim dam olish mintaqasi ajratilmagan Tashqi suv bilan ta‟minlanish Suv sepish moslamalari Mavjud Oyoq kiyim yuvish Bor, lekin foydalanilmaydi. Suv ichish uchun fontanchalar YOpiq Xonalar guruhi 1-qavat: -ma‟muriy – xo„jalik - sport zali - oshxona - mexnat darsi Talabga mos ravishda Kichik sinflar Alohida blok 2-3-qavat: - kutubxona - tibbiyot kabineti - faollar zali Talabga mos ravishda 5-7 sinflar uchun sinfxonalar va kabinetlar Alohida blok Sinfxonаlari Normada bitta sinfga 25 tagacha o„quvchi 35-40 o„quvchi 1 nafar o„quvchi uchun maydon - 2 m2 1,25 m2/1 o„quvchi Sport zali - soni - 1-2 1-360 m2 maydon: 160 dan 360 m2 gacha 1-288 m2 199 Involta Ilmiy Jurnali Vol. 1 No.4 (2022) balandligi – 6 m 6 m Sport zal tarkibi sport zali Mavjud (2) Snaryadli Mavjud (2) - kiyim almashtirish xonasi (2) Mavjud (2), lekin jihozlanmagan - dush (2) Mavjud, lekin ishlamaydi. - xojatxona (2) Mavjud, lekin ishlamaydi. - instruktor xonasi Mavjud (2) Tibbiy punkt -Vrach xonasi – 9-14 m2 9 m2 -stamotolog -12 m2 12 m2 - muolaja xonasi-14 m2 14 m2 - sanitariya tarmog„i Bor Oshxonalar kompleksi - tarqatish xonasi Bor - idish-tovoq yuvish xonasi Bor - yordamchi xonalar Bor - tushlik qilish zali 0,7 m2 /1 o„ringa 0,65 m2/1 o„ringa - YUvinish kranlari (25 o„ringa 1 dona) 30 o„ringa 1 dona Epidemik tarbiga davriy ravishda rioya qilish kerak Rioya qilinmagan Maktab jihozi 1-5-sinflar–4 guruhdagi markirovkalangan rangli partalar Bir turdagi markirovkalanmagan partalar 6-10 sinflar – stollar va stullar Bir xil turdagi alohida stol va stullar Bolalarni partada (stol) Bo„y, eshitish va ko„rish Bo„y inobatga Involta Ilmiy Jurnali Vol. Maktabning ta’lim va tarbiya sharoitining solishtirma tavsifnomasi 1 No.4 (2022) Vol. 1 No.4 (2022) 200 Involta Ilmiy Jurnali Vol. 1 No.4 (2022) o„tirishi o„tkirligi inobatga olingan xolda olinmagan. Binoning yoritilganligi Tabiy - TYOK - 1,5% 0,7-1% Sun‟iy– 200 dan 500 lk gacha (lyuminessent lampada) 150 dan 300 lk gacha Xavo harorati (yilning sovuq mavsumida) - sinflar- 17-21 °S 10 dan 15 °S gacha - sport zali - 14-15 °S 10-15 °S gacha Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vol. 1 No.4 (2022) Maktabning er maydoni va binosining sanitariya me‟yor va qoidalari bilan solishtirma tavsifnomasi shuni ko„rsatadiki, belgilangan ko„rsatkichlar va ularning mos kelmasligi natijasida quyidagi muammolar hosil bo„lishi olib keldi. Bu esa o„z o„rnida er maydoning noto„g„ri taqsimlanish oqibatida binoning qurilish % oshganligi, sport maydonining etarli emasligi va undagi jihozlarning etishmasligi, ichki va tashqi suv taminotining etishmasligi, asosan issiq suvning etishmasligi, sanitar-texnik qurilmalarning ish sharoitida emasligi, sinflardagi bolalar soning ortiqcha ekanligi eng asosiysi maktab jihozlarining o„quvchilarning bo„yiga va salomatlik holatiga, ya‟ni ko„rish va eshitish tizimi organlari faoliyati va ularning darajasi, shuningdek sinfxonalarda joylashtirilishining gigienik talablarga mos kelmasligi oqibatida o„quvchilar oraisda jismoniy rivojlanishidan orqada qolish va qomatidagi o„zgarishlarning yuzaga kelish va xonalarning mikroiqlim va yoritilganlik darajasini etishmasligi holatlariga olib kelganligi aniqlandi. Maktab jixozlarini o„quvchilarning bo„yiga mos ravishda tanlash va foydalanishni tavsiya etish kerak. Distanitsiya va differensiyaning o„lchamlarini davriy ravishda maktab vrachi va o„qituvchilar tomonidan nazorat qilish kerak. Kun davomida davriy ravishda ertalabki badantarbiya, chiniqtirish muolajalari, jismoniy tarbiya va tavsiya etilgan 18 ta sport turlaridan yoshi, jinsi va salomatlik holatiga mos sport to„garaklarida shug„ullanishi tavsiya etiladi. 201 Involta Ilmiy Jurnali Vol. 1 No.4 (2022) 8. Straker L, et al. Mobile technology dominates school children‟s IT use in an advantaged school community and is associated with musculoskeletal and visual symptoms. Ergonomics. 2018;61(5):658–69. Involta Ilmiy Jurnali Vol. 1 No.4 (2022) Vol. 1 No.4 (2022) 1. Zorina I.G. Vliyanie faktorov vnutri shkolnoy sredы na umstvennuyu rabotosposobnost i sostoyanie zdorovya uchaщixsya pri raznыx formax obucheniya: Avtoref.dis. …kand.med.nauk. –Ekaterinburg, 2002. -18 s. 2. Kamilova R.T., Iskandarova SH.T., SHayxova G.I., Salixova N.S., Ermatov N.J. Gigienicheskie rekomendatsii po organizatsii rejima obucheniya i vospitaniya uchaщixsya obщeobrazovatelnыx shkol // Byulleten Assotsatsii vrachey Uzbekistana.-2004.- №3.-S.99-105. 3. Semeniщenkova T.A. Gigienicheskie aspektы formirovaniya prenevroticheskix otkloneniy u starshix shkolnikov // Gig.i san. -2003. -№4. –s.34- 37. 4. Suxarev A.G., Mixaylova S.A. Sostoyanie zdorovya detskogo naseleniya v napryajennыx ekologicheskix i sotsialnыx usloviyax // Gig.i san. -2004. -№1. – s.47-51. 5. SHayxova G.I. i dr. Bolalar va o„smirlar gigienasi. Tashkent.-2004.-S.416. 6. Ermatov N.J. Hozirgi islohotlar davrida zamonaviy maktablarda o„quv jarayonining tashkillashtirilishini gigienik baholash //O„zbekiston tibbiyot jurnali. №1, 2008. 67-71 sahifalar. 7. Alexander D, Lewis L . Condition of America‟s Public School Facilities: 2021-13. First Look. NCES 2014-022. National Center for Education Statistics, 2014. 8. Straker L, et al. Mobile technology dominates school children‟s IT use in an advantaged school community and is associated with musculoskeletal and visual symptoms. Ergonomics. 2018;61(5):658–69. 202
https://openalex.org/W4390890642	https://repozytorium.uwb.edu.pl/jspui/bitstream/11320/15589/1/I_Lojewska_Repozytorium_Uniwersytetu_w_Bialymstoku_w_liczbach.pdf	Polish	null	Repozytorium Uniwersytetu w Białymstoku w liczbach	Wydawnictwo Uniwersytetu w Białymstoku eBooks	2,023	cc-by	4,397	Iwona Łojewska Biblioteka Uniwersytecka im. Jerzego Giedroycia i.sztabkowska@uwb.edu.pl ORCID: 0000-0003-1190-7153 DOI: 10.15290/10latRUB.2023.12 DOI: 10.15290/10latRUB.2023.12 2 Repozytorium Uniwersytetu w Białymstoku zarejestrowało się w serwisie OpenDOAR 24.03.2014 roku. 1 Zob. OpenDOAR, dane z 21 lipca 2023 roku. 2 Repozytorium Uniwersytetu w Białymstoku zarejestrowało się w serwisie OpenDOAR 24.03.2014 roku. 1 Zob. OpenDOAR, dane z 21 lipca 2023 roku. 1 Zob. OpenDOAR, dane z 21 lipca 2023 roku. Repozytorium Uniwersytetu w Białymstoku w liczbach Przedmiotem badań jest analiza statystyczna Repozytorium Uniwersytetu w Białymstoku (RUB) w czasie 10 lat funkcjonowania. Scharakteryzowano RUB pod kątem zasobu bazy, typu dokumentów archiwizowanych na platformie oraz zakresu tematycznego materiałów. Analiza obejmowała również ocenę wkładu po- szczególnych jednostek Uniwersytetu w Białymstoku (UwB) w rozwój Repozytorium (porównanie pod względem liczby obiektów oraz przedstawienie listy Top 10 auto- rów). Kolejne kluczowe elementy, które zostały poddane charakterystyce, to: data wydania i język publikacji zdeponowanych w Repozytorium oraz identyfikatory DOI nadane obiektom cyfrowym. Przy wykorzystaniu narzędzia Google Analytics przed- stawiono geograficzną lokalizację użytkowników RUB według krajów, co pozwoliło na uświadomienie międzynarodowego zasięgu i oddziaływania Repozytorium. Słowa kluczowe: repozytorium instytucjonalne, Repozytorium Uniwersytetu w Białymstoku (RUB), zasób repozytorium, typy dokumentów, otwarte publika- cje, użytkownicy Jubileusz dziesięciolecia Repozytorium Uniwersytetu w Białymstoku (RUB) stanowi nie tylko okazję do świętowania, lecz także do głębokich refleksji nad osiągnięciami, które udało nam się zdobyć w ciągu dekady funkcjonowania. To wyjątkowy moment, który zachęca nas do dokonania wszechstronnego przeglądu oraz analizy statystycznej, aby zbadać jak rozwijała się nasza plat- forma zarówno na tle krajowego, jak i światowego środowiska repozytoriów 137 137 instytucjonalnych. Serwis OpenDOAR, który pełni rolę globalnego katalogu repozytoriów otwartego dostępu, zawiera obecnie dane o imponującej liczbie 6036 baz z całego świata. Wśród nich największy zbiór stanowią repozytoria instytucjonalne, których jest obecnie 5364 (co stanowi 88,87% wszystkich archiwów)1. 138 ymstoku. Wspólna droga do otwierania nauki 923 655 322 301 190 183 181 178 177 160 159 146 138 115 110 106 100 86 85 67 65 62 62 60 57 55 55 52 51 1135 0 200 400 600 800 1000 1200 Stany Zjednoczone Ameryki Japonia Wielka Brytania Niemcy Peru Hiszpania Turcja Indonezja Brazylia Francja Chorwacja Włochy Polska Kolumbia Ukraina Indie Kanada Australia Argentyna Norwegia Chiny Portugalia Serbia Tajwan Holandia Austria Meksyk Republika Południowej Afryki Republika Korei Inne Wykres 1. Repozytoria według krajów według danych z 21 lipca 2023 roku (źródło: opracowanie własne na podstawie OpenDOAR). Repozytorium Uniwersytetu w Białymstoku. Ws Wykres 1. Repozytoria według krajów według danych z 21 lipca 2023 roku (źródło: opracowanie własne na podstawie OpenDOAR). W momencie tworzenia RUB liczba wszystkich repozytoriów otwartego dostępu, obecnych w serwisie OpenDOAR, wynosiła 2481, a repozytoriów instytucjonalnych było wśród nich 2046 (dane z 22 października 2013 roku2). Powyższe dane wskazują, jak dynamicznie rozwija się w ostatnich latach ruch open access, a szczególnie zielona droga, czyli otwarte repozytoria. 3 Dokładnie 13 993, dane z 24 lipca 2023 roku. 4 Projekt realizowany od 14 lutego do 31 grudnia 2014 roku. Repozytorium Uniwersytetu w Białymstoku w liczbach Jest to odzwierciedlenie rosnącej świadomości naukowej społeczności i instytucji edukacyjnych na temat znaczenia szerokiego dostępu do wiedzy oraz potrze- by propagowania badań i osiągnięć naukowych na arenie międzynarodowej. W Polsce, według danych z OpenDOAR, funkcjonuje obecnie 138 baz. Wśród tych serwisów znajdują się zarówno repozytoria naukowe, jak również bi- blioteki cyfrowe i bazy wiedzy. Liczba ta wzrosła prawie dwukrotnie w ciągu ostatnich 10 lat (z 78 w roku 2013). Dane te nie tylko oddają ogromny postęp, jaki nastąpił w dziedzinie open access, ale również rzucają światło na rosnącą rolę Polski w budowaniu globalnej sieci badawczej. To znak, że nasza krajowa społeczność naukowa w pełni rozumie wartość współdzielenia wiedzy i otwar- tego dostępu do wyników badań. 426 218 167 141 108 79 78 75 1189 Stany Zjednoczone Ameryki = 17% Wielka Brytania = 9% Niemcy = 7% Japonia = 6% Hiszpania = 4% Francja = 3% Polska = 3% Brazylia = 3% Inne = 48% Wykres 2. Repozytoria open access według krajów – dane z 22 października 2013 roku (źródło: opracowanie własne na podstawie OpenDOAR). Wykres 2. Repozytoria open access według krajów – dane z 22 października 2013 roku (źródło: opracowanie własne na podstawie OpenDOAR). Przez ostatnią dekadę obserwowaliśmy wyjątkowo dynamiczny wzrost liczby publikacji zgromadzonych w bazie, co stanowi wyraz naszego zaangażo- wania w promowanie i udostępnianie naukowych osiągnięć. Obecnie możemy pochwalić się poziomem blisko 14 tysięcy dokumentów (wykres 3)3. 139 140 Repozytorium Uniwersytetu w Białymstoku. Wspólna droga do otwierania nauki 151 2097 3300 4387 5487 6618 7877 9049 11296 13133 13993 0 2000 4000 6000 8000 10000 12000 14000 16000 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 Wykres 3. Zasób Repozytorium Uniwersytetu w Białymstoku na przestrzeni lat (źródło: opracowanie własne). Już w ciągu trzymiesięcznego okresu próbnego (ostatni kwartał 2013 roku), w którym Repozytorium funkcjonowało w wewnętrznej sieci Uczelni, osiągnę- liśmy liczbę 151 prac. Świadczy to o skutecznej akcji promocyjnej naszej bazy w środowisku akademickim. Bardzo wyraźny wzrost zasobu odnotowaliśmy w roku 2014, gdy Biblioteka Uniwersytecka realizowała projekt ministerial- ny „Poszerzenie zasobów naukowych Biblioteki Uniwersyteckiej im. Jerzego Giedroycia w Białymstoku poprzez rozwój repozytorium instytucjonalnego Uniwersytetu w Białymstoku”4. Według złożonego wniosku, pierwotnie zapla- nowano zdeponowanie 900 publikacji naukowych (liczba ustalona na podstawie prac wprowadzonych w pierwszym roku funkcjonowania przez inne repozyto- ria w Polsce). Jednak nasze osiągnięcia przekroczyły te oczekiwania – udało się nam uzyskać ponad dwukrotnie wyższą liczbę wprowadzonych dokumen- tów – 1946 prac. Repozytorium Uniwersytetu w Białymstoku w liczbach W kolejnych latach, tj. od 2015 do 2020 roku, utrzymał się stały, rosnący trend, w którym corocznie dodawano ponad tysiąc nowych 4 Projekt realizowany od 14 lutego do 31 grudnia 2014 roku. 0 151 2097 3300 4387 5487 6618 7877 9049 11296 13133 13993 0 2000 4000 6000 8000 10000 12000 14000 16000 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 Wykres 3. Zasób Repozytorium Uniwersytetu w Białymstoku na przestrzeni lat (źródło: opracowanie własne). 140 ierania nauki 151 2097 3300 4387 5487 6618 7877 9049 11296 13133 13993 0 2000 4000 6000 8000 10000 12000 14000 16000 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 Wykres 3. Zasób Repozytorium Uniwersytetu w Białymstoku na przestrzeni lat (źródło: opracowanie własne). 140 Wykres 3. Zasób Repozytorium Uniwersytetu w Białymstoku na przestrzeni lat (źródło: opracowanie własne). Już w ciągu trzymiesięcznego okresu próbnego (ostatni kwartał 2013 roku), w którym Repozytorium funkcjonowało w wewnętrznej sieci Uczelni, osiągnę- liśmy liczbę 151 prac. Świadczy to o skutecznej akcji promocyjnej naszej bazy w środowisku akademickim. Bardzo wyraźny wzrost zasobu odnotowaliśmy w roku 2014, gdy Biblioteka Uniwersytecka realizowała projekt ministerial- ny „Poszerzenie zasobów naukowych Biblioteki Uniwersyteckiej im. Jerzego Giedroycia w Białymstoku poprzez rozwój repozytorium instytucjonalnego Uniwersytetu w Białymstoku”4. Według złożonego wniosku, pierwotnie zapla- nowano zdeponowanie 900 publikacji naukowych (liczba ustalona na podstawie prac wprowadzonych w pierwszym roku funkcjonowania przez inne repozyto- ria w Polsce). Jednak nasze osiągnięcia przekroczyły te oczekiwania – udało się nam uzyskać ponad dwukrotnie wyższą liczbę wprowadzonych dokumen- tów – 1946 prac. W kolejnych latach, tj. od 2015 do 2020 roku, utrzymał się stały, rosnący trend, w którym corocznie dodawano ponad tysiąc nowych prac. Kolejny dynamiczny wzrost nastąpił w latach 2021 oraz 2022. W roku 2021 osiągnęliśmy rekordową liczbę 2247 zgromadzonych prac. Na ten wynik wpłynęła pokaźna liczba publikacji pokonferencyjnych, które zostały prze- słane do Redakcji RUB. Natomiast wzrost zasobu w roku 2022 był związany z realizacją kolejnego projektu „Poszerzenie zasobów naukowych Biblioteki Uniwersyteckiej im. Jerzego Giedroycia w Białymstoku poprzez rozwój re- pozytorium instytucjonalnego Uniwersytetu w Białymstoku”5, który zakładał wprowadzenie 1200 publikacji wydanych przez Wydawnictwo Uniwersytetu w Białymstoku i wydawnictwo Temida 2. W roku 2022, w ramach powyższe- go projektu, zdeponowano w RUB 998 publikacji. Warto podkreślić, że nasze starania nie kończą się na tych osiągnięciach. Biblioteka Uniwersytecka bę- dzie kontynuowała projekt, który przyczyni się do dalszego upowszechniania i otwierania zasobów naukowych naszej Uczelni. 5 Projekt realizowany od października 2021 do końca lutego 2023 roku. Etap związany z de- ponowaniem prac rozpoczął się w marcu 2022 roku. 7 CEON Agregator, http://agregator.ceon.pl/Welcome.action (dostęp: 20.07.2023). 6 Projekt „Rozbudowa otwartych zasobów naukowych Repozytorium Uniwersytetu w B łymstoku – kontynuacja” będzie realizowany przez 14 miesięcy od października 2023 ro 5 Projekt realizowany od października 2021 do końca lutego 2023 roku. Etap związany z de- ponowaniem prac rozpoczął się w marcu 2022 roku. 6 Projekt „Rozbudowa otwartych zasobów naukowych Repozytorium Uniwersytetu w Bia łymstoku – kontynuacja” będzie realizowany przez 14 miesięcy od października 2023 roku. 7 CEON Agregator, http://agregator.ceon.pl/Welcome.action (dostęp: 20.07.2023). łymstoku kontynuacja będzie realizowany przez 14 miesięcy od października 2023 roku. 7 CEON Agregator, http://agregator.ceon.pl/Welcome.action (dostęp: 20.07.2023). Repozytorium Uniwersytetu w Białymstoku w liczbach Dzięki tym wysiłkom, w naj- bliższych miesiącach RUB wzbogaci się o kolejne 900 publikacji autorstwa pracowników Uniwersytetu6. Iwona Łojewska . Repozytorium Uniwersytetu w Białymstoku w liczbach Łączna wartość dofinansowania, które otrzymała Biblioteka Uniwersytecka na rozwój uczelnianego repozytorium, w ramach trzech projektów wyniosła 277 550 zł. Od lat Repozytorium Uniwersytetu w Białymstoku utrzymuje swoją pozy- cję w czołówce repozytoriów instytucjonalnych w Polsce według liczby doku- mentów. Rysunek 1 przedstawia zestawienie ze strony Agregatora CEON7, który jest wspólnym punktem dostępu do zasobów polskich repozytoriów. Na czele listy znajdują się serwisy pochodzące z dużych ośrodków badawczych o dłu- giej historii i ugruntowanej pozycji w kraju. W pierwszej trójce od lat mamy AMUR (pierwsze repozytorium instytucjonalne, które powstało w Polsce) oraz Repozytorium Uniwersytetu Łódzkiego. Baza tworzona na Uniwersytecie Jagiellońskim zdeklasowała pozostałych graczy, zdobywając pierwsze miejsce i stając się liderem, którego pozycji trudno będzie zagrozić. W tym zestawieniu RUB zajmuje obecnie zaszczytne siódme miejsce, co potwierdza duży wkład białostockiej Uczelni w zapewnieniu swobodnego dostępu do treści naukowych. 141 Dla porównania Rysunek 2 prezentuje stan Agregatora CEON w listopadzie 2014. Widzimy, że białostockie repozytorium od samego początku stanowiło istotny element polskiego krajobrazu repozytoriów instytucjonalnych. 142 Rysunek 1. Strona Agregatora CEON (lipiec 2023). epozytorium Uniwersytetu w Białymstoku. Wspólna droga do otw Rysunek 2. Strona Agregatora CEON (listopad 2014). Rysunek 2. Strona Agregatora CEON (listopad 2014). Wykres 4 prezentuje typy dokumentów archiwizowanych w RUB. Fundamentem zasobu są artykuły naukowe (7018 obiektów), które stanowią obecnie połowę wszystkich prac8. Już w początkowej fazie tworzenia na naszej Uczelni repozytorium zakładaliśmy, że zgromadzimy w nim tytuły periody- ków ukazujących się na Uniwersytecie. Powstał do tego oddzielny zespół – Czasopisma naukowe. Było to istotne ze względu na rozproszenie informacji o poszczególnych czasopismach oraz ich zawartości na stronach wydziałów, czy też na amatorsko tworzonych stronach domowych czasopism. Adresy tych witryn ulegały zmianie, a zdeponowanie artykułów w wersji cyfrowej w RUB gwarantowało (poprzez stosowanie unikalnych identyfikatorów URI) wieczy- stą archiwizację oraz indeksowanie w zewnętrznych serwisach, co pomaga w ich odnalezieniu przez innych badaczy za pomocą wyszukiwarek nauko- wych. Nie wszystkie numery czasopism posiadały swoje wersje elektroniczne, a jeżeli dostępne były pliki PDF, to często zawierały cały numer czasopisma. Repozytorium pozwalało na zdeponowanie poszczególnych artykułów opi- sanych za pomocą metadanych, co ułatwia ich wyszukiwanie i identyfikację oraz zwiększa dostępność do treści publikacji. 8 Jeszcze pięć lat temu artykuły naukowe stanowiły 72% zasobu RUB. W ostatnich la- tach można zauważyć wyraźny wzrost liczby innych typów dokumentów. Porównaj: A. Gałecka-Golec, I. Łojewska, Repozytorium Uniwersytetu w Białymstoku jako narzę- dzie gromadzenia, upowszechniania i promowania dorobku uczelni, [w:] S. Skórka (red.), E. Piotrowska (współpr.), Książki w plikach – publikowanie, udostępnianie i użytkowanie, Kraków 2021. Repozytorium Uniwersytetu w Białymstoku w liczbach Zdecydowaliśmy się również, po konsultacjach z władzami Uczelni i redakcjami czasopism, że artykuły obec- ne w RUB otrzymają identyfikatory DOI (Digital Object Identifier), które będą kierowały do adresu URL artykułu zdeponowanego w naszym Repozytorium. Dotyczyło to nie tylko numerów ukazujących się na bieżąco, ale również arty- kułów archiwalnych. Obecnie na platformie repozytorium znajdują się 24 tytuły czasopism wydawanych na Uniwersytecie w Białymstoku. Dla każdego tytułu przypisany jest oddzielny podzespół, gdzie można znaleźć podstawowe informa- cje o czasopiśmie: krótki opis, dane redaktora naczelnego, kontakt do redakcji, adres strony internetowej, numer ISSN, nazwę wydawcy, punktację ministerial- ną za poszczególne lata. Artykuły są pogrupowane w osobne kolekcje odpo- wiadające poszczególnym numerom czasopism. Sukcesywnie rośnie kolekcja rozdziałów z książek deponowanych w Repozytorium (obecnie 4525 obiektów, co stanowi 32% zasobu). Wzrost liczby tego rodzaju dokumentów związany jest 143 144 7018 709 4525 392 1349 0 1000 2000 3000 4000 5000 6000 7000 8000 Artykuły naukowe Książki Rozdziały z książek Prace doktorskie Inne Wykres 4. Zasób RUB według typów dokumentów (źródło: opracowanie własne). Wykres 4. Zasób RUB według typów dokumentów (źródło: opracowanie własne). z zamieszczaniem w ostatnich latach dużej liczby materiałów pokonferencyjnych oraz projektem realizowanym w latach 2021–2023, w którym rozdziały z ksią- żek stanowiły główny typ dokumentów. Książki (monografie autorskie, książki redakcyjne, monografie pokonferencyjne) stanowią obecnie 5% zasobu. Są to w głównej mierze prace opublikowane w oficynach uczelnianych: 226 książek z Wydawnictwa Uniwersytetu w Białymstoku, 129 z Wydawnictwa Temida 2, 35 tytułów z Trans Humany oraz 4 z Wydawnictwa Prawo i Partnerstwo. Rozprawy doktorskie, stanowiące ważny nurt akademickiego dorobku, również zajmują istotne miejsce w naszym Repozytorium. Obecnie stanowią one 3% całkowitej kolekcji. Z 394 dysertacji obronionych od października 2013 roku, 185 prac jest dostępnych tylko dla uprawnionych użytkowników korzystających z komputerów Biblioteki Uniwersyteckiej, podczas gdy z 209 rozpraw doktor- skich mogą korzystać wszyscy przez Internet. Pozostałe publikacje zdeponowane w Repozytorium, takie jak recenzje książek, prezentacje, sprawozdania, wstępy, indeksy, wspomnienia itp., zostały przydzielone do kategorii Inne dokumenty i stanowią 10% całości. z zamieszczaniem w ostatnich latach dużej liczby materiałów pokonferencyjnych oraz projektem realizowanym w latach 2021–2023, w którym rozdziały z ksią- żek stanowiły główny typ dokumentów. Książki (monografie autorskie, książki redakcyjne, monografie pokonferencyjne) stanowią obecnie 5% zasobu. Są to w głównej mierze prace opublikowane w oficynach uczelnianych: 226 książek z Wydawnictwa Uniwersytetu w Białymstoku, 129 z Wydawnictwa Temida 2, 35 tytułów z Trans Humany oraz 4 z Wydawnictwa Prawo i Partnerstwo. 9 Grafika stworzona w aplikacji WordCloud, https://www.wordclouds.com/. Repozytorium Uniwersytetu w Białymstoku w liczbach Rozprawy doktorskie, stanowiące ważny nurt akademickiego dorobku, również zajmują istotne miejsce w naszym Repozytorium. Obecnie stanowią one 3% całkowitej kolekcji. Z 394 dysertacji obronionych od października 2013 roku, 185 prac jest dostępnych tylko dla uprawnionych użytkowników korzystających z komputerów Biblioteki Uniwersyteckiej, podczas gdy z 209 rozpraw doktor- skich mogą korzystać wszyscy przez Internet. Pozostałe publikacje zdeponowane w Repozytorium, takie jak recenzje książek, prezentacje, sprawozdania, wstępy, indeksy, wspomnienia itp., zostały przydzielone do kategorii Inne dokumenty i stanowią 10% całości. Repozytorium charakteryzuje się bogactwem treści oraz różnorodnością dziedzin wiedzy, które znajdują się w jego zasobach. Analizując zakres tematycz- ny publikacji zgromadzonych w RUB wykorzystano słowa kluczowe użyte przez Rysunek 3. Zasób RUB według słów kluczowych (źródło: opracowanie własne). Iwona Łojewska . Repozytorium Uniwersytetu w Białymstoku w l Iwona Łojewska . Repozytorium Uniwersytetu w Białymstoku w liczbach Rysunek 3. Zasób RUB według słów kluczowych (źródło: opracowanie własne). autorów do opisu swoich prac. Rysunek 39 prezentuje 30 tematów, które wystę- pują w bazie najczęściej. Recenzje książek są stałym elementem wielu tytułów czasopism zarchiwizowanych w Repozytorium. To słowo kluczowe występuje w metadanych aż 330 razy. Badania nad procesem kształcenia również stano- wią istotny obszar zainteresowań autorów prac. Termin education występuje 89 razy, edukacja – 55 oraz edukacja międzykulturowa – 50. Słowa kluczowe związane z regionem, takie jak Białystok (74 wystąpienia), Podlasie (68), hi- storia Podlasia (45), są również licznie obecne w metadanych. Odnoszą się one do lokalnej perspektywy i skupiają na historii badanego obszaru. Również pojęcia identity (78 wystąpień), tożsamość (53), multiculturalism (51), prawo- sławie (45) oraz wielokulturowość (45) sugerują obecność badań i analiz zwią- zanych z kulturą, religią oraz różnorodnością kulturową regionu. Rozszerzając analizę na pozostałe terminy, takie jak Poland (95), poetry (83), sustainable development (76), family (66), Russia (63), space (62), można założyć, że RUB jest z pewnością wartościowym źródłem wiedzy dla różnych grup odbiorców. autorów do opisu swoich prac. Rysunek 39 prezentuje 30 tematów, które wystę- pują w bazie najczęściej. Recenzje książek są stałym elementem wielu tytułów czasopism zarchiwizowanych w Repozytorium. To słowo kluczowe występuje w metadanych aż 330 razy. Badania nad procesem kształcenia również stano- wią istotny obszar zainteresowań autorów prac. Termin education występuje 89 razy, edukacja – 55 oraz edukacja międzykulturowa – 50. Słowa kluczowe związane z regionem, takie jak Białystok (74 wystąpienia), Podlasie (68), hi- storia Podlasia (45), są również licznie obecne w metadanych. Odnoszą się one do lokalnej perspektywy i skupiają na historii badanego obszaru. Repozytorium Uniwersytetu w Białymstoku w liczbach Również pojęcia identity (78 wystąpień), tożsamość (53), multiculturalism (51), prawo- sławie (45) oraz wielokulturowość (45) sugerują obecność badań i analiz zwią- zanych z kulturą, religią oraz różnorodnością kulturową regionu. Rozszerzając analizę na pozostałe terminy, takie jak Poland (95), poetry (83), sustainable development (76), family (66), Russia (63), space (62), można założyć, że RUB jest z pewnością wartościowym źródłem wiedzy dla różnych grup odbiorców. Struktura repozytorium ma budowę hierarchiczną, w pewnym stopniu odzwierciedlającą strukturę Uniwersytetu w Białymstoku. Podczas 10 lat 145 funkcjonowania RUB wchodzące w jej skład elementy były zmieniane i dosto- sowywane do aktualnych potrzeb. Wiązało się to między innymi ze zmianami w samej strukturze Uczelni (zmiana wydziałów, instytutów). Biorąc pod uwagę liczbę publikacji ze względu na jednostki uczelniane zauważamy, że zasób na- szej bazy opiera się na dziedzinach humanistycznych i społecznych. Najwięcej prac zdeponowano w zespole Wydział Filologiczny (1936 dokumentów). Wiąże się to między innymi z liczbą czasopism wydawanych na tym wydzia- le – sześć tytułów – oraz wieloma organizowanymi konferencjami, których wyniki w formie publikacji przekazywane są do RUB. Pracownicy naukowi z Wydziału Filologicznego są najbardziej aktywnymi depozytariuszami na- szej bazy. Można było to dostrzec w czasie ostatniej ewaluacji działalności 146 120 82 119 245 107 79 166 45 42 36 48 15 702 1936 9 1042 8 436 1618 0 500 1000 1500 2000 Biblioteki Instytut Filozofii Instytut Informatyki Instytut Socjologii Instytut Studiów Kulturowych Instytut Zarządzania Katedra Teologii Katolickiej Katedra Teologii Prawosławnej Szkoły Doktorskie Wydział Biologii Wydział Chemii Wydział Ekon.-Inf. w Wilnie Wydział Ekonomii i Finansów Wydział Filologiczny Wydział Fizyki Wydział Historii i Stosunków Międzynarodowych Wydział Matematyki Wydział Nauk o Edukacji Wydział Prawa Wykres 5. Liczba publikacji według jednostek Uniwersytetu w Białymstoku (źródło: opracowanie własne). Repozytorium Uniwersytetu w Białymstoku. Wspólna droga do otwierania nauki 120 82 119 245 107 79 166 45 42 36 48 15 702 1936 9 1042 8 436 1618 0 500 1000 1500 2000 Biblioteki Instytut Filozofii Instytut Informatyki Instytut Socjologii Instytut Studiów Kulturowych Instytut Zarządzania Katedra Teologii Katolickiej Katedra Teologii Prawosławnej Szkoły Doktorskie Wydział Biologii Wydział Chemii Wydział Ekon.-Inf. w Wilnie Wydział Ekonomii i Finansów Wydział Filologiczny Wydział Fizyki Wydział Historii i Stosunków Międzynarodowych Wydział Matematyki Wydział Nauk o Edukacji Wydział Prawa Wykres 5. Liczba publikacji według jednostek Uniwersytetu w Białymstoku (źródło: opracowanie własne). Wykres 5. Liczba publikacji według jednostek Uniwersytetu w Białymstoku (źródło: opracowanie własne). naukowej za lata 2017−2021, podczas której filolodzy systematycznie depono- wali swoje publikacje. Repozytorium Uniwersytetu w Białymstoku w liczbach Kolejne jednostki, których pracownicy deponują najwię- cej publikacji, to Wydział Prawa (1618 prac) oraz Wydział Historii i Stosunków Międzynarodowych (1042 publikacji). Warto również zwrócić uwagę na mniej- sze jednostki, których zasób jest pokaźny. Katedra Teologii Katolickiej, któ- ra obecnie składa się tylko z trzech pracowników, ma zdeponowanych 166 dokumentów. Natomiast 120 publikacji znajduje się w zespole Biblioteki. Dostrzegamy tu znaczący wkład bibliotekarzy w rozwój RUB, którzy prze- cież nie zajmują się na co dzień działalnością naukową i publikacyjną. Wśród przedstawicieli jednostek z nauk ścisłych i przyrodniczych widzimy mniej- sze zainteresowanie możliwością deponowania w uczelnianym Repozytorium. W tych dziedzinach wiedzy podstawowym kanałem komunikacji naukowej jest publikowanie w czasopismach, a archiwizacja w repozytoriach nie jest tak powszechna. Iwona Łojewska . Repozytorium Uniwersytetu w Białymstoku w liczbach p W pierwszej dziesiątce autorów, których prac mamy w Repozytorium najwięcej, jest trzech filologów i trzech histo- ryków. Już od pierwszego roku funkcjonowania bazy na czele zestawienia jest dr hab. Józef Maroszek, prof. UwB, który zdeponował na platformie aż 165 prac. Jako autor oraz wieloletni redaktor czasopi- sma historycznego „Studia Podlaskie” jest propagatorem idei otwartego dostępu i rozu- mie potrzebę jak najszersze- go udostępniania publikacji w Internecie. Pozostali dwaj autorzy na liście top 10 to: Cezary Kulesza (Wydział Prawa) oraz ks. Andrzej Proniewski (Katedra Teologii Katolickiej). Wśród na- zwisk widocznych na liście mamy również dwóch autorów spoza kręgu pra- cowników Uniwersytetu w Białymstoku. Są to Yasunari Shidama oraz Hiroyuki Okazaki – naukowcy z Japonii systematycznie publikujący w kwartalniku „Formalized Mathematics”, wydawanym na naszej Uczelni. Rysunek 4. Top 10 autorów w RUB (źródło: https://repozytorium.uwb.edu.pl/jspui/). Rysunek 4. Top 10 autorów w RUB źródło: https://repozytorium.uwb.edu.pl/jspui/). W pierwszej dziesiątce autorów, których prac mamy w Repozytorium najwięcej, jest trzech filologów i trzech histo- ryków. Już od pierwszego roku funkcjonowania bazy na czele zestawienia jest dr hab. Józef Maroszek, prof. UwB, który zdeponował na platformie aż 165 prac. Jako autor oraz wieloletni redaktor czasopi- sma historycznego „Studia Podlaskie” jest propagatorem idei otwartego dostępu i rozu- mie potrzebę jak najszersze- go udostępniania publikacji w Internecie. Pozostali dwaj autorzy na liście top 10 to: Cezary Kulesza (Wydział Prawa) oraz ks. Andrzej Proniewski (Katedra Teologii Katolickiej). Wśród na- zwisk widocznych na liście mamy również dwóch autorów spoza kręgu pra- cowników Uniwersytetu w Białymstoku. Są to Yasunari Shidama oraz Hiroyuki Okazaki – naukowcy z Japonii systematycznie publikujący w kwartalniku „Formalized Mathematics”, wydawanym na naszej Uczelni. Rysunek 4. Top 10 autorów w RUB (źródło: https://repozytorium.uwb.edu.pl/jspui/). Repozytorium Uniwersytetu w Białymstoku w liczbach W pierwszej dziesiątce autorów, których prac mamy w Repozytorium najwięcej, jest trzech filologów i trzech histo- ryków. Już od pierwszego roku funkcjonowania bazy na czele zestawienia jest dr hab. Józef Maroszek, prof. UwB, który zdeponował na platformie aż 165 prac. Jako autor oraz wieloletni redaktor czasopi- sma historycznego „Studia Podlaskie” jest propagatorem idei otwartego dostępu i rozu- mie potrzebę jak najszersze- go udostępniania publikacji w Internecie. Pozostali dwaj auto Prawa) oraz ks. Andrzej Proniew zwisk widocznych na liście ma cowników Uniwersytetu w Biały Okazaki – naukowcy z Japoni Formalized Mathematics” wyd Rysunek 4. Top 10 autorów w RUB (źródło: https://repozytorium.uwb.edu.pl/jspui/). Rysunek 4. Top 10 autorów w RUB (źródło: https://repozytorium.uwb.edu.pl/jspui/). 147 Zakres chronologiczny dokumentów zarchiwizowanych w RUB obejmu- je lata 1974–2023. Warto zaznaczyć, że publikacje najnowsze, wydane od 2020 wynoszą 19% wszystkich prac naukowych obecnych w bazie. Jest to szczególnie ważne, ponieważ użytkownicy bardzo często szukają aktualnych danych i publikacji. Największy zbiór – 8980 obiektów cyfrowych – stano- wią prace z lat 2010–2019. Jest to 64% całego zasobu Repozytorium. Liczba publikacji z lat 2000–2009 wynosi 1996, a 3% zarchiwizowanych obiektów (329 dokumentów) powstało w latach 90. ubiegłego wieku. Pozostały zasób to 40 publikacji z lat 1980–1989 oraz dwie z lat 70. XX wieku. Podsumowując, zasób naszego Repozytorium zawiera materiały wydane na przestrzeni 50 lat, co stanowi bogate źródło informacji dla badań i celów edukacyjnych. Szczególnie cenna jest znaczna liczba publikacji najnowszych. 148 tu w Białymstoku. Wspólna droga do otwierania nauki 2646 8980 1996 329 40 2 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 2020–2023 2010–2019 2000–2009 1990–1999 1980–1989 1970-1979 Wykres 6. Zasób RUB według daty wydania publikacji (źródło: opracowanie własne). Repozytorium Uniwersytetu w Białymstoku. Wspólna droga do otw Wykres 6. Zasób RUB według daty wydania publikacji (źródło: opracowanie własne). Kluczowym narzędziem dla efektywnego zarządzania publikacjami na- ukowymi, które ułatwia odnajdywanie, prawidłowe cytowanie i analizę wpły- wu w świecie nauki, są identyfikatory DOI. Od 2014 roku Redakcja RUB zarejestrowała, w firmie Crossref, 7319 identyfikatorów DOI dla publikacji dostępnych w Repozytorium. Największy zbiór obiektów cyfrowych, które otrzymały numery DOI to artykuły z czasopism – 6936, następnie materiały konferencyjne (poszczególne rozdziały z publikacji pokonferencyjnej oraz całe książki) – 201. Dla książek redakcyjnych zarejestrowaliśmy 176 numerów DOI (podobnie jak w przypadku materiałów konferencyjnych unikalny identyfikator cyfrowy otrzymały poszczególne rozdziały oraz publikacja redakcyjna jako całość). Repozytorium Uniwersytetu w Białymstoku w liczbach Monografiom autorskim przypisano 5 numerów DOI oraz 1 publikacji w formie preprintu (wstępna wersja publikacji naukowej przed wydaniem). Identyfikatory są finansowane z subwencji Rektora. Kluczowe warunki otrzy- mania identyfikatorów do publikacji to afiliacja autora oraz umieszczenie ma- teriałów w wersji cyfrowej w RUB. Iwona Łojewska . Repozytorium Uniwersytetu w Białymstoku w liczbach Blisko 75% obiektów cyfrowych zarchiwizowanych w Repozytorium opublikowano w języku polskim, natomiast 16% prac (2289 publikacji) re- prezentuje język angielski. Warto zwrócić uwagę na obecność dużego zbioru prac w języku rosyjskim – 560 dokumentów oraz białoruskim – 366 prac. Na te wartości wpłynęły zapewne artykuły publikowane w trzech czasopismach uczelnianych: „Białorutenistyka Białostocka”, „Linguodidactica” oraz „Studia Wschodniosłowiańskie” oraz wieloletnia działalność publikacyjna pracowni- ków Instytutu Filologii Wschodniosłowiańskiej. Wśród zasobu Repozytorium znajduje się także niewielki odsetek, tj. 2% obiektów, napisanych w innych językach. 10479 2289 560 366 116 98 45 25 12 3 0 2000 4000 6000 8000 10000 12000 polski angielski rosyjski białoruski francuski inny niemiecki włoski hiszpański litewski Wykres 7. Zasób RUB według języka publikacji (źródło: opracowanie własne). 10479 2289 560 366 116 98 45 25 12 3 0 2000 4000 6000 8000 10000 12000 polski angielski rosyjski białoruski francuski inny niemiecki włoski hiszpański litewski Wykres 7. Zasób RUB według języka publikacji (źródło: opracowanie własne). 149 150 Repozytorium Uniwersytetu w Białymstoku. Wspólna droga do otwierania nauki Wykres 8. Lokalizacja użytkowników RUB według kraju (źródło: Google Analytics) Repozytorium Uniwersytetu w Białymstoku. Wspólna droga do otwierania nauki Wykres 8. Lokalizacja użytkowników RUB według kraju (źródło: Google Analytics). Analizując informacje o kraju pochodzenia użytkowników RUB, wy- korzystano serwis Google Analytics. Do wykonania wykresu użyto danych za ostatnie pięć lat (analiza na podstawie 1 5304 896 sesji przeprowadzonych między 1 czerwca 2018 a 30 czerwca 2023 roku). Statystyki użytkowania wskazują, że najwięcej sesji (50,71%) zostało przeprowadzonych z terytorium Polski. Ponad 20% użytkowników RUB to mieszkańcy Stanów Zjednoczonych, a kolejne lokalizacje to Rosja (3,17%), Niemcy (2,33%) i Ukraina (0,87%). Dowodzi to, że zasięg RUB nie ogranicza się jedynie do granic naszego kraju. Nasze zasoby stanowią źródło informacji również dla użytkowników z różnych zakątków świata. Iwona Łojewska . Repozytorium Uniwersytetu w Białymstoku w licz Iwona Łojewska . Repozytorium Uniwersytetu w Białymstoku w liczbach Podsumowując, systematyczny rozwój Repozytorium Uniwersytetu w Białymstoku na przestrzeni 10 lat to dynamiczny proces, który przyczynił się do wzrostu ilości zgromadzonych zasobów, zwiększenia widoczności naszej instytucji oraz wzmocnienia jej pozycji w świecie nauki. Repozytorium Uniwersytetu w Białymstoku w liczbach Repozytorium stało się kluczowym narzędziem promującym badania Uniwersytetu i wspierającym wymianę wiedzy w społeczności akademickiej. Było to możliwe dzięki zaan- gażowaniu i owocnej współpracy środowiska uniwersyteckiego: pracowników naukowych, doktorantów, studentów, zespołu redakcyjnego odpowiedzialnego za obsługę bazy, administratora systemu, współpracowników odpowiedzialnych za digitalizację zbiorów, redaktorów czasopism akademickich i pracowników wydawnictw uczelnianych. Jubileusz dziesięciolecia to więc nie tylko moment radości i dumy z dotychczasowych osiągnięć, ale także punkt wyjścia do dal- szych wyzwań i celów. Dzięki współpracy, innowacji i stałemu dążeniu do doskonalenia mamy nadzieję, że kolejna dekada przyniesie jeszcze większe sukcesy i wyjątkowe możliwości dla naszej społeczności akademickiej, a po- przez uczelniane Repozytorium zasili zasoby globalnej sieci otwartej nauki. CEON Agregator, http://agregator.ceon.pl/Welcome.action (dostęp: 20.07.2023). Gałecka-Golec A., Łojewska I., Repozytorium Uniwersytetu w Białymstoku jako narzędzie gromadzenia, upowszechniania i promowania dorobku uczelni, [w:] S. Skórka (red.), E. Piotrowska (współpr.), Książki w plikach – publi- kowanie, udostępnianie i użytkowanie, Kraków 2021. Bibliografia 151
https://openalex.org/W2962172702	https://sciencepubco.com/index.php/ijet/article/download/12478/8054	English	null	Experimental and numerical evaluations of Kirkuk field soil treated with waste shredded tire	International journal of engineering & technology	2,018	cc-by	6,574	1. Introduction Waste material is any kind of material that is the output of human and industrial action with no living value. The increasing numbers and categories of waste materials, lack of landfill spaces, and shortage of natural earth materials emphasize on the necessity of discovering advanced ways of reusing and recycling these waste materials [1]. In addition, reutilizing with subsequent reuse of waste materials can decrease the need for natural resources, which can eventually direct to a more sustainable environment [2]. The tire chips are generally between 12 mm to 50 mm in size whereas tire shreds are larger [3]. The specific gravity of tire chips and tire shreds varies from 1.02 to 1.26 depending on the quantity of steel belt in the tire [8-10]. y The problem of waste tires is growing daily all over the world. For instance, in the United States, more than 250 million waste tires are stockpiled and currently 5 billion tires are stockpiled [3], and more than 28 million passenger car tires are discarded in Canada [4]. Based on the data of the department of land transport and in the case of Thailand specifically, an accumulated number of regis- tered cars by the 31st of December 2009 was almost 27 million [5]. Thus, if each vehicle needs changing tires every 3 years, then each year the waste tires would approximate 36 million. Consider- ing these enormous numbers, it can be noticed that Thailand and any other country having a similar situation needs to think sensi- bly about how to manage the waste tires in the near future. In ad- dition, stockpiling of waste scrap tires have the potential of fire hazard leading to environmental harm and a good breeding home for disease-carrying insects and vermin [6]. Hence, finding new and useful ways to reuse and recycle large volumes of waste tires are required. Tire waste can be used as a lightweight material in different forms such as chips, powder, and shredded tires where Waste tires products represented by tire shreds, tire bales, crumb and ground rubber is used in a variety of engineering purposes such as embankments, backfills, subgrades, erosion control, and asphalt mixture design [11-17]. The waste tire maintains a high damping characteristic that has the benefit to be used in the em- bankment and railroad track to decrease the disturbance of close by residents [10-18]. Abstract In this study, an experimental and numerical investigation of Kirkuk real field soil treated with waste tire has been examined. Field soil samples from Kirkuk city have been collected and tested experimentally to evaluate the basic soil properties. The field soil has been treated with waste shredded tires and up to 10%. A series of direct shear tests under different normal stresses and unconfined compres- sion tests with two different rates have been performed on both untreated and waste tire treated soils. For the untreated soil, the maxi- mum shear stress measured by the direct shear test increased by 150% when the normal shear stress increased from 50 kPa to 150 kPa. For the 5% and 10% waste tire treated soils, the maximum shear stresses measured by the direct shear test increased by 110% and 105% when the normal stress increased from 50 kPa to 150 kPa respectively. The peak uniaxial stress measured by the unconfined compression test increased by 83% and 98% as the waste tire treatment increased from 0% to 10% for both testing rates of 0.125 mm/min and 0.25 mm/min respectively. Finally, finite element method using three different models represented by elastic, hyperbolic and Mohr-Coulomb elastic-plastic models have been used to model unconfined compression tests for both untreated and 10% waste tire treated soils. For both untreated and waste tire treated soils, the elastic model over predicted the shear stress versus shear strain relationship whereas the elastic-plastic model had a very good agreement with the experimental data. However, the hyperbolic model had a good prediction for the initial part of the shear stress versus shear strain relationship for both untreated and waste tire treated soils with an overestimation for the second part of the experimental data. Keywords: Field Kirkuk Soil; Waste Tire; Direct Shear; Unconfined Compression; Finite Element; Elastic Model; Hyperbolic Model; Mohr-Coulomb. Keywords: Field Kirkuk Soil; Waste Tire; Direct Shear; Unconfined Compression; Finite Element; Elastic Model; Hyperbolic Mo rkuk Soil; Waste Tire; Direct Shear; Unconfined Compression; Finite Element; Elastic Model; Hyperbolic Model; Mohr-Coulomb. any type of these forms is used above or below the ground water table [7]. Scrap tires have been used in geotechnical application particularly as embankment materials since scrap tires are cheaper than other alternatives. An estimation of over 13.5 million tons of scrap tires is stockpiled of every single year worldwide [8]. Experimental and numerical evaluations of Kirkuk field soil treated with waste shredded tire Aram Mohammed Raheem * Ph.D., Civil Engineering Department, University of Kirkuk, Kirkuk, Iraq Corresponding author E-mail: engaram@yahoo.com Ph.D., Civil Engineering Department, University of Kirkuk, Kirkuk, Iraq Corresponding author E-mail: engaram@yahoo.com Abstract Plac- ing 15 to 30 cm of shredded waste tires can preserve the subgrade soil from thawing in spring. In addition, the waste tires have a high permeability that allows draining the water from beneath the roads avoiding the road surface damage. Copyright © 2018 Aram Mohammed Raheem. This is an open access article distributed under the Creative Commons permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Engineering & Technology Website: www.sciencepubco.com/index.php/IJET doi: 10.14419/ijet.v7i3.12478 Research paper International Journal of Engineering & Technology, 7 (3) (2018) 1768-1775 International Journal of Engineering & Technology Website: www.sciencepubco.com/index.php/IJET doi: 10.14419/ijet.v7i3.12478 Research paper 1. Introduction The main objective of this study is to check the effectiveness of waste tires on the behavior of field soil obtained from Kirkuk city. The main objective of this study is to check the effectiveness of waste tires on the behavior of field soil obtained from Kirkuk city. Particularly, it is required to measure the effect of waste tires on the shear strength behavior of field soil from Al-Sayada location near the campus of the University of Kirkuk. In addition, finite element analysis has been incorporated using three different mod- els represented by elastic, hyperbolic and Mohr-Coulomb models to study the behavior of untreated and waste treated field soil. : Disturbed Samples at Depth of 0.5 M below the Ground Level. 3) Laboratory experiments The water content of the soil samples has been determined accord- ing to ASTM 2216 [22]. The specific gravity of the soil was calcu- lated based on the ASTM D854-02 [23]. The particle size distribu- tion of the field soil has been evaluated using ASTM D422-63 [24]. Atterberg limits have been determined according to ASTM D4318-00 [25]. The relationship between water content and dry unit weight was carried out according to ASTM D698-00a [26]. The shear strength of untreated and waste tire treated soils have been tested using direct shear device according to ASTM D3080 [27]. In addition, the unconfined strength of untreated and waste tire treated soils have been performed based on ASTM D2166 [28]. 4) Waste tire 1) Site The soil for the study has been chosen from the University of Kir- kuk location at Al-Sayada as shown in Figs. 1 and 2 respectively. 2) D illi d li y 2) Drilling and sampling ) g p g The soil was collected from Kirkuk city (University of Kirkuk), which is located in the Kirkuk Governorate at the north of Iraq. The samples were taken from a depth of 0.5 m below the natural ground level using simple manual tools as shown in Fig. 3. The top layer of soil was removed, and then disturbed samples were obtained and transported to the soil mechanics laboratory of the Civil Engineering Department at the University of Kirkuk. 4) Waste tire The waste tires have been shredded in very small sizes using me- chanical tools in order to make the size of the shredded tires as the same as the particle size of the filed soil. A different percentage of waste shredded tires has been mixed with the field soil and up to 10% of the total weight of the tested soil samples. Fig. 1: The Map of Kirkuk City. 5) Finite element analysis (FEA) 1. Introduction Ahmed [9] performed several triaxial tests on tire chips mixed with Ottawa sand and found that the apparent cohesion increases while friction angle decreases with increasing tire chips in the mix. Adding 25% of the tire chips to sand with size ranges from 20 mm to 80 mm increases the shear strength (obtained from di- rect shear test) slightly at low normal stresses [10]. Foose et al. [8] conducted a study on sand reinforced with shredded waste tires with sizes of 6 mm to 50 mm in direct shear test and revealed that Mohr strength envelopes were non-linear and were affected by International Journal of Engineering & Technology 1769 Fig. 2: The Location of the University of Kirkuk. shred contents, normal stress and sand matrix unit weight. It was observed from the stress-strain relationship of a mixture of sand and tire chips that the dilatancy behavior of rubber sand is in be- tween pure sand and pure chips [3]. Triaxial compression tests on sand tire mixture have shown that the shear strength is increased and the compressibility is decreased when there is an increase in the proportion of tire chips or the density [6]. Ghazavi and Sakhi [19] revealed that the affecting parameters on the shear strength characteristics of the sand-shred mixture are sand matrix unit weight, normal stress, shred width, shred content, and aspect ratio of tire shreds. It was reported that initial stiffness decreases with increasing tire chip content in the mixture and increases linearly with the confining pressure [20]. In addition, scrap tires could be used as cushions around building to absorb the vibrating energy generated by an earthquake [21]. Waste tires have been a serious problem facing the entire world for the last 20 to 30 years. By some estimation, Kirkuk city damps a very high percentage of waste tires with no reusing or recycling. Around the world, several previous studies have been attempted to solve this problem in particular treating the soil with waste tires. No studies have addressed the waste tire problem in Iraq generally and Kirkuk specifically. In addition, all the earlier studies have focused on the experimental aspects with no numerical investiga- tion of the behavior of waste tire treated soil. Fig. 2: The Location of the University of Kirkuk. Fig. 2: The Location of the University of Kirkuk. Fig. 3: Disturbed Samples at Depth of 0.5 M below the Ground Level. Where Rf = failure ratio, a value typically between 0.5 and 0.9. Soil strength can be related to confining stress through the Mohr- Coulomb yield criterion: (3) (σ1-σ3)f = (2c cos∅+2σ3 sin∅)/ (1-sin∅) (3) Where c = soil cohesive intercept;  = soil friction angle; and 3 = minor principal stress, which is related to the confining stress imposed on the soil. The failure envelope is considered to be curved by proposing another parameter Δ, which is the change in friction angle because of an increase in 3. Table 2: Physical Soil Properties Property Value Water content 8.4% Specific gravity 2.68 Effective size, D10 0.1 mm Coefficient of uniformity, Cu 11.8 Coefficient of curvature, Cc 0.76 Liquid limit 23.9 Plastic limit 4.9 Table 2: Physical Soil Properties Property Value Water content 8.4% Specific gravity 2.68 Effective size, D10 0.1 mm Coefficient of uniformity, Cu 11.8 Coefficient of curvature, Cc 0.76 Liquid limit 23.9 Plastic limit 4.9 Fig. 4: Grain Size Distribution of the Field Soil. Fig. 5: Liquid Limit Evaluation. The initial tangent modulus is proportional to the confining stress according to: Ei=KPa (σ3/Pa)n (4) (4) 5) Finite element analysis (FEA) [29] proposed the hyperbolic model that used to validate the nonlinear elastic model with a reasonably representative for the soil behaviour. The hyperbolic model pa- rameters can be determined using triaxial compression tests. The hyperbolic model cannot model neither the strain softening behav- iour nor plastic failure. In addition, the hyperbolic model cannot characterize the dilatant behaviour or the lateral compression and axial extension. Based on the hyperbolic model, the deviatoric stress (1-3) is related to axial strain  as follows: (1-σ3) = ε / (1/ Ei + ε / (σ1-σ3)ult) (1) (1) where (1-3)ult = ultimate (asymptotic) value of deviatoric stress; and Ei = initial tangent Young’s modulus. The ultimate deviatoric stress is related to the deviatoric stress at “failure” (soil strength) through: 5) Finite element analysis (FEA) A two dimensional finite element analysis of untreated and waste tire treated field soil have been investigated to verify the experi- ment test results and understand the deformation of the soil mass where axisymmetric condition was exhibited. The real untreated and waste tire treated soils have been modelled using the data obtained from the laboratory soil experiments. The behaviour of the untreated and tire treated real field soils were simulated using different soil models such as elastic, hyperbolic and elastic-plastic models. The simulation concentrates on modelling the unconfined compression tests performed on both untreated and tire treated field soils. A two dimensional finite element analysis of untreated and waste tire treated field soil have been investigated to verify the experi- ment test results and understand the deformation of the soil mass where axisymmetric condition was exhibited. The real untreated and waste tire treated soils have been modelled using the data obtained from the laboratory soil experiments. The behaviour of the untreated and tire treated real field soils were simulated using different soil models such as elastic, hyperbolic and elastic-plastic models. The simulation concentrates on modelling the unconfined compression tests performed on both untreated and tire treated field soils. The simplest used soil model is the linear elastic model where the stresses are directly proportional to the strains. The proportionality constants are both Young's Modulus “E” and Poisson's Ratio “ν”. The hyperbolic model is used to model the nonlinear elastic stress- strain behaviour of the materials. It provides an adequate predic- tion of the soil at fairly low shear stress levels. The hyperbolic model is developed in the manner that the soil stiffness increases Fig. 1: The Map of Kirkuk City. International Journal of Engineering & Technology 1770 Hyperbolic (Nonlinear- Elastic) Unit weight (kN/m3) 20 19 Young’s modulus, E (kN/m2) 5000 15000 Poisson’s ratio, v 0.4 0.4 Cohesion (kN/m2) 10 15 Angle of internal friction, o 25 30 Failure ratio, Rf 0.7 0.7 Elastic-Plastic (Mohr- Coulomb) Unit weight (kN/m3) 20 19 Young’s modulus, E (kN/m2) 5000 15000 Poisson’s ratio, v 0.4 0.4 Cohesion (kN/m2) 10 15 Angle of internal friction, o 25 30 Dilation angle, o 20 20 with increasing confining pressure and decreases with increasing shear stress level. The soil stiffness exhibits a very low value at the failure. Duncan et al. 1) Physical soil properties ) y p p The collected field soil samples have been tested in the laboratory and the results are summarized in Table 2. In addition, the particle size distribution of field soil has been shown in Fig. 4. The liquid limit evaluation is shown in Fig. 5. The soils can be classified as clayey sands “sand-clay mixtures” (SC) according to USCS (uni- fied soil classification system). The compaction curve for the test- ed field soil have been presented in Fig. 6. The maximum and minimum dry densities for the tested field soil were 1.6 gm /cm3 and 2.0 gm/cm3 respectively, whereas the optimum moisture con- tent was 21%. (σ1-σ3)f=Rf (σ1-σ3)ult (2) (2) (σ1-σ3)f=Rf (σ1-σ3)ult Ei=KPa (σ3/Pa)n Fi 4 G i Si Di t ib ti f th Fi ld S il Where K = modulus number, n = modulus exponent, and Pa = reference stress (numerically equal to the atmospheric pressure) in the desired units for Ei. By differentiating both equations (1) and (4), the tangent modulus can be given as: Ei= [1-(Rf (1-sin∅) (σ1-σ3))/ (2c.cos∅+2σ3sin∅)] 2KPa (σ3/Pa)n (5) (5) Ei= [1-(Rf (1-sin∅) (σ1-σ3))/ (2c.cos∅+2σ3sin∅)] 2KPa (σ3/Pa)n (5) In this study, an elastic-plastic Mohr-Coulomb model was selected to represent the non-linear behavior of the untreated and tire treat- ed field soils. The limiting states of the stresses are defined by means of the soil undrained shear strength. The initial stresses of the soil were generated using Jaky’s formula which defines the at rest earth pressure coefficient (Ko = 1- sin) where  is the friction angle in terms of total stress. In the finite element modeling, an axisymmetric condition for a soil sample size of 5.5 cm diameter and 11 cm height with 8- noded quadrilateral elements having total number of elements equal to 33, total number of nodes equal to 128 and average ele- ment size of 1 mm have been used. The boundary conditions were chosen where the vertical boundaries are constrained horizontally and free vertically while the bottom horizontal boundary is fully fixed. Loading was applied in very small increments up to the total load. Values of soil parameters used in this analysis are summa- rized in Table 1. Fig. 4: Grain Size Distribution of the Field Soil. Fig. 5: Liquid Limit Evaluation. Table 1: Soil Parameters for Finite Element Analysis Model Properties Untreated field soil Tire treated soil Linear-Elastic Unit weight (kN/m3) 20 19 Young’s modulus, E (kN/m2) 5000 15000 Poisson’s ratio, v 0.4 0.4 Fig. 5: Liquid Limit Evaluation. 1771 Fig. 7: The Variation of the Shear Stress With the Horizontal Displace- ment for the Untreated and Waste Tire Treated Soils with Different Ap- plied Normal Stresses of (A) 0% Waste Tire Treatment, (B) 5% Waste Tire Treatment, and (C) 10% Waste Tire Treatment. b b A c a International Journal of Engineering & Technology 1771 Fig. 6: The Compaction Curve for the Tested Field Soil. Fig. Ei=KPa (σ3/Pa)n 7: The Variation of the Shear Stress With the Horizontal Displace- ment for the Untreated and Waste Tire Treated Soils with Different Ap- plied Normal Stresses of (A) 0% Waste Tire Treatment, (B) 5% Waste Tire Treatment, and (C) 10% Waste Tire Treatment. b A c b A Fig. 6: The Compaction Curve for the Tested Field Soil. c 2) Direct shear results 2) Direct shear results The shear stress variation with the horizontal displacement for different applied normal stress for untreated and waste tire treated soils is shown in Fig. 7. It is clearly shown that the shear stress increases with increasing the normal stress for both untreated and treated field soils and up to 10% waste tire treatment. For the un- treated soil, the maximum shear stress increased by 150% when the normal shear stress increased from 50 kPa to 150 kPa. For the 5% waste tire treated soil, the maximum shear stress increased by 110% when the normal stress increased from 50 kPa to 150 kPa. For the 10% waste tire treated soil, the maximum shear stress increased by 105% when the normal stress increased from 50 kPa to 150 kPa. Applying higher normal stresses on both untreated and waste tire treated soils increase the shear stress since the particles become closer leading to higher tangential stresses. Fig. 7: The Variation of the Shear Stress With the Horizontal Displace- ment for the Untreated and Waste Tire Treated Soils with Different Ap- plied Normal Stresses of (A) 0% Waste Tire Treatment, (B) 5% Waste Tire Treatment, and (C) 10% Waste Tire Treatment. The shear stress variation with the horizontal displacement for the same applied normal stress of untreated and waste tire treated soils is shown in Fig. 8. For the applied normal stress of 50 kPa, the maximum shear stress increased by 67% when the waste tire treatment increased from 0% to 10%. For the applied normal stress of 100 kPa, the maximum shear stress increased by 119% when the waste tire treatment increased from 0% to 10%. For the applied normal stress of 150 kPa, the maximum shear stress in- creased by 36% when the waste tire treatment increased from 0% to 10%. It is effectively shown that the waste tire has the ability to increase the shear strength (maximum shear stress) of the field soil with the maximum impact of 119% when the applied normal stress and waste tire treatment are 100 kPa and 10% respectively. b a a maximum shear stress increased by 67% when the waste tire treatment increased from 0% to 10%. For the applied normal stress of 100 kPa, the maximum shear stress increased by 119% when the waste tire treatment increased from 0% to 10%. 2) Direct shear results For the applied normal stress of 150 kPa, the maximum shear stress in- creased by 36% when the waste tire treatment increased from 0% to 10%. It is effectively shown that the waste tire has the ability to increase the shear strength (maximum shear stress) of the field soil with the maximum impact of 119% when the applied normal stress and waste tire treatment are 100 kPa and 10% respectively. b a a b a a b 1772 International Journal of Engineering & Technology Fig. 10: The Failure Pattern of the Untreated and Waste Tire Treated Soils in the Unconfined Compression Test. 4) Finite elements results ) D f d M h Fig. 8: The Variation of the Shear Stress with the Horizontal Displacement for the Untreated and Waste Tire Treated Soils with Different Applied Normal Stresses of (A) Normal Applies Stress = 50 Kpa, (B) Normal Applies Stress = 100 Kpa, And (C) Normal Applies Stress = 150 Kpa. c c Fig. 8: The Variation of the Shear Stress with the Horizontal Displacement for the Untreated and Waste Tire Treated Soils with Different Applied Normal Stresses of (A) Normal Applies Stress = 50 Kpa, (B) Normal Applies Stress = 100 Kpa, And (C) Normal Applies Stress = 150 Kpa. Fig. 10: The Failure Pattern of the Untreated and Waste Tire Treated Soils in the Unconfined Compression Test. p 4) Finite elements results a) Deformed Mesh 3) Unconfined compression results (A) (B) Fig. 11: The Deformed Mesh for Unconfined Compression Test Using Elastic Model for (A) Untreated Soil, and (B) 10% Waste Tire Treated Soil. The deformed mesh for unconfined compression test using the elastic model for both untreated and 10% waste tire treated soils have shown in Fig. 11. When the elastic model is used, both un- treated and waste tire treated soils exhibited similar deformed shapes. The variation of the uniaxial compression stress with the vertical strain for untreated and waste tire treated field soils tested under different rates are shown in Fig. 9. The uniaxial stress increased with increasing the vertical strain up to the peak value then started to decrease up to the failure. The peak uniaxial stress increased by 83% and 98% as the waste tire treatment increased from 0% to 10% for both testing rates of 0.125 mm/min and 0.25 mm/min respectively. The peak uniaxial stresses were 23 kPa and 42 kPa for 0% and 10% waste tire treatment at a testing rate of 0.125 mm/min whereas the peak uniaxial stresses were 16.1 kPa and 31.9 kPa for 0% and 10% waste tire treatment at a testing rate of 0.25 mm/min. Hence, it is clearly indicated that the lower testing rate has given higher peak uniaxial stress for both untreated and waste tire treated field soils. For the both testing rates of 0.125 mm/min and 0.25 mm/min, the peak uniaxial stresses occurred at a strain range of 0.5% to 0.65%. (A) (B) The failure patterns of the untreated and waste tire treated soils for the unconfined compression tests have shown in Fig. 10. It has noticed that the untreated and waste tire treated soils follow differ- ent failure patterns where the waste tire strengthen the core of the soils compared to the untreated soils. (B) (B) Fig. 11: The Deformed Mesh for Unconfined Compression Test Using Elastic Model for (A) Untreated Soil, and (B) 10% Waste Tire Treated Soil. Fig. 9: The Variation of the Uniaxial Stress with the Vertical Strain for the Untreated and Waste Tire Treated Soils with Different Testing Rates of (A) Testing Rate = 0.125 Mm/Min, and (B) Testing Rate = 0.25 Mm/Min. Fig. 11: The Deformed Mesh for Unconfined Compression Test Using Elastic Model for (A) Untreated Soil, and (B) 10% Waste Tire Treated Soil. 3) Unconfined compression results The deformed mesh for unconfined compression test using the elastic model for both untreated and 10% waste tire treated soils have shown in Fig. 11. When the elastic model is used, both un- treated and waste tire treated soils exhibited similar deformed shapes. a b Fig. 9: The Variation of the Uniaxial Stress with the Vertical Strain for the Untreated and Waste Tire Treated Soils with Different Testing Rates of (A) Testing Rate = 0.125 Mm/Min, and (B) Testing Rate = 0.25 Mm/Min. Fig. 11: Elastic M Soil. The de elastic have sh treated shapes. a b a b Fig. 11: The Deformed Mesh for Unconfined Compression Test Using Elastic Model for (A) Untreated Soil, and (B) 10% Waste Tire Treated Soil. The deformed mesh for unconfined compression test using the elastic model for both untreated and 10% waste tire treated soils have shown in Fig. 11. When the elastic model is used, both un- treated and waste tire treated soils exhibited similar deformed shapes. Fig. 9: The Variation of the Uniaxial Stress with the Vertical Strain for the Untreated and Waste Tire Treated Soils with Different Testing Rates of (A) Testing Rate = 0.125 Mm/Min, and (B) Testing Rate = 0.25 Mm/Min. 1773 International Journal of Engineering & Technology (A) (B) Fig. 12: The Deformed Mesh for Unconfined Compression Test Using Hyperbolic Model for (A) Untreated Soil, and (B) 10% Waste Tire Treated Soil. (A) (B) (B) Fig. 13: The Deformed Mesh for Unconfined Compression Test Using Elastic-Plastic Model for (A) Untreated Soil, and (B) 10% Waste Tire Treated Soil. Fig. 13: The Deformed Mesh for Unconfined Compression Test Using Elastic-Plastic Model for (A) Untreated Soil, and (B) 10% Waste Tire Treated Soil. (B) (B) b) Shear stress-shear strain The finite element method has been used to predict the shear stress versus shear strain relationship for unconfined compression test using elastic, hyperbolic and elastic-plastic models for both un- treated and 10% waste tire treated soil as shown in Figs. 14 and 15 respectively. For both untreated and waste tire treated soils, the elastic model over predicted the shear stress versus shear strain relationship whereas the elastic-plastic model had a very good agreement with the experimental data. For both untreated and waste tire treated soils, the hyperbolic model had a good predic- tion for the initial part of the shear stress versus shear strain rela- tionship with an overestimation for the second part of the experi- mental data. c) Maximum vertical displacement-time c) Maximum vertical displacement-time The variation of the maximum vertical displacement with the time for both untreated and 10% waste tire treated soils were predicted using finite element method with three different models as shown in Figs. 16 and 17 respectively. For both untreated and waste tire treated soils, the elastic model underestimated the maximum verti- cal displacement versus time relationship whereas the elastic- plastic model had a very good agreement with the experimental data. For the untreated soil, the hyperbolic model has predicted the early initial part of the experimental data well with an overestima- tion for the rest of the experimental data. For the 10% waste tire treated soil, the hyperbolic model has underestimated most of the experimental data with a good agreement with the final experi- mental value. Fig. 12: The Deformed Mesh for Unconfined Compression Test Using Hyperbolic Model for (A) Untreated Soil, and (B) 10% Waste Tire Treated Soil. In addition, the deformed mesh for unconfined compression test using the elastic-plastic model for both untreated and 10% waste tire treated soils have shown in Fig. 13. When the elastic-plastic model is used, both untreated and waste tire treated soils adopted similar deformed shapes. (A) (A) (A) (A) mental value. Fig. 14: Comparison between Finite Element Predictions with Experi- mental Data for the Shear Stress-Shear Strain Relationship of Untreated Soil Using Three Different Models. Fig. 14: Comparison between Finite Element Predictions with Experi- mental Data for the Shear Stress-Shear Strain Relationship of Untreated Soil Using Three Different Models. Fig. 14: Comparison between Finite Element Predictions with Experi- mental Data for the Shear Stress-Shear Strain Relationship of Untreated Soil Using Three Different Models. International Journal of Engineering & Technology 1774 Fig. 15: Comparison between Finite Element Predictions with Experi- mental Data for the Shear Stress-Shear Strain Relationship of 10% Waste Tire Treated Soil Using Three Different Models. Fig. 16: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Vertical Displacement Relationship of Untreated Soil Using Three Different Models. Fig. 18: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Horizontal Displacement Relationship of Untreated Soil Using Three Different Models. Fig. 19: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Horizontal Displacement Relationship of 10% Waste Tire Treated Soil Using Three Different Models. 4 Conclusions Fig. c) Maximum vertical displacement-time 15: Comparison between Finite Element Predictions with Experi- mental Data for the Shear Stress-Shear Strain Relationship of 10% Waste Tire Treated Soil Using Three Different Models. Fig. 18: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Horizontal Displacement Relationship of Untreated Soil Using Three Different Models. Fig. 18: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Horizontal Displacement Relationship of Untreated Soil Using Three Different Models. Fig. 15: Comparison between Finite Element Predictions with Experi- mental Data for the Shear Stress-Shear Strain Relationship of 10% Waste Tire Treated Soil Using Three Different Models. Fig. 19: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Horizontal Displacement Relationship of 10% Waste Tire Treated Soil Using Three Different Models. Fig. 16: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Vertical Displacement Relationship of Untreated Soil Using Three Different Models. Fig. 19: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Horizontal Displacement Relationship of 10% Waste Tire Treated Soil Using Three Different Models. Fig. 16: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Vertical Displacement Relationship of Untreated Soil Using Three Different Models. d) Maximum horizontal displacement-time d) Maximum horizontal displacement-time The variation of the maximum horizontal displacement with the time for both untreated and 10% waste tire treated soils have been predicted using finite element method with three different models as shown in Figs. 18 and 19 respectively. For both untreated and waste tire treated soils, the elastic and hyperbolic models overestimated the maximum horizontal displacement versus time relationships whereas the elastic model had a good agreement with the experimental data. Based on the experimental and numerical results of this study, the following conclusions can be drawn: 1) The grain size distribution of the Kirkuk field soil has shown that the soil can be classified as sand-clay mixtures with maximum and minimum dry densities of 1.6 gm /cm3 and 2.0 gm/cm3 respectively. 2) For the untreated field soil, the maximum shear stress ob- tained from direct shear tests increased by 150% when the normal shear stress increased from 50 kPa to 150 kPa. For the 5% and 10% waste tire treated soils, the shear stress measured from direct shear tests increased by 110% and 105% when the normal stress increased from 50 kPa to 150 kPa respectively. Fig. 17: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Vertical Displacement Relationship of 10% Waste Tire Treated Soil Using Three Different Models. 3) It is remarkably shown that the waste tire has the ability to increase the shear strength of the field soil with the maxi- mum impact of 119% when the applied normal stress of the direct shear test was 100 kPa at 10% waste tire treatment. 4) The peak uniaxial stress obtained from unconfined compres- sion test increased by 83% and 98% as the waste tire treat- ment increased from 0% to 10% for both testing rates of 0.125 mm/min and 0.25 mm/min respectively. 5) It is clearly shown that the lower testing rate of the uncon- fined compression test has given higher peak uniaxial stress for both untreated and waste tire treated field soils. 5) It is clearly shown that the lower testing rate of the uncon- fined compression test has given higher peak uniaxial stress for both untreated and waste tire treated field soils. Fig. 17: Comparison between Finite Element Predictions with Experi- mental Data for the Maximum Vertical Displacement Relationship of 10% Waste Tire Treated Soil Using Three Different Models. 4. Conclusions d) Maximum horizontal displacement-time References [1] Lepcha K. H., Agnihotri A. K., Priyadarshee A., Yadav M. (2014). “Application of tire chips in reinforcement of soil: a review,” Jour- nal of Civil Engineering and Environmental Technology, 1(5), pp. 51-53. [23] ASTM D854-02. (2002). “Specific gravity of Soil Solids by Water Pycnometer,” ASTM International, West Conshohocken, PA. [24] ASTM D422-63. (2007). “Particle-size Analysis of Soils,” ASTM International, West Conshohocken, PA. [2] Chen H., Wong Q. (2006). “The behavior of soft soil stabilization- nusing cement,” Bulletin of Engineering Geology and the Envi- ronmenta by Springerlink. [25] ASTM D4318-00. (2000). “Liquid limit, Plastic limit and Plasticity Index of Soils,” ASTM International, West Conshohocken, PA. 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(1993). “Laboratory study on properties of rubber soils,” Report No. FHWA/IN/JHRP – 93/4, Purdue University, West Lafayette, IN. y , [10] Edil T.B., Bosscher P.J. (1994). d) Maximum horizontal displacement-time 6) Both untreated and waste tire treated soils exhibited similar deformed shapes when elastic or elastic-plastic models are used. However, the upper zone for the untreated soil is af- fected more that the upper zone for 10% waste tire treated soils when the hyperbolic model is used. 6) Both untreated and waste tire treated soils exhibited similar deformed shapes when elastic or elastic-plastic models are used. However, the upper zone for the untreated soil is af- fected more that the upper zone for 10% waste tire treated soils when the hyperbolic model is used. 7) For both untreated and waste tire treated soils, the elastic model over predicted the shear stress versus shear strain re- lationship whereas the elastic-plastic model had a very good agreement with the experimental data. However, the hyper- 7) For both untreated and waste tire treated soils, the elastic model over predicted the shear stress versus shear strain re- lationship whereas the elastic-plastic model had a very good agreement with the experimental data. However, the hyper- 1775 International Journal of Engineering & Technology bolic model had a good prediction for the initial part of the shear stress versus shear strain relationships with an overes- timation for the second part of the experimental data. [16] Zornberg J.G., Christopher B.R., Oosterbaan M.D. (2005). "Tire bails in highway applications: feasibility and properties evaluation," Colorado Department of Transportation Research Branch, (CDOT- DTD-R-2005-2). 8) For both untreated and waste tire treated soils, the elastic model underestimated the maximum vertical displacement versus time relationship whereas the elastic-plastic model had a very good agreement with the experimental data. [17] Ashmanwy A., McDonald R., Carreon D., Atalay F. (2006). "Stabi- lization of marginal soils using recycled materials," Florida De- partment of Transportation, (BD-544-4). [18] Feng Z. and Sutter K.G. (2000). "Dynamic properties of granulated rubber/sand mixtures," Geotechnical Testing J., GTJODJ, 23 (3), pp. 338-344. 9) For both untreated and waste tire treated soils, the elastic and hyperbolic models overestimated the maximum horizontal displacement versus time relationships whereas the elastic model had a good agreement with the experimental data. 9) For both untreated and waste tire treated soils, the elastic and hyperbolic models overestimated the maximum horizontal displacement versus time relationships whereas the elastic model had a good agreement with the experimental data. [19] Ghazavi M., Sakhi M.A. Acknowledgements [20] Dutta R.K., Rao V. G. (2007).”Regression model for predicting the behavior of sand reinforced with waste plastic,” Turkish Journal of Engineering and Environmental Sciences, 31(2), pp. 119-126. Engineering and Environmental Sciences, 31(2), pp. 119-126. 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https://openalex.org/W4288731646	https://www.frontiersin.org/articles/10.3389/fmars.2022.903134/pdf	English	null	Size Dependent Transport of Floating Plastics Modeled in the Global Ocean	Frontiers in marine science	2,022	cc-by	9,125	INTRODUCTION Plastics are everywhere in the global ocean, with observations of accumulation near coastlines (Lavers & Bond, 2017; Olivelli et al., 2020), in offshore regions spanning from the surface (Law et al., 2010) to the deep sea (Egger et al., 2020b) and from equatorial (Garcia et al., 2020) to polar waters (Cózar et al., 2017; Suaria et al., 2020; Ross et al., 2021). Characteristics of plastics such as size, shape and polymer differ among observational reports and often depend on where and how sampling was conducted. For example, plastics with density larger than seawater naturally accumulate on the seabed while positively buoyant plastics can be transported by currents and winds to different regions around the world (van Sebille et al., 2020). The floating fraction is particularly problematic due to its negative impact on a wide array of marine species (Gall and Thompson, 2015), its role transporting invasive species across different regions (García-Gómez et al., 2021) and its constant degradation into smaller particles from UV induced weathering and fragmentation (Andrady, 2011). Early attempts to quantify the total mass of plastic at the surface of the ocean coupled data collected from sea surface nets with dispersal and accumulation model outputs for floating marine debris (Cozar et al., 2014; Eriksen et al., 2014; van Sebille et al., 2015). One conclusion of these studies was that there was less small debris at the surface than expected based on the amount of large debris. These results suggested size selective removal processes for floating plastics at the surface of the ocean. Sampling bias related to mesh size contributes to this discrepancy (Lindeque et al., 2020), but is insufficient to explain the several orders of magnitude gap (Cozar et al., 2014). Plastics are everywhere in the global ocean, with observations of accumulation near coastlines (Lavers & Bond, 2017; Olivelli et al., 2020), in offshore regions spanning from the surface (Law et al., 2010) to the deep sea (Egger et al., 2020b) and from equatorial (Garcia et al., 2020) to polar waters (Cózar et al., 2017; Suaria et al., 2020; Ross et al., 2021). Characteristics of plastics such as size, shape and polymer differ among observational reports and often depend on where and how sampling was conducted. doi: 10.3389/fmars.2022.903134 ORIGINAL RESEARCH published: 20 July 2022 doi: 10.3389/fmars.2022.903134 ORIGINAL RESEARCH published: 20 July 2022 ORIGINAL RESEARCH Edited by: Edited by: José Antonio López-López, University of Cádiz, Spain Reviewed by: Giovanni Besio, University of Genoa, Italy Andrés Cózar, University of Cadiz, Spain Correspondence: Doug Klink d.klink@theoceancleanup.com Specialty section: This article was submitted to Marine Pollution, a section of the journal Frontiers in Marine Science Received: 23 March 2022 Accepted: 07 June 2022 Published: 20 July 2022 Citation: Klink D, Peytavin A and Lebreton L (2022) Size Dependent Transport of Floating Plastics Modeled in the Global Ocean. Front. Mar. Sci. 9:903134. doi: 10.3389/fmars.2022.903134 Edited by: José Antonio López-López, University of Cádiz, Spain Reviewed by: Giovanni Besio, University of Genoa, Italy Andrés Cózar, University of Cadiz, Spain Correspondence: Doug Klink d.klink@theoceancleanup.com Specialty section: This article was submitted to Marine Pollution, a section of the journal Frontiers in Marine Science Received: 23 March 2022 Accepted: 07 June 2022 Published: 20 July 2022 Keywords: marine debris, variable size, whole ocean, computational model, Lagrangian trajectories, three- dimensional, parallel hardware, GPU University of Cádiz, Spain Reviewed by: Giovanni Besio, University of Genoa, Italy Andrés Cózar, University of Cadiz, Spain Correspondence: Doug Klink Size Dependent Transport of Floating Plastics Modeled in the Global Ocean Doug Klink 1, Axel Peytavin 1 and Laurent Lebreton 1,2 1 The Ocean Cleanup Foundation, Rotterdam, Netherlands , 2 The Modelling House Limited, Raglan, New Zealand Plastic has been detected in the ocean in most locations where scientists have looked for it. While ubiquitous in the environment, plastic pollution is heterogeneous, and plastics of varying composition, shape, and size accumulate differently in the global ocean. Many physical and biological processes influence the transport of plastics in the marine environment. Here we focus on physical processes and how they can naturally sort floating plastics at the ocean surface and within its interior. We introduce a new open-source GPU-accelerated numerical model, ADVECT, which simulates the three- dimensional dispersal of large arrays of modelled ocean plastics with varying size, shape, and density. We use this model to run a global simulation and find that buoyant particles are sorted in the ocean according to their size, both at the surface due to wind-driven drift and in the water column due to their rising velocity. Finally, we compare our findings with recent literature reporting the size distribution of plastics in the ocean and discuss which observations can and cannot be explained by the physical processes encoded in our model. INTRODUCTION For example, plastics with density larger than seawater naturally accumulate on the seabed while positively buoyant plastics can be transported by currents and winds to different regions around the world (van Sebille et al., 2020). The floating fraction is particularly problematic due to its negative impact on a wide array of marine species (Gall and Thompson, 2015), its role transporting invasive species across different regions (García-Gómez et al., 2021) and its constant degradation into smaller particles from UV induced weathering and fragmentation (Andrady, 2011). Specialty section: This article was submitted to Marine Pollution, a section of the journal Frontiers in Marine Science Specialty section: This article was submitted to Marine Pollution, a section of the journal Frontiers in Marine Science Received: 23 March 2022 Accepted: 07 June 2022 Published: 20 July 2022 Received: 23 March 2022 Accepted: 07 June 2022 Published: 20 July 2022 Early attempts to quantify the total mass of plastic at the surface of the ocean coupled data collected from sea surface nets with dispersal and accumulation model outputs for floating marine debris (Cozar et al., 2014; Eriksen et al., 2014; van Sebille et al., 2015). One conclusion of these studies was that there was less small debris at the surface than expected based on the amount of large debris. These results suggested size selective removal processes for floating plastics at the surface of the ocean. Sampling bias related to mesh size contributes to this discrepancy (Lindeque et al., 2020), but is insufficient to explain the several orders of magnitude gap (Cozar et al., 2014). Keywords: marine debris, variable size, whole ocean, computational model, Lagrangian trajectories, three- dimensional, parallel hardware, GPU Citation: Klink D, Peytavin A and Lebreton L (2022) Size Dependent Transport of Floating Plastics Modeled in the Global Ocean. Front. Mar. Sci. 9:903134. doi: 10.3389/fmars.2022.903134 July 2022 \| Volume 9 \| Article 903134 Frontiers in Marine Science \| www.frontiersin.org Size Dependent Ocean Plastic Transport Klink et al. the entire three-dimensional ocean domain. Debris is modeled as sphere-like particles with variable size, density, and shape. Transport is modeled as a combination of advection due to ocean currents, sinking/rising due to buoyancy, windage based on emerged surface area, near-surface vertical mixing due to wind, and random diffusion in three dimensions. Particles are constrained to the ocean, meaning they cannot be entrained into bathymetry or beached onto shorelines. To properly sample the new per-particle parameters, simulations must process a huge number of particles, which is computationally challenging. One way to increase computational capability is to use GPUs to compute particle trajectories in parallel (van Sebille et al., 2018), as GPU computing power has been significantly outpacing that of CPUs in the last decade (Sun et al., 2019). With these considerations in mind, we designed ADVECT to compute its simulations on GPUs via the OpenCL framework (Stone et al., 2010). Though others efforts have used GPUs to model Lagrangian trajectories in the ocean (Gelderloos et al., 2016), ADVECT is the first to do so in an offline manner (i.e., without being coupled to an ocean model). The other primary computational concern in offline Lagrangian particle tracking is the ever-increasing size of model inputs, particularly the three- dimensional ocean currents (van Sebille et al., 2018). To address this challenge, ADVECT dynamically divides its simulation into temporal chunks based on available memory, allowing support for arbitrarily large datasets. These characteristics differentiate ADVECT from other models and represent a new computational approach to offline particle tracking in the ocean. Differences in relative concentration of larger debris from coastal environments to offshore areas also suggest different fates for different sizes of floating plastics (Ryan, 2015; Morales-Caselles et al., 2021). The scarcity of small plastic particles found in trawl nets deployed in the Eastern North Pacific Ocean also varied between different locations of the subtropical gyre, with smaller particles (0.5 - 5 mm) appearing more abundant in the center of the accumulation zone (Egger et al., 2020a).h This abundance could be explained by several mechanisms. Model Description Particles are modeled as sphere-like objects with three key properties: size, shape, and density. Shape is parameterized using Corey Shape Factor (hereafter CSF), which ranges from 0 to 1, and is defined as: CSF a bc = (1) (1) Where a, b, and c are the short, medium, and long dimensions of the particle respectively (Dietrich, 1982). Decreasing the CSF below 1 can be envisioned as either flattening or elongating an initially spherical particle. Many definitions of size exist when speaking of irregularly shaped particles. Our model uses nominal radius, defined as the radius of a sphere with equivalent volume as the particle for consistency with our settling velocity equations (see equation 2). Particles are advected through a time-evolving 3D velocity field of ocean currents according to a 3D adaptation of the second-order Taylor-expansion scheme used in (L. C.-M. Lebreton et al., 2012). The full equations can be found in Supplementary Material. This scheme eliminates erroneous radial drift from particle trajectories along circular streamlines (Black & Gay, 1990). Citation: First, through biofouling, floating plastics can increase in density and eventually lose buoyancy (Fazey & Ryan, 2016); this process impacts small floating particles more rapidly due to their large surface area to volume ratio. However, the oligotrophic environment in subtropical gyres may prevent the growth of organisms responsible for biofouling and substantially increase sinking timescales (Lobelle et al., 2021), explaining the abundance of smaller floating particles in these regions. Second, the transport of floating plastics in the ocean likely varies with size, shape and density of debris. For example, these quantities impact the rising velocity of debris, which in turn alters transport due to turbulent mixing (Kukulka et al., 2012) or wind driven drift current (van der Mheen et al., 2020). Small particles should experience a smaller wind-driven drift current, and indeed, the gradient of concentration observed accross the North Pacific subtropical gyre was better represented in a dispersal model simulation when debris was advected with no wind drag (L. Lebreton et al., 2018). Thus, physical processes may also explain the variation in debris size reported by observations at sea. p y Understanding the transport and long-term fate of plastics, and most particularly floating plastics, in the ocean is necessary for risk assessments and the design of efficient mitigation strategies. While field observations show that different plastics will know different fates in the ocean, attempts at quantifying the mass of plastics accumulated at the surface of the ocean generally consider plastics as a whole and fail to differentiate transport as a function of debris characteristics such as size, shape and density. In this study we investigate how size-dependent physical transport processes influence the distribution of positively buoyant plastics in the global ocean. We present a new GPU- accelerated numerical model, ADVECT, which computes three dimensional trajectories of plastic particles with individually varying size, shape, and density. These trajectories are derived from advection due to oceanic currents, surface wind-driven drift, wave induced vertical mixing, and vertical transport due to buoyancy. We conduct a simple experiment with randomly spawned particles with a wide range of sizes, shapes and densities and show how the modelled global ocean physically distributes and sorts particles based on their size at the surface, ocean interior and seabed over a duration of 20 years. We then discuss our findings and compare with recent field observations. Experimental Setuph The simulation was run for 20 years, from January 1, 1993 to December 31, 2012, with a timestep of one hour. Three million particles are released at uniformly random locations on the ocean surface, at uniformly random times during the first 10 years of the simulation. The simulation then carries on for an additional 10 years with no new releases. Particle properties were set randomly according to the following distributions: Particles at the ocean surface are further transported horizontally using wind velocity estimated at 10 cm above sea level. The transport is derived from the particle’s emerged surface area according to Richardson (1997) and van der Mheen et al. (2020). Wind velocity at 10 cm above sea level is estimated from 10-meter wind data by assuming a log-wind profile (Charnock, 1955). nominalradius m density U kg m CSF U U ~ ~ ( , ) ~ (. ( , ) 10 800 1000 6 0 3 − − 15 1, ) (5) nominalradius m density U kg m CSF U U ~ ~ ( , ) ~ (. ( , ) 10 800 1000 6 0 3 − − 15 1, ) (5) Near-surface particles are mixed vertically by the action of wind-driven waves. Local significant wave height is estimated from 10-meter wind velocity according to Kukulka et al. (2012). Rather than being transported according to equation (2), buoyant near-surface particles are randomly displaced according to a concentration profile predicted by Kukulka et al. (2012, eq. 4). The shape of this profile is determined by both a particle’s rising velocity and wind speed. Further details on this algorithm can be found in Supplementary Material. (5) Zonal and meridional ocean currents are sourced from the GLORYS12V1 ocean reanalysis (Lellouche et al., 2018) at 1/12 degree horizontal resolution, 50 depth levels, and daily temporal sampling. GLORYS12V1 is based on NEMO coupled with atmospheric forcing from ERA-interim/ERA5 and assimilates many observational datasets. We compute our own vertical ocean currents from the provided data by integrating the continuity equation via the adjoint method (Luettich Richard et al., 2002). Our seawater density data is gridded at ¼ degree horizontal resolution, 75 depth levels, and monthly temporal sampling. It is calculated using GSW-Python (Firing et al., 2021) from monthly-mean ocean salinity and temperature, which are sourced from the Global Ocean Ensemble Physics Reanalysis, a product of Copernicus Marine Environment Monitoring Service (CMEMS, doi:10.48670/moi-00024). METHODS 5-5 mm (Khatmullina & Isachenko, 2017) (Note: W* is undefined for CSF ≤.15, so ADVECT does not support particles with CSF ≤.15). We neglect any inertial effects, and assume the particle is always traveling at this terminal sinking (rising) velocity due to the relatively large model timesteps in practice, which are generally on the order of hours. (4) Where ao = 1e-7 m2s-2, N0 = 5e-4 s-1, B = b = -1000 m, and z is positive-up.i Particles cannot leave the ocean, whose domain is defined to match that of the ocean current data. This means ADVECT does not include the processes of beaching or sedimentation. Instead, when particles are pushed against coastline or bathymetry, they flow along the boundary. This boundary handling scheme was chosen because beaching and seafloor entrainment processes are not well-understood (van Sebille et al., 2020). Experimental Setuph 10-meter wind data are sourced from the ERA5 atmospheric reanalysis (Hersbach et al., 2020) at ¼ degree horizontal resolution and daily-mean temporal sampling. All forcing datasets span the same temporal range as the experiment. Transport due to sub-mesoscale eddies is parameterized through random eddy diffusion, represented as a Wiener process. In the ocean, sub-mesoscale eddy diffusivity varies temporally as well as with region and depth (Cole et al., 2015). This temporal and regional variation is not resolved by ocean models by definition, and observations are scarce. However, there are some observations and theories showing significant and systematic variation of diffusivity with depth, as well as a marked difference between vertical and horizontal diffusivities (Gargett, 1984; Groeskamp et al., 2020). As such, we decompose eddy diffusion into its horizontal and vertical components and define a vertical diffusivity profile for each. The horizontal diffusivity profile is based on sub-mesoscale eddy diffusivity observations, and is defined to decay with depth as follows: K z K z h h z ( ) = = ( )     0 1 2 * ζ (3) (3) METHODS Simulations are performed using a newly developed software, ADVECT (open-source code at DOI: 10.5281/zenodo.6301282). ADVECT simulates Lagrangian advection of marine debris in Each particle moves vertically according to a buoyancy-driven terminal sinking (or rising) velocity ws, calculated according to Dietrich (1982): July 2022 \| Volume 9 \| Article 903134 Frontiers in Marine Science \| www.frontiersin.org 2 Klink et al. Size Dependent Ocean Plastic Transport is based on estimates of vertical eddy diffusivity from bottle casts in the North Pacific (Gargett, 1984). It is defined as follows: is based on estimates of vertical eddy diffusivity from bottle casts in the North Pacific (Gargett, 1984). It is defined as follows: w g W s s = − ( ) ρ ρ ρ ν * 3 (2) (2) K z a N e v o z B b ( ) = −+     0 2 (4) Where ρ is the density of the particle, ρs is the density of surrounding seawater, g is the acceleration due to gravity, ν is the kinematic viscosity of the surrounding seawater, and W* is a “dimensionless settling velocity.” W* depends on the particle’s radius, shape, and density, as well as the density and kinematic viscosity of surrounding seawater, and is calculated from further equations in Dietrich (1982) which we omit here for brevity. Dietrich’s method has been verified to accurately model ws for spherical and cylindrical plastic particles ranging in diameter from. 5-5 mm (Khatmullina & Isachenko, 2017) (Note: W* is undefined for CSF ≤.15, so ADVECT does not support particles with CSF ≤.15). We neglect any inertial effects, and assume the particle is always traveling at this terminal sinking (rising) velocity due to the relatively large model timesteps in practice, which are generally on the order of hours. Where ρ is the density of the particle, ρs is the density of surrounding seawater, g is the acceleration due to gravity, ν is the kinematic viscosity of the surrounding seawater, and W* is a “dimensionless settling velocity.” W* depends on the particle’s radius, shape, and density, as well as the density and kinematic viscosity of surrounding seawater, and is calculated from further equations in Dietrich (1982) which we omit here for brevity. Dietrich’s method has been verified to accurately model ws for spherical and cylindrical plastic particles ranging in diameter from. RESULTS After 20 years of model simulation, we observe significant variations in the distribution of buoyant particles in our ocean model based on particle characteristics. The size of particles had the largest influence on particle transport and fate. At the near After 20 years of model simulation, we observe significant variations in the distribution of buoyant particles in our ocean model based on particle characteristics. The size of particles had the largest influence on particle transport and fate. At the near Where z is measured in meters, Kh(z=0) = 50 m2s-1, a typical value from estimates over the Argentinian shelf (Capet et al., 2008), and the half-life depth ζ is set to 1000 m based on Groeskamp et al. (2020). The vertical diffusivity profile also decays with depth and July 2022 \| Volume 9 \| Article 903134 Frontiers in Marine Science \| www.frontiersin.org Size Dependent Ocean Plastic Transport Klink et al. surface (Figure 1), small particles (diameter < 1 mm) appear to be highly concentrated in a few select areas, leaving the rest of the ocean to be dominated by larger particles (diameter > 1 mm). The Pacific and Atlantic subtropical gyres each have a gradient of size, with smaller particles accumulating toward the tropics and larger particles towards the poles. Smaller particles also dominate in the Eastern Indian Ocean, the West Greenland sea, and the majority of the Mediterranean. Large particles dominate in a small but highly concentrated coastal zone in the Eastern Mediterranean, creating a gradient in size across the basin; a similar and stronger gradient is present in the Red Sea. Since there is still a great diversity of size among the large particles (>1 mm), one might expect a pattern similar to Figure 1 to emerge when analyzing the distribution of particles within this size class. However, this is not the case. Instead, particles larger than 1 mm appear not to preferentially accumulate in specific regions based on their size, but instead are distributed somewhat uniformly across the ocean surface (Figure S8). general pattern found by models and observations (Cozar et al., 2014; Eriksen et al., 2014; van Sebille et al., 2015), with high concentrations in subtropical gyres and near-zero concentration in the equatorial divergence zone. The ocean interior exhibits significantly lower concentration than the surface, with a much more uniform spatial pattern. RESULTS There is some evidence of fallout below subtropical gyres, particularly in the South Pacific, but the most concentrated zones tend to be near coastlines, as well as the Western portions of the North and South Atlantic Oceans, and the Labrador Basin. The seafloor exhibits a similar spatial pattern to the interior, although particle concentrations are almost universally around two orders of magnitude greater. Figure 2D shows the size distribution of particle size within each ocean domain. After 20 years, roughly half the particles below 100 micrometers are lost from the surface to the interior and seafloor. This figure also shows that essentially zero particles larger than 100 micrometers escape the surface. The interior distribution slightly favors larger particles compared to the distribution at the seafloor. Figure 2D demonstrates that particles smaller than 100 micrometers can be removed from the ocean surface by downwelling currents, wind-driven mixing, and eddy diffusion, whose mean downward transport velocity is small, but still larger than the rising velocity of such small particles. g With densities ranging between 800 kg m−3 and 1,000 kg m−3 (reflecting the variation among buoyant plastic polymers; Kooi & Koelmans, 2019), the vast majority of large particles (diameter > 1mm) remained at the surface (Figure 2). However, smaller particles with their smaller rising velocities were more easily entrained into deeper layers, resulting in larger particles dominating the surface distribution. Figures 2A–C show the spatial variation in sub-1mm particle concentration within three non-overlapping depth domains: respectively the “Ocean Surface” (the region above -10 m depth), the “Ocean Seafloor” (the region within 50 m of local bathymetry, not extending past -10 m depth in shallow regions), and the “Ocean Interior” (the region between the other two domains). As expected with buoyant particles, the surface is by far the domain with the highest overall concentration, and the spatial pattern in this domain follows the Analysis of Results In this study, we present ADVECT, a new three-dimensional dispersal model for plastic in the global ocean. With this numerical model, we show how physical transport mechanisms alone can disperse particles of different characteristics to different oceanic regions. For example, the observed size distributions at FIGURE 1 \| Size sorting of floating plastic particles in the top 10 meters of the global ocean. Distribution is an average over the 20th model year. Cells with < 5 samples are marked as “no data.” Contours show highest-concentration regions, containing 50% (thin line) and 25% (thick line) of the global particle total, respectively (c.f. Figure 2A). Diameter is defined as 2 * nominal radius. FIGURE 1 \| Size sorting of floating plastic particles in the top 10 meters of the global ocean. Distribution is an average over the 20th model year. Cells with < 5 samples are marked as “no data.” Contours show highest-concentration regions, containing 50% (thin line) and 25% (thick line) of the global particle total, respectively (c.f. Figure 2A). Diameter is defined as 2 * nominal radius. July 2022 \| Volume 9 \| Article 903134 4 Frontiers in Marine Science \| www.frontiersin.org Klink et al. Size Dependent Ocean Plastic Transport A B C D FIGURE 2 \| Modelled distribution of small plastic particles (diameter < 1 mm) across different depth domains, average of 20th model year. (A) Horizontal distribution at the “Ocean Surface.” (B) Horizontal distribution in the “Ocean Interior.” (C) Horizontal distribution at the “Ocean Seafloor.” (D) Comparison of size distribution between domains. Diameter is defined as 2nominal radius. C D D C FIGURE 2 \| Modelled distribution of small plastic particles (diameter < 1 mm) across different depth domains, average of 20th model year. (A) Horizontal distribution at the “Ocean Surface.” (B) Horizontal distribution in the “Ocean Interior.” (C) Horizontal distribution at the “Ocean Seafloor.” (D) Comparison of size distribution between domains. Diameter is defined as 2nominal radius. The distributions found at depth (Figure 2) can be explained in large part by the mean upwelling/downwelling (Figure S5), as well as its variability (Figure S6). Firstly, it is important to note that only the very smallest particles escape the surface (Figure 2D); their small size results in a near-zero rising velocity, allowing them to be entrained by downwelling and diffusion. Analysis of Results The relative uniformity of concentration in the ocean interior and seabed implies that the dominant transport mechanism is diffusion, since our modeled diffusion does not vary geographically. Further, the mean downwelling under the subtropical gyres (roughly 1e-7 m/s) is not sufficiently strong to transport modeled particles to the seabed. Velocities of that order could only transport neutrally buoyant particles 100s of meters over the timespan of this simulation (20 years). Yet, Figure 2 shows many regions of increased concentration in both the interior and at the seafloor, including the Western North Atlantic, Argentine Basin, Labrador Sea, coast of South Africa, and coast of Somalia. Figure S4 shows that in these regions, downwelling is highly variable, with standard deviations on order 1e-5 m/s. Transient downwellings of this magnitude are strong enough to transport particles to the seafloor within the timespan of our simulation. the surface (Figure 1) can be mainly explained by surface wind patterns, due to the greater impact of wind on larger particles, both due to their increased surface area and stronger tendency to float at the surface as a result of their high rising velocity. Together, the yearly average wind pattern of the ERA5 wind data (Figure S3) and its temporal variability (Figure S4) can explain many features of the surface distribution of modelled particles presented in Figure 1. One such feature is the gradient of size across each subtropical gyre. The strong and consistent trade winds pass over the more equatorial region of each gyre, sweeping the larger objects away and leaving a region dominated by micro (<1 mm) debris. The Indian Ocean has a different pattern, with the eastern gyre retaining a high concentration of micro (<1 mm) debris, while macro (>1 mm) debris is dominating the rest of the basin. Figures S3, S4 show a consistent south-easterly wind over the eastern gyre, which again serves to selectively remove large debris. The modelled size gradient across the Mediterranean and Red seas is similarly explained by consistent winds from the west and northwest, respectively. Finally, modelled micro (<1 mm) debris also dominates the western Greenland sea. Not only are there consistent Northwest winds blowing off Greenland (Figure S3), but Figure S5 shows a consistent downwelling in this region. Analysis of Results The combination of these factors can explain the aggregation of micro (<1 mm) debris in this region of our model, as particles of all sizes are attracted towards Eastern Greenland by the action of the downwelling, but the larger debris is pushed away by the prevailing wind. Our model results also show a hundredfold higher concentration of particles at the seafloor than in the interior (Figure 2). This could be due to several factors. First, we observe that modeled particles accumulate at the seafloor whenever there is downwelling in the deepest layers of our ocean current July 2022 \| Volume 9 \| Article 903134 Frontiers in Marine Science \| www.frontiersin.org 5 Size Dependent Ocean Plastic Transport Klink et al. data. Second, the movement of particles may be restricted by bathymetric obstacles, which may help trap the particles near the seabed. Though it is reasonable to expect both these processes may occur in the global ocean, it is important to recognize the limited resolution of our ocean current data at the seabed: roughly 10km horizontally at the equator, and 500m vertically in the deep ocean. This limited resolution combined with complex bathymetry can lead to a great deal of uncertainty in the modeled flows near the seafloor. observations. Ryan attributed their observations to biofouling and argued that windage alone was insufficient to explain their results. Since we do not model biofouling, but windage alone, the fact that our respective results disagree lends support to his conclusions. Several studies have sampled the vertical distribution of plastic in the ocean. Egger et al. (2020b) analyzed debris from a wide size range (.5 – 50 mm) taken at various depths in the North Pacific and found a three or four order of magnitude decrease in concentration from the surface to the ocean interior. Figure 2 shows a four or five order of magnitude decrease between the concentration of sub-1 mm particles at the surface and in the ocean interior in the North Pacific, which is roughly in accord with their result. Further, half of the samples they found below the surface were in their smallest measured size class (.5-1.5 mm) (Table S4 in Egger et al., 2020b). This higher prevalence of objects with smaller size agrees with our modeled results. Yet, our results suggest that few objects larger than 0.1 mm should be found below the surface, in disagreement with their observations. Comparison to Field Observations Comparison to Field Observations Our predicted spatial variation in the size distribution of debris (Figure 1) is in good agreement with several observational studies, though it disagrees with some others. This is expected, since ADVECT does not model all physical processes which transport plastic debris in the ocean. The agreement or disagreement between our results and observations is useful in understanding what mechanisms are or are not able to explain a pattern of observations. First, Egger et al. (2020a) reported on a scarcity of sub-5 mm debris in the Gulf of Alaska that decreases towards the center of the subtropical gyre. Our results agree with these observational findings as the proportion of small objects in Figure 1 increases steadily from the Gulf of Alaska to the center of the subtropical gyre. While Egger and Nijhof suggested a micro-debris removal mechanism as the cause of this size scarcity, our model demonstrates that size-dependent windage may be sufficient to explain the observations. Next, (Morales- Caselles et al., 2021), found that small fragments were notably more frequent in oceanic accumulation zones, in contrast to large objects found in coastal waters, a pattern our results share (Figure 1). They suggest the size-dependent sorting they observed could be explained by larger objects with higher buoyancy being ejected from accumulation zones due to their larger windage; our results show that these processes can indeed produce the sorting they observed. Additionally, in the Hawaiian Archipelago, O’ahu and Hawai’i are known as hotspots for microplastic accumulation (Young & Elliott, 2016; Rey et al., 2021) while the Northwestern Hawaiian Islands are known as hotspots of larger debris, notably derelict fishing gear (Dameron et al., 2007). Our results share this pattern, with Figure 1 showing very small debris in the waters around the main Hawaiian Islands, but much larger debris to the northwest. Finally, Ryan (2015) observed the distribution of macro-debris (1 cm – 1 m) off the coast of South Africa, finding that the debris tended to be larger further from the coast. Our results suggest a modest increase in debris size along their cruise track (Figure 1), but in general do not agree well with their Pabortsava and Lampitt (2020) also examined the vertical distribution of micro (<1 mm) debris along a transect through the South and North Atlantic. They reported a strong preference for smaller objects in the ocean interior, with the average size near 100 micrometers. Comparison to Field Observations This roughly agrees with our prediction (Figure 2D), suggesting that hydrodynamical processes alone could explain this result. However, Pabortsava and Lampitt also found that the concentration of micro (<1 mm) debris at 50-100 meter depth is similar to that at 10 meters, and still within an order of magnitude at 100-270 meters. Our results differ significantly, predicting a hundredfold decrease in concentration from 10 to 100 meters in the North and South Atlantic (see Figure S5). This suggests ADVECT over-estimates the impact of wind-driven mixing below 10 meters. Analysis of Results However, we note that the larger (5 – 50 mm) objects they found in deeper water were fibrous fragments from ropes and nets. These are not representative of the pseudo spherical particles we modeled, and fibers are expected to have the smallest rising velocities of all shapes (see section below discussing size definitions). We also acknowledge that larger debris could be sinking due to processes not modeled in ADVECT. Some possibilities could be strong downwelling due to sub-mesoscale eddies, unresolved by our forcing data (Lévy et al., 2018), or density-increasing mechanisms such as biofouling or chemical weathering (van Sebille et al., 2020). ll In terms of model convergence, we found the horizontal distribution of particles at all depth levels (Figure 2) stabilizes around year 15, five years after the final particles are released. The vertical distribution of particles through the water column also stabilizes around year 15 (Figure S8). This means the distributions we present in Figures 1, 2 are close to being converged. However, the number of particles below -4000 m in the final year of our simulation is notably still increasing. This tells us that under our modeled processes, it can take decades for surface debris to significantly accumulate in the deep ocean. Frontiers in Marine Science \| www.frontiersin.org Discussion of Size Definitions Our results show that size has a strong impact on the distribution of plastic objects at sea. Comparisons with observational studies, however, are confounded by the numerous and vague methodologies used to report the size of plastic particles collected at sea. Observational studies often report size by binning objects into “size classes” based on the catchments of differently sized meshes. While this is a natural and efficient way to describe size observationally, it is challenging to use this description to estimate an object’s three-dimensional form, which is necessary to accurately model its rising velocity. We find that the volume of July 2022 \| Volume 9 \| Article 903134 Frontiers in Marine Science \| www.frontiersin.org 6 Size Dependent Ocean Plastic Transport Klink et al. an object has the strongest impact on its rising velocity, stronger than its shape or density. Unfortunately, given a measurement of an irregularly shaped object’s “size class,” estimates of volume vary dramatically depending on the object’s shape. For example, the volume of a 1 cm diameter sphere could be 50x larger than the volume of a 1 cm-long filament or 20x larger than a 1 cm-wide flake. Yet, all these objects might be caught by a 1 cm trawl, and thus recorded in the same size class. In order to test our model against observational data, it is imperative for studies to record enough information on objects to recover their volume. For example, (Pabortsava & Lampitt, 2020) recorded images of all their samples, and were later able to recover their volume by coupling the 2D areas in the images with assumptions of simple three-dimensional morphologies. Such a method is not too labor-intensive, and can result in good volume estimates. process well. For example, the model introduced in (Kooi et al., 2017) has been incorporated into a global circulation model (Lobelle et al., 2021), yet its defouling dynamics are mainly driven by a constant 39% decrease in algal mass per day, regardless of environmental conditions. For these reasons, we feel the fouling and defouling of ocean plastics must be studied in a wider range of conditions before it can be included in our global model. Though we do not include bioufouling, our model can still determine what observations are explainable without it (e.g. size-selective sorting at surface, removal of sub-100 micron particles from surface), and what measurements are unexplainable without some such density-increasing process (e.g. Limitations of Model We intend to improve our model as research on the transport of ocean plastic continues. For example, an improved methodology for incorporating wind-driven mixing based on (Kukulka et al., 2012) into a Lagrangian particle tracking model such as ours is currently under review (Onink et al., 2021b); we plan to update our methodology to reflect theirs, as it has stronger physical motivation. Also, the work of (Ruiz et al., 2004) suggests wind-driven mixing could paradoxically serve to increase the concentration of micro (<1 mm) debris at the surface, since turbulent conditions can increase the rising velocity of these small particles. This effect could be a crucial element of mixing dynamics, and we intend to investigate its impact by introducing it into our model. ADVECT does not model particle removal at boundaries, both at coastlines and the seabed, yet real marine plastic is often stranded on land or entrained in seafloor sediments (van Sebille et al., 2020). This behavior is omitted for the sake of simplicity, and because permanent beaching and sedimentation can be modeled in post-processing by simply truncating trajectories, e.g. (Kaandorp et al., 2020). We do not perform any such analyses here in order to limit the scope of the study. We believe coastal processes are a key component in the lifecycle of ocean plastics and emphasize that the results of this study must be interpreted with their absence in mind. Additionally, ADVECT does not attempt to incorporate every transport mechanism for ocean plastics. We focused on the few mechanisms which we believe to be most relevant for the three-dimensional transport of plastic in the ocean; for this reason, we notably left out transport by marine Stokes Drift, both because it can be approximated by artificially increasing windage (which our model supports), and because its effect is relatively small compared to transport due to currents and wind (van Sebille et al., 2020). We also focused on mechanisms which are well-developed and empirically validated; for this reason, we omitted biofouling, and interactions with biota more generally.i In addition to updating the existing dynamics, we intend to expand ADVECT in two important directions. First, while the importance of subsurface plastic is becoming clear, there remains a huge amount of debris, we suspect, at the sea- land boundary (L. Lebreton et al., 2019; Onink et al., 2021a). Discussion of Size Definitions presence of >.1mm particles of initially buoyant polymers in the ocean interior). These findings can help to focus future research. Frontiers in Marine Science \| www.frontiersin.org REFERENCES Eriksen, M., Lebreton, L. C. M., Carson, H. S., Thiel, M., Moore, C. J., Borerro, J. C., et al. (2014). Plastic Pollution in the World’s Oceans: More Than 5 Trillion Plastic Pieces Weighing Over 250,000 Tons Afloat at Sea. PLoS One 9 (12), e111913. doi: 10.1371/journal.pone.0111913 Amaral-Zettler, L. A., Zettler, E. R., Mincer, T. J., Klaassen, M. A. and Gallager, S. M. (2021). Biofouling Impacts on Polyethylene Density and Sinking in Coastal Waters: A Macro/Micro Tipping Point? Water Res. 201, 117289. doi: 10.1016/J.WATRES.2021.117289 Fazey, F. M. C. and Ryan, P. G. (2016). Biofouling on Buoyant Marine Plastics: An Experimental Study Into the Effect of Size on Surface Longevity. Environ. Pollut. 210, 354–360. doi: 10.1016/j.envpol.2016.01.026 f Pollut. 210, 354–360. doi: 10.1016/j.envpol.2016.01.026 Andrady, A. L. (2011). Microplastics in the Marine Environment. Mar. pollut. Bull. 62 (8). 1596-1605, doi: 10.1016/j.marpolbul.2011.05.030 Firing, E., Filipe, Barna, A. and Abernathey, R. (2021). TEOS-10/GSW-Python: V3.4.1.Post0. Zenodo. doi: 10.5281/ZENODO.5214122hh Black, K. P. and Gay, S. L. (1990). “A Numerical Scheme for Determining Trajectories in Particle Models,” in Acanthaster and the Coral Reef: A Theoretical Perspective. Ed. Bradbury, R. (Springer Berlin Heidelberg), 151–156. doi: 10.1007/978-3-642-46726-4_9 Gall, S. C., and Thompson, R. C. (2015). The Impact of Debris on Marine Life. Marine Pollution Bulletin 92, 170–179. doi: 10.1016/j.marpolbul.2014.12.041 Garcia, T. M., Campos, C. C., Mota, E. M. T., Santos, N. M. O., Campelo, R. P., de, S., et al. (2020). Microplastics in Subsurface Waters of the Western Equatorial Atlantic (Brazil). Mar. Pollut. Bull. 150, 110705. doi: 10.1016/j. marpolbul.2019.110705 Capet, X., Campos, E. J., and Paiva, A. M. (2008). Submesoscale activity over the Argentinian shelf. Geophys. Res. Lett. 35, L15605. doi: 10.1029/2008GL034736 García-Gómez, J. C., Garrigós, M. and Garrigós, J. (2021). Plastic as a Vector of Dispersion for Marine Species With Invasive Potential. A Review. Front. Ecol. Evol. 9. doi: 10.3389/fevo.2021.629756f Charnock, H. (1955). Wind Stress on a Water Surface. Q. J. R. Meteorological. Soc. 81 (350), 639–640. doi: 10.1002/qj.49708135027 Cole, S. T., Wortham, C., Kunze, E. and Owens, W. B. (2015). Eddy Stirring and Horizontal Diffusivity From Argo Float Observations: Geographic and Depth Variability. Geophysical. Res. Lett. 42 (10), 3989–3997. doi: 10.1002/2015GL063827 Gargett, A. E. (1984). Vertical Eddy Diffusivity in the Ocean Interior. J. Mar. Res. 42 (2), 359–393. doi: 10.1357/002224084788502756 Gelderloos, R., Szalay, A. S., Haine, T. W. N. and Lemson, G. (2016). DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. SUPPLEMENTARY MATERIAL DK and LL designed the study and model. DK and AP wrote the source code. DK ran the model simulations and analyzed the results. DK and LL wrote the manuscript and prepared the figures. All authors reviewed 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/fmars.2022.903134/ full#supplementary-material ACKNOWLEDGMENTS The authors are thankful to all donors who support The Ocean Cleanup. We would also like to acknowledge Tim Jansen for his early contribution in formulating the three-dimensional advection equations as part of his master’s thesis: A Lagrangian 3D Dispersal Model for Plastic Debris in the Eastern North Pacific. Finally, we thank Dr. Matthias Egger for his input and review of an earlier version of the manuscript. FUNDING if possible. Comparing in-situ density to pristine density on plastic samples will also be helpful in identifying the role of biofouling or other density-altering mechanisms and will help determine not just where plastic objects are, but how and why they arrived there. All funding for this research was provided by The Ocean Cleanup. All funding for this research was provided by The Ocean Cleanup. Limitations of Model Further, the impact of this debris is potentially outsized, given the importance of coastal regions for both humans and ecosystems. Therefore, we would like to incorporate more coastal dynamics and model the effect of beaching in particular. Though recent research has made strong headway towards a preliminary understanding of the nearshore behavior of plastic debris (L. Lebreton et al., 2019; Olivelli et al., 2020; Morales-Caselles et al., 2021; Onink et al., 2021b; Ryan & Perold, 2021), these processes are still not well observed on a global scale. Coastal processes are complex, and more observation is essential to assess what mechanisms most strongly govern the nearshore behavior of plastic debris, and thus what mechanisms should be incorporated into global dispersal models such as ADVECT. g y We would like to specifically discuss our decision to omit biofouling from ADVECT. Bioufouling has been an active area of research recently (Fazey & Ryan, 2016; Kaiser et al., 2017; Kooi et al., 2017; Lobelle et al., 2021), and it most likely plays an important role in removing floating plastic objects from the sea surface. However, we find the current models to be insufficiently developed to include at this time. In particular, the behavior of fouled objects below the euphotic zone is uncertain; while several studies roughly agree in the rate at which pristine objects accumulate biology and subsequently sink (Fazey & Ryan, 2016; Kaiser et al., 2017; Amaral-Zettler et al., 2021), few studies have investigated what happens to objects after they are removed from the surface. In fact, we are only aware of a single study on this topic (Ye & Andrady, 1991). Different assumptions about the rate of subsurface defouling lead to drastically different three dimensional trajectories for modeled particles, and due to a lack of observations, current biofouling models cannot resolve this Second, as research progresses, we intend to include a biofouling framework. However, we must again emphasize the need for further observational studies which extend beyond the ocean surface and the importance of recording more detailed and diverse particle properties. Particularly, in order to inform future modeling, observational studies should record all properties which impact the rising velocity of objects: namely their volume, shape, and in-situ density, July 2022 \| Volume 9 \| Article 903134 Frontiers in Marine Science \| www.frontiersin.org 7 Size Dependent Ocean Plastic Transport Klink et al. REFERENCES Technol. Lett. 6 (9), 551–557. doi: 10.1021/acs.estlett.9b00379 Richardson, P. L. (1997). Drifting in the Wind: Leeway Error in Shipdrift Data. Deep. Sea. Res. Part I.: Oceanographic. Res. Papers. 44 (11), 1877–1903. doi: 10.1016/S0967-0637(97)00059-9 Ross, P. S., Chastain, S., Vassilenko, E., Etemadifar, A., Zimmermann, S., Quesnel, S.-A., et al. (2021). Pervasive Distribution of Polyester Fibres in the Arctic Ocean Is Driven by Atlantic Inputs. Nat. Commun. 12 (1), 106. doi: 10.1038/ s41467-020-20347-1 Kooi, M., Nes, E. H., Scheffer, M. and Koelmans, A. A. (2017). 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Sustainability. 4 (6), 484–493. doi: 10.1038/s41893-021-00720-8 Conflict of Interest: DK, AP, and LL were employed by The Ocean Cleanup at the time of this research, a not-for-profit organization developing and scaling technology to rid the ocean of plastic. LL was employed by The Modelling House Limited. Olivelli, A., Hardesty, B. D. and Wilcox, C. (2020). Coastal Margins and Backshores Represent a Major Sink for Marine Debris: Insights From a Continental-Scale Analysis. Environ. Res. Lett. 15 (7). 484–493 doi: 10.1088/1748-9326/ab7836fk 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. Onink, V., Jongedijk, C. E., Hoffman, M. J., van Sebille, E. and Laufkötter, C. (2021a). 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REFERENCES Microplastic Pollution on Island Beaches, Oahu, Hawai`I. PLoS One 16 (2), e0247224. doi: 10.1371/ JOURNAL.PONE.0247224 July 2022 \| Volume 9 \| Article 903134 Frontiers in Marine Science \| www.frontiersin.org 9
https://openalex.org/W4361239919	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_BCR-ABL_Induced_Deregulation_of_the_IL-33_ST2_Pathway_in_CD34_Progenitors_from_Chronic_Myeloid_Leukemia_Patients/22398374/1/files/39844094.pdf	English	null	Supplementary Figure 2 from BCR-ABL–Induced Deregulation of the IL-33/ST2 Pathway in CD34(+) Progenitors from Chronic Myeloid Leukemia Patients	null	2,023	cc-by	532	Supplementary Figure S2 Supplementary Figure S2 B 103) p<0.004 A 4+ cells Days in culture p<0.02 p<0.04 p<0.005 Days in culture total cell number (x1 HD CML-CP fold-increase in CD34 ncorporation .D. value) p<0.05 ncorporation D. value) Days in culture y ncorporation .D. value) D p<0.03 p<0.02 C BrdU i (O. p<0.02 p<0.02 Days in culture BrdU in (O. BrdU i (O. - IL-33 - + + + - IgG - + - - + + anti-ST2 Supplementary Figure S2. CML-CP CD34(+) cells proliferate in response to IL-33. (A-D) Enriched primary CD34(+) cells (2.5x104 per well) from CML-CP patients or HDs were incubated for several days in the presence of IL-33 (50 ng/mL) with or without neutralizing anti- human ST2 antibody (anti-ST2, 1 μg/ml) or isotype-matched control (IgG, 1 μg/ml). (A) Viable cell recovery was calculated by dividing viable cell counts from 3-day cultures with IL-33 by those from cultures without IL-33. Each symbol represents one HD or CML-CP patient. Mann- y p p Whitney non-parametric test. (B) CML-CP CD34(+) cells were incubated with (⎯) or without (- --) IL-33 and were collected at day 3, 5 or 8 and viable cells were counted. (C) CD34(+) cell proliferation was determined by an 18-h pulse with BrdU on day 2, 4 or 7 and quantified by measuring the absorbance (O.D.). Data are means ± SEM from three separate experiments. Wilcoxon non-parametric test. (D) CD34(+) cell proliferation was determined by an 18-h pulse with BrdU on day 2 and quantified by measuring the absorbance (O.D.). Data are means ± SEM from three separate experiments. Wilcoxon non-parametric test. B 103) Days in culture p<0.02 p<0.04 p<0.005 Days in culture total cell number (x1 Days in culture y p<0.004 A 4+ cells HD CML-CP fold-increase in CD34 B A s ncorporation .D. value) p<0.05 ncorporation D. value) C BrdU i (O. p<0.02 p<0.02 Days in culture BrdU in (O. ncorporation .D. value) D p<0.03 p<0.02 BrdU i (O. - IL-33 - + + + - IgG - + - - + + anti-ST2 D C Supplementary Figure S2. CML-CP CD34(+) cells proliferate in response to IL-33. (A-D) Enriched primary CD34(+) cells (2.5x104 per well) from CML-CP patients or HDs were incubated for several days in the presence of IL-33 (50 ng/mL) with or without neutralizing anti- human ST2 antibody (anti-ST2, 1 μg/ml) or isotype-matched control (IgG, 1 μg/ml). Supplementary Figure S2 (A) Viable cell recovery was calculated by dividing viable cell counts from 3-day cultures with IL-33 by those from cultures without IL-33. Each symbol represents one HD or CML-CP patient. Mann- y p p Whitney non-parametric test. (B) CML-CP CD34(+) cells were incubated with (⎯) or without (- --) IL-33 and were collected at day 3, 5 or 8 and viable cells were counted. (C) CD34(+) cell proliferation was determined by an 18-h pulse with BrdU on day 2, 4 or 7 and quantified by measuring the absorbance (O.D.). Data are means ± SEM from three separate experiments. Wilcoxon non-parametric test. (D) CD34(+) cell proliferation was determined by an 18-h pulse with BrdU on day 2 and quantified by measuring the absorbance (O.D.). Data are means ± SEM from three separate experiments. Wilcoxon non-parametric test.
https://openalex.org/W2730182725	https://europepmc.org/articles/pmc5498884?pdf=render	English	null	Colonic metastasis from breast carcinoma: a case report	World journal of surgical oncology	2,017	cc-by	3,414	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tsujimura et al. World Journal of Surgical Oncology (2017) 15:124 DOI 10.1186/s12957-017-1193-5 Tsujimura et al. World Journal of Surgical Oncology (2017) 15:124 DOI 10.1186/s12957-017-1193-5 Background symptoms, and therefore, she underwent colonoscopy twice at this hospital. Colonoscopy indicated a bulge and mucosal inflammatory changes at the ileocecum. Biopsy of the lesion was performed twice; however, malignancy was not noted. Moreover, she was diagnosed with chole- lithiasis, and she underwent laparoscopic cholecystec- tomy in November 2015. During the operation, white spots were noted at the greater omentum, abdominal wall, small intestinal serosa, and mesentery as intraperi- toneal findings, and biopsies of the white spots at the greater omentum and abdominal wall did not indicate malignancy. The primary areas of metastasis from breast carcinoma are the bones, lungs, pleura, liver, and brain. Gastro- intestinal (GI) tract metastasis from breast carcinoma is relatively rare, and colonic metastasis from breast carcinoma is very rare [1, 2]. Here, we report a case of colonic metastasis from breast carcinoma. * Correspondence: tsujikazuma1978@yahoo.co.jp 1Department of Surgery, Tomishiro Central Hospital, 25 Azaueda, Tomishiro-shi, Okinawa 901-0243, Japan Full list of author information is available at the end of the article Colonic metastasis from breast carcinoma: a case report Kazuma Tsujimura1*, Tsuyoshi Teruya1, Masaya Kiyuna2, Kuniki Higa1, Junko Higa3, Kouji Iha1, Kiyoshi Chinen1, Masaya Asato1, Yasukatsu Takushi1, Morihito Ota1, Eijirou Dakeshita1, Atsushi Nakachi1, Akira Gakiya1 and Hiroshi Shiroma3 Background: Colonic metastasis from breast carcinoma is very rare. Here, we report a case of colonic metastasis from breast carcinoma. Case presentation: The patient was a 51-year-old woman. She had upper abdominal pain, vomiting, and diarrhea, repeatedly. We performed abdominal contrast-enhanced computed tomography (CT) to investigate these symptoms. The CT scan revealed a tumor in the ascending colon with contrast enhancement and showed an expanded small intestine. For further investigation of this tumor, we performed whole positron emission tomography-computed tomography (PET-CT). The PET-CT scan revealed fluorodeoxyglucose uptake in the ascending colon, mesentery, left breast, and left axillary region. Analysis of biopsy samples obtained during colonoscopy revealed signet ring cell-like carcinoma. Moreover, biopsy of the breast tumor revealed invasive lobular carcinoma. Therefore, the preoperative diagnosis was colonic metastasis from breast carcinoma. Open ileocecal resection was performed. The final diagnosis was multiple metastatic breast carcinomas, and the TNM classification was T2N1M1 Stage IV. Conclusions: We presented a rare case of colonic metastasis from breast carcinoma. PET-CT may be useful in the diagnosis of metastatic breast cancer. When analysis of biopsy samples obtained during colonoscopy reveals signet ring cell-like carcinoma, the possibility of breast cancer as the primary tumor should be considered. Keywords: Breast carcinoma, Colonic metastasis, Positron emission tomography-computed tomog Abstract Background: Colonic metastasis from breast carcinoma is very rare. Here, we report a case of colonic metastasis from breast carcinoma. Case presentation The patient was a 51-year-old woman without a relevant medical history. She presented at another hospital be- cause of upper abdominal pain, vomiting, and diarrhea in July 2015. She was diagnosed with acute enteritis. Subsequently, she repeatedly experienced the same She again had the same symptoms in January 2016, and she was admitted to the same hospital for further examination and treatment. Eventually, for close examination, she was transferred to our hospital. In our hospital, abdominal contrast-enhanced computed tomography (CT) was performed to investigate her Page 2 of 6 Page 2 of 6 Page 2 of 6 Tsujimura et al. World Journal of Surgical Oncology (2017) 15:124 symptoms. The CT scan revealed a tumor in the ascend- ing colon with contrast enhancement and showed an ex- panded small intestine (Fig. 1). For further investigation, we performed whole positron emission tomography- computed tomography (PET-CT) using Siemens True Point Biograph 16 (Siemens, Erlangen, Germany). The PET-CT scan revealed fluorodeoxyglucose (FDG) uptake in the ascending colon, mesentery, left breast, and left axillary region, with maximum standardized uptake values (SUVmax) of 5.0, 2.8, 6.4, and 2.1, respectively (Fig. 2a–d). Additionally, colonoscopy indicated swelling and stenosis of the ascending colon (Fig. 3), and we could not pass the endoscope to the ileocecum. Biopsies of the swelling and stenosis of the ascending colon were performed. Hematoxylin and eosin (HE) staining of the biopsy samples revealed signet ring cell-like carcinoma (Fig. 4). Upper gastrointestinal endoscopic examination showed normal findings. Moreover, we performed ultra- sonography (US) and biopsy of the left breast tumor identified using PET-CT. US revealed an ill-defined, ir- regular, and hypervascular hypoechoic tumor measuring 36 × 22 × 22 mm (Fig. 5). HE staining of the breast tumor sample revealed invasive lobular carcinoma, and cadherin staining was negative (Fig. 6). In March Fig. 1 Contrast-enhanced computed tomography (CT). A contrast- enhanced CT image reveals a tumor with contrast enhancement and shows an expanded small intestine Fig. 1 Contrast-enhanced computed tomography (CT). A contrast- enhanced CT image reveals a tumor with contrast enhancement and shows an expanded small intestine a c b d Fig. 2 Positron emission tomography-computed tomography (PET-CT). a A whole PET-CT image shows fluorodeoxyglucose (FDG) uptake in the ascending colon. The maximum standardized uptake value (SUVmax) is 5.0. b A whole PET-CT image shows FDG uptake in the mesentery. The SUVmax is 2.8. Case presentation c A whole PET-CT image shows FDG uptake in the left breast. The SUVmax is 6.4. d A whole PET-CT image shows FDG uptake in the left axillary region. The SUVmax is 2.1 a c c a c a b d Fig. 2 Positron emission tomography-computed tomography (PET-CT). a A whole PET-CT image shows fluorodeoxyglucose (FDG) uptake in the ascending colon. The maximum standardized uptake value (SUVmax) is 5.0. b A whole PET-CT image shows FDG uptake in the mesentery. The SUVmax is 2.8. c A whole PET-CT image shows FDG uptake in the left breast. The SUVmax is 6.4. d A whole PET-CT image shows FDG uptake in the left axillary region. The SUVmax is 2.1 b d Fig. 2 Positron emission tomography-computed tomography (PET-CT). a A whole PET-CT image shows fluorodeoxyglucose (FDG) uptake in the ascending colon. The maximum standardized uptake value (SUVmax) is 5.0. b A whole PET-CT image shows FDG uptake in the mesentery. The SUVmax is 2.8. c A whole PET-CT image shows FDG uptake in the left breast. The SUVmax is 6.4. d A whole PET-CT image shows FDG uptake in the left axillary region. The SUVmax is 2.1 Tsujimura et al. World Journal of Surgical Oncology (2017) 15:124 Page 3 of 6 Fig. 3 Colonoscopy. Colonoscopy shows swelling and stenosis of the ascending colon Fig. 5 Ultrasonography (US). A US image of the breast shows an ill-defined, irregular, and hypervascular hypoechoic tumor measuring 36 × 22 × 22 mm Fig. 5 Ultrasonography (US). A US image of the breast shows an ill-defined, irregular, and hypervascular hypoechoic tumor measuring 36 × 22 × 22 mm Fig. 3 Colonoscopy. Colonoscopy shows swelling and stenosis of the ascending colon 2016, the patient continued to experience abdominal pain and vomiting. Abdominal contrast-enhanced CT was performed again, and it revealed continuous ileus due to an ileocecal mass; therefore, open ileocecal re- section was scheduled. With regard to tumor markers, before operation, the CA125 level was high (141 U/ ml), while the CEA (3.2 ng/ml), CA19-9 (11.4 U/ml), and AFP (<2.0 ng/ml) levels were normal. The pre- operative diagnosis was colonic metastasis from breast carcinoma. mesentery, and throughout the observed area. Biopsies of the spots were performed simultaneously. A surgical specimen from the ileocecal region showed a normal mucosal layer, and a tumor was noted at the submucosal region. Case presentation Because the tumor involved the entire circumfer- ence of the intestinal tract and developed in an inward direction, there was stenosis of the ileocecum. The tumor size was 3 cm in the longitudinal direction (Fig. 7a, b). Histological examination revealed invasive lobular carcinoma (Fig. 8a). The positive rates of estro- gen receptor, progesterone receptor, human epidermal Open ileocecal resection was performed, and white spots were noted at the peritoneum, pouch of Douglas, Fig. 4 Hematoxylin and eosin (HE) staining. HE staining of the biopsy samples obtained during colonoscopy reveals signet ring cell-like carcinoma Fig. 6 Hematoxylin and eosin (HE) staining. HE staining of the biopsy sample of the breast tumor reveals invasive lobular carcinoma Fig. 4 Hematoxylin and eosin (HE) staining. HE staining of the biopsy samples obtained during colonoscopy reveals signet ring cell-like carcinoma Fig. 6 Hematoxylin and eosin (HE) staining. HE staining of the biopsy sample of the breast tumor reveals invasive lobular carcinoma Fig. 4 Hematoxylin and eosin (HE) staining. HE staining of the biopsy samples obtained during colonoscopy reveals signet ring cell-like carcinoma Fig. 4 Hematoxylin and eosin (HE) staining. HE staining of the biopsy samples obtained during colonoscopy reveals signet ring cell-like carcinoma Fig. 6 Hematoxylin and eosin (HE) staining. HE staining of the biopsy sample of the breast tumor reveals invasive lobular carcinoma Page 4 of 6 Tsujimura et al. World Journal of Surgical Oncology (2017) 15:124 Page 4 of 6 colonic metastasis from breast carcinoma is extremely rare, with a frequency of approximately 3% in a review of the literature [5]. a b Fig. 7 Surgical specimen. a A surgical ileocecal specimen shows a normal mucosal layer. The tumor involves the entire circumference of the intestinal tract and develops in an inward direction. The tumor size is 3 cm in the longitudinal direction. b A surgical ileocecal specimen shows a tumor in the submucosal region a a In the present case, there was continuous ileus due to an ileocecal mass, and open ileocecal resection was per- formed. Before the operation, PET-CT was useful for preoperative diagnosis, and the preoperative diagnosis was colonic metastasis from breast carcinoma. Gener- ally, FDG-PET-CT is not recommended as the primary diagnostic procedure in breast cancer. Its sensitivity ranges from 48 to 96% and specificity from 73 to 100%. However, it has the potential to be useful for the detec- tion of distant metastases. Case presentation In such a case, its sensitivity has been reported to range from 80 to 100% and specifi- city from 75 to 100% [6]. In another report, for detecting distant metastatic disease, the sensitivity and specificity of FDG-PET-CT were shown to be greater than the sen- sitivity and specificity of conventional imaging [7]. How- ever, FDG-PET-CT is not suitable for the detection of primary tumors owing to its low sensitivity for tumors measuring 0–10 mm [6]. In this case, we performed PET-CT to further investigate a tumor in the ascending colon that was identified using abdominal contrast- enhanced CT. As mentioned previously, PET-CT is not recommended as a primary diagnostic procedure in breast cancer. However, in this case, as distant metasta- sis was noted and the primary breast cancer was rela- tively large, we could make a preoperative diagnosis of colonic metastasis from breast carcinoma. b Fig. 7 Surgical specimen. a A surgical ileocecal specimen shows a normal mucosal layer. The tumor involves the entire circumference of the intestinal tract and develops in an inward direction. The tumor size is 3 cm in the longitudinal direction. b A surgical ileocecal specimen shows a tumor in the submucosal region b Fig. 7 Surgical specimen. a A surgical ileocecal specimen shows a normal mucosal layer. The tumor involves the entire circumference of the intestinal tract and develops in an inward direction. The tumor size is 3 cm in the longitudinal direction. b A surgical ileocecal specimen shows a tumor in the submucosal region In this case, histological examination revealed invasive lobular carcinoma. Among the different histological types of breast carcinoma, it has been reported that lobular carcinoma and mucinous carcinoma metastasize relatively frequently to the GI tract [3, 8, 9]. One histo- logical feature of colonic metastasis from breast carcin- oma is the limited existence in the submucosa, muscles, and chorionic membrane layer. Especially, the tumor markedly invades the muscle layer, and the mucosal layer is normal [8, 10, 11]. As the mucosal layer is nor- mal, it is essential to perform deep sampling during col- onoscopy. In this case, biopsy during colonoscopy was performed thrice; however, a correct pathological diag- nosis was not obtained. According to our findings, co- lonic metastasis from breast carcinoma is asymptomatic in the early stage, and symptoms due to stenosis occur only in the advanced stage, making early detection diffi- cult. Case presentation In this case, the tumor size was small in the longi- tudinal direction, but the tumor involved the entire circumference of the intestinal tract and developed in an inward direction. Therefore, there was continuous ileus due to stenosis of the ileocecum. growth factor receptor 2, and Ki-67 staining were almost 100, 2–3, 0, and 5–10%, respectively (Fig. 8b–e). The final diagnosis was multiple metastatic breast carcin- omas, and the TNM classification was T2N1M1 stage IV. The histological grade of this tumor according to the College of American Pathologists (CAP) was grade 2 (score 7). On postoperative day 8, anastomotic leakage oc- curred, and we performed open drainage and ileos- tomy. Subsequently, her general condition was good. We started treatment with letrozole. The duration of hospitalization was long, and she was discharged 1.5 months after open ileocecal resection. At 9 months after the operation, we continue to administer letro- zole, and she has stable disease. Submit your next manuscript to BioMed Central and we will help you at every step: p Informed consent was obtained from the patient for the publication of this case report. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: Competing interests The authors declare that they have no competing interests. Discussion Invasive lobular carcinoma is associated with mucus in the intracellular region, and the histological features may indicate signet ring cell-like carcinoma [12]. When GI metastasis from breast carcinoma is relatively rare and has been reported to occur in 4–18% of dissemi- nated breast cancer patients [3, 4]. Especially, the rate of Tsujimura et al. World Journal of Surgical Oncology (2017) 15:124 Page 5 of 6 a b c d e Fig. 8 (See legend on next page.) a b c c d d e e Tsujimura et al. World Journal of Surgical Oncology (2017) 15:124 Page 6 of 6 (See figure on previous page.) Fig. 8 Histological examination. The findings of histological examination indicate invasive lobular carcinoma. a Hematoxylin and eosin staining of the tumor (×400). b Estrogen receptor (ER) staining of the tumor (×200). The positive rate of ER was 100%. c Progesterone receptor (PgR) staining of the tumor (×400). The positive rate of PgR was 2–3%. d Human epidermal growth factor receptor 2 (Her-2) staining of the tumor (×400). The positive rate of Her-2 was 0%. E Ki-67 staining of the tumor (×400). The positive rate of Ki-67 was 5–10% analysis of biopsy samples obtained during colonos- copy reveals signet ring cell-like carcinoma, colonic metastasis from breast carcinoma should be suspected. With regard to the treatment of colonic metastasis from breast carcinoma, even though tem- porizing treatment, narrow colon is resected and nutritional condition may be improved. Moreover, treatment success with chemotherapy and hormo- notherapy after operation has been reported [13]. Tomishiro Central Hospital, Okinawa, Japan. 3Department of Surgery, Nanbu Hospital, Okinawa, Japan. Received: 30 March 2017 Accepted: 22 June 2017 Abbreviations d CT: Computed tomography; ER: Estrogen receptor; FDG: Fluorodeoxyglucose; GI: Gastrointestinal; HE: Hematoxylin and eosin; Her-2: Human epidermal growth factor receptor 2; PET-CT: Positron emission tomography-computed tomography; PgR: Progesterone receptor; SUVmax: Maximum standardized uptake value; US: Ultrasonography. 7. Constantinidou A, Martin A, Sharma B, Johnston SRD. Positron emission tomography/computed tomography in the management of recurrent/ metastatic breast cancer: a large retrospective study from the Royal Marsden Hospital. Ann Oncol. 2011;22:307–14. 7. Constantinidou A, Martin A, Sharma B, Johnston SRD. Positron emission tomography/computed tomography in the management of recurrent/ metastatic breast cancer: a large retrospective study from the Royal Marsden Hospital. Ann Oncol. 2011;22:307–14. Marsden Hospital. Ann Oncol. 2011;22:307–14. 8. Koos L, Field RE. Metastatic carcinoma of breast simulating Crohn’s disease. Int Surg. 1979;65:359–62. 8. Koos L, Field RE. Metastatic carcinoma of breast simulating Crohn’s disease. Int Surg. 1979;65:359–62. References Dh S 1. Dhar S, Kulaylat MN, Gordon K, Lall P, Doerr RJ. Solitary papillary breast carcinoma metastasis to the large bowel presenting as primary coloncarcinoma: case report and review of the literature. Am Surg. 2003;69 799–803. 2. Law WL, Chu KW. Scirrhous colonic metastasis from ductal carcinoma of the breast: report of a case. Dis Colon Rectum. 2003;46:1424–7. Conclusions 3. Cormier WJ, Gaffey TA, Welch JM, Welch JS, Edmonson JH. Linitis plastica caused by metastatic lobular carcinoma of the breast. Mayo Clin Proc. 1980; 55:747–53. PET-CT may be useful in the diagnosis of metastatic breast cancer. When analysis of biopsy samples obtained during colonoscopy reveals signet ring cell-like carcin- oma, the possibility of breast cancer as the primary tumor should be considered. 4. Taal BG, den Hartog Jager FCA, Steinmetz R, Peterse H. The spectrum of gastrointestinal metastases of breast carcinoma: II. The colon and rectum. Gastrointest Endosc. 1992;38:136–41. 4. Taal BG, den Hartog Jager FCA, Steinmetz R, Peterse H. The spectrum of gastrointestinal metastases of breast carcinoma: II. The colon and rectum. Gastrointest Endosc. 1992;38:136–41. 5. Klein MS, Sherlock P. Gastric and colonic metastases from breast cancer. Am J Dig Dis. 1972;17:881–6. 6. Warning K, Hildebrandt MG, Kristensen B, Ewertz M. Utility of 18FDG-PET/CT in breast cancer diagnostics–a systematic review. Dan Med Bull. 2011;58: A4289. Acknowledgements W f ll k l We gratefully acknowledge the advice and support of Dr. Takayoshi Toda and Ms. Shoko Nakaema. We would like to thank Editage (http://www.editage.jp/) for English language editing. 9. Fayemi AO, Ali M, Braun EV. Metastatic carcinoma simulating linitis plastica of the colon. Am J Gastroenterolo. 1979;71:311–4. We gratefully acknowledge the advice and support of Dr. Takayoshi Toda and Ms. Shoko Nakaema. We would like to thank Editage (http://www.editage.jp/) for English language editing. 10. Weisberg A. Metastatic adenocarcinoma of the breast masquerading as Crohn’s disease of the colon. American J Proctol Gastro. 1982;33:10. Ethics approval and consent to participate Ethics approval and consent to participate Approval for the study was obtained from the institutional review board of Tomishiro Central Hospital. Funding 11. Rabau MY, Alon RJ, Werbin N, Yossipov Y. Colonic metastases from lobular carcinoma of the breast. Dis Colon Rectum. 1988;31:401–2. Funding The authors declare that there was no funding for this case report. Funding The authors declare that there was no funding for this case report. carcinoma of the breast. Dis Colon Rectum. 1988;31:401–2. 12. Livolsi VA. Signet ring carcinoma of the female breast: a clinicopathologic analysis of 24 cases. Cancer. 1981;48:1830–7. 12. Livolsi VA. Signet ring carcinoma of the female breast: a clinicopathologic analysis of 24 cases. Cancer. 1981;48:1830–7. Availability of data and materials We do not wish to share our patient data. Because this is a case report and privacy of this participant should be protected. Availability of data and materials Availability of data and materials We do not wish to share our patient data. Because this is a case report and privacy of this participant should be protected. Availability of data and materials We do not wish to share our patient data. Because this is a case report and privacy of this participant should be protected. 13. Clavien PA, Laffer U, Torhost J, Harder F. Gastro-intestinal metastases as first clinical manifestation of the dissemination of a breast cancer. Eur J Surg Oncol. 1990;16:121–6. 13. Clavien PA, Laffer U, Torhost J, Harder F. Gastro-intestinal metastases as first clinical manifestation of the dissemination of a breast cancer. Eur J Surg Oncol. 1990;16:121–6. Authors’ contributions All authors participated in the treatment of this patient. All authors read and approved the final manuscript. Authors’ contributions All authors participated in the treatment of this patient. All authors read and approved the final manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4392434824	https://jonsat.nstri.ir/article_1603_7cb85902110dfa372ff8bac850f97b6a.pdf	Chinese	null	تولید موتانت‌های بنفشه آفریقایی (Saintpaulia ionantha) با استفاده از تابش اشعه گاما	Majallah-i ̒ulūm va funūn-i hastah/ī	2,024	cc-by	8,007	Production of African violet mutants (Saintpaulia ionantha) using gamma radiation A. Sharifi1, Z. Sargazi Moghaddam2, M. Kharrazi1, A. Khadem1, M. Moradian1 1. Horticultural Plants Biotechnology Department, Industrial Biotechnology Institute, ACECR- Mashhad Branch, P.O.BOX: 917751376, Mashhad - Iran 2. Plant Breeding and Biotechnology, Ferdowsi University of Mashhad, P.O.BOX: 9177948974, Mashhad – Iran Research Article Received: 19.4.2023, Revised: 29.7.2023, Accepted: 19.9.2023 ﻮﻟﯿﺪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ )Saintpaulia ionantha ( ﺑﺎ اﺳﺘﻔﺎده از ﺗﺎﺑﺶ اﺷﻌﻪ ﮔﺎﻣﺎ ﺗﻮﻟﯿﺪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ )Saintpaulia ionantha ( ﺑﺎ اﺳﺘﻔﺎده از ﺗﺎﺑﺶ اﺷﻌﻪ ﮔﺎﻣﺎ اﺣﻤﺪ ﺷﺮﯾﻔﯽ 1 ، زﻫﺮا ﺳﺮﮔﺰي ﻣﻘﺪم2، ﻣﻬﺪﯾﻪ ﺧﺮازي1 ، آزاده ﺧﺎدم1، ﻣﺮﯾﻢ ﻣﺮادﯾﺎن1 1 ،. ﮔﺮوه ﺑﯿﻮﺗﮑﻨﻮﻟﻮژي ﮔﯿﺎﻫﺎن ﺑﺎﻏﺒﺎﻧﯽ، ﭘﮋوﻫﺸﮑﺪه ﺑﯿﻮﺗﮑﻨﻮﻟﻮژي ﺻﻨﻌﺘﯽ، ﺟﻬﺎد داﻧﺸﮕﺎﻫﯽ ﺧﺮاﺳﺎن رﺿﻮي ﺻﻨﺪوق ﭘﺴﺘﯽ: 917751376 ، ﻣﺸﻬﺪ - اﯾﺮان 2 ،. ﺑﯿﻮﺗﮑﻨﻮﻟﻮژي و ﺑﻪ ﻧﮋادي ﮔﯿﺎﻫﯽ، داﻧﺸﮕﺎه ﻓﺮدوﺳﯽ ﻣﺸﻬﺪ ﺻﻨﺪوق ﭘﺴﺘﯽ: 9177948974 ، ﻣﺸﻬﺪ- اﯾﺮان Email: a-sharifi@jdm.ac.ir ﻣﻘﺎﻟﻪ ي ﭘﮋوﻫﺸﯽ :ﺗﺎرﯾﺦ درﯾﺎﻓﺖ ﻣﻘﺎﻟﻪ30 /1 / 1402 ﺗ ﺎرﯾﺦ ﺑﺎز ﻧﮕﺮي ﻣﻘﺎﻟﻪ: 7 /5 / 1402 ﺗ ﺎرﯾﺦ ﭘﺬﯾﺮش ﻣﻘﺎﻟﻪ: 28 /6/ 1402 اﯾﻦ ﺗﺤﻘﯿﻖ ﺑﺎ ﻫﺪف ﺑﺮرﺳﯽ اﻣﮑﺎن اﯾﺠﺎد ﺗﻨﻮع و دﺳﺖ ﯾﺎﺑﯽ ﺑﻪ ژﻧﻮﺗﯿﭗ ﻫﺎي ﺟﺪﯾﺪ ﺑﺎ اﺳﺘﻔﺎده از اﺷﻌﻪ ﮔﺎﻣﺎ در ﮐﺸﺖ درون ﺷﯿﺸﻪ اي ﮔﯿﺎه ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ اﻧﺠﺎم ﺷﺪ. در اﯾﻦ ﺑﺮرﺳﯽ اﺑﺘﺪا رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎ در ﺷﺮاﯾﻂ اﯾﻦ وﯾﺘﺮو ﮐﺸﺖ و ﺳﭙﺲ ﺑﺎ دُزﻫﺎي ﻣﺨﺘﻠﻒ اﺷﻌﻪ ﮔﺎﻣﺎ ﺗﯿﻤﺎر ﺷﺪﻧﺪ. ﻧﺘﺎﯾﺞ ﻧﺸﺎن داد ﮐﻪ اﻓﺰاﯾﺶ دُز اﺷﻌﻪ ﮔﺎﻣﺎ ﺳﺒﺐ ﮐﺎﻫﺶ درﺻﺪ ﺑﺎززاﯾﯽ رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎ، ﺗﻌﺪاد ﮔﯿﺎﻫﭽﻪ ﺑﺎززا ﺷﺪه و ﺗﻌﺪاد ﺑﺮگ آن ﻫﺎ ﺷﺪ. ﻫﻢ ﭼﻨﯿﻦ ا ﻓﺰاﯾﺶ دُز اﺷﻌﻪ ﮔﺎﻣﺎ ﺳﺒﺐ اﻓﺰاﯾﺶ ﺗﻨﻮع و ﺗﻐﯿﯿﺮات ﻣﻮرﻓﻮﻟﻮژﯾﮏ در ﮔﯿﺎﻫﺎن ﺑﺎززا ﺷﺪه ﮔﺮدﯾﺪ. ﺑﻪ ﻃﻮري ﮐﻪ در ﺑﺮگ ﻫﺎ ﺗﻐﯿﯿﺮ رﻧﮓ، ﺷﮑﻞ، اﻧﺪازه و ﭘﯿﭽﺶ ﻣﺸﺎﻫﺪه ﺷﺪ و ﺑﺮﺧﯽ ﮔﻞ ﻫﺎ ﮐﻢ ﭘَﺮ ﺷﺪﻧﺪ و ﯾﺎ ﺗﻐﯿﯿﺮ ﺷﮑﻞ ﯾﺎﻓﺘﻨﺪ. ارزﯾﺎﺑﯽ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﻧﻤﻮﻧﻪ ﻫﺎي ﺗﯿﻤﺎر ﺷﺪه ﺑﺎ اﺳﺘﻔﺎده از ﻧﺸﺎﻧﮕﺮ ISSR و8 آﻏﺎزﮔﺮ در اﻧﺠﺎم واﮐﻨﺶ PCR اﻧﺠﺎم ﺷﺪ ﮐﻪ در ﻣﺠﻤﻮع71 ﺑﺎﻧﺪ ﻗﺎﺑﻞ اﻣﺘﯿﺎزدﻫﯽ اﯾﺠﺎد ﺷﺪ. ﺑﺮرﺳﯽ دﻧﺪروﮔﺮام ﺣﺎﺻﻞ از ﺗﺠﺰﯾﻪ ﺧﻮﺷﻪ اي ﺑﺎ اﺳﺘﻔﺎده از ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ﺟﺎﮐﺎرد و روش اﻟﮕﻮرﯾﺘﻢ UPGMA ﺗﻨﻮع ﺑﺎﻻﯾﯽ ﺑﯿﻦ در را ﻧﻤﻮﻧﻪ ﻫﺎي ﻣﻮرد ﺑﺮرﺳﯽ ﻧﺸﺎن . داد ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي اﯾﺠﺎد ﺷﺪه ﺑ ﺎ ﯾﮏ ﺳﻄﺢ ﺗﯿﻤﺎر در ﺳﻄﺢ ﺗﺸﺎﺑﻪ 51 /0 درﺻﺪ ﺑﻪ4 ﮔﺮوه اﺻﻠﯽ ﺗﻘﺴﯿﻢ ﺑﻨﺪي ﺷﺪﻧﺪ و داﻣﻨﻪ ﺗﺸﺎﺑﻪ ژﻧﺘﯿﮑﯽ از 98 /0 - 45 /0 ﻣﺘﻐﯿﺮ ﺑﻮد. در ﻧﻬﺎﯾﺖ5 ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﺣﺎﺻﻞ از دُزﻫﺎي ﻣﺨﺘﻠﻒ اﺷﻌﻪ ﮔﺎﻣﺎ ﺑﺎ ﻣﻘﺎﯾﺴﻪ ﺗﻐﯿﯿﺮات ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ و ﻣﻮﻟﮑﻮﻟﯽ ﻣﻮرد ﺗﺄﯾﯿﺪ ﻗﺮار ﮔﺮﻓﺖ. ﻧﺘﺎﯾﺞ اﯾﻦ ﺗﺤﻘﯿﻖ ﻧﺸﺎن داد ﮐﻪ ﻣﯽ ﺗﻮان از ﺗﺎﺑﺶ ﮔﺎﻣﺎ و اﻟﻘﺎي ﺗﻨﻮع در ﺷﺮاﯾﻂ ﮐﺸﺖ ﺑﺎﻓﺖ ﺑﻪ ﻋﻨﻮان راﻫﺒﺮدي ﻣﻨﺎﺳﺐ ﺟﻬﺖ اﻓﺰاﯾﺶ ﺗﻨﻮع در اﯾﻦ ﮔﯿﺎه اﺳﺘﻔﺎده ﻧﻤﻮد . Journal of Nuclear Science, Engineering and Technology Vol. 45 (4), Serial Number 109, 2024 1 . ﻣﻘﺪﻣﻪ اﺻﻼح ﺟﻬﺶ زا در ﮔﯿﺎﻫﺎن ﺑﻪ ﻋﻨﻮان ﯾﮑﯽ از راه ﻫﺎي اﻓﺰاﯾﺶ ﺗﻨﻮع در ﺟﻤﻌﯿﺖ ﻫﺎي اﺻﻼح ﺷﺪه از ﻃﺮﯾﻖ ﺗﻼﻗﯽ ﻣﯽ ﺑﺎﺷﺪ ]1 [ . ﺟﻬﺶ در ﮔﯿﺎﻫﺎن ﺑﻪ دو ﺻﻮرت ﺧﻮدﺑﻪ ﺧﻮدي و ﯾﺎ ﺑﺎ اﺳﺘﻔﺎده از ﻋﻮاﻣﻞ ﺟﻬﺶ زا اﯾﺠﺎد ﻣﯽ ﺷﻮد ﮐﻪ در اﯾﻦ ﻣﯿﺎن، ﭘﺎﯾﯿﻦ ﺑﻮدن ﻧﺮخ ﺟﻬﺶ ﻃﺒﯿﻌﯽ ﻣﻨﺠﺮ ﺑﻪ ﻋﺪم ﮐﺎرﺑﺮد اﯾﻦ روش در ﺗﻮﻟﯿﺪ ارﻗﺎم ﮔﯿﺎﻫﯽ ﺟﺪﯾﺪ ﺷﺪه اﺳﺖ. ﺑﺮرﺳﯽ ﻫﺎي ﺑﺴﯿﺎري ﺑﻪ ﻣﻨﻈﻮر اﻓﺰاﯾﺶ اﻟﻘﺎي ﺟﻬﺶ ﺑﺎ ﺑﻪ ﮐﺎرﮔﯿﺮي ﻋﻮاﻣﻞ ﺟﻬﺶ زاي ﻣﺨﺘﻠﻒ ﺻﻮرت ﮔﺮﻓﺘﻪ اﺳﺖ ﮐﻪ ﻧﺘﺎﯾﺞ اﯾﻦ ﺗﺤﻘﯿﻘﺎت ﻧﺸﺎن دﻫﻨﺪه اﺛﺮ ﻣﻌﻨﯽ دار ﮐﺎرﺑﺮد ﻋﻮاﻣﻞ ﺟﻬﺶ زا ﺑﺮ اﻟﻘﺎي ﺟﻬﺶ در ﺳﻠﻮل ﻫﺎي ﮔﯿﺎﻫﯽ اﺳﺖ. از اﯾﻦ رو در ﺑﺮﻧﺎﻣﻪ ﻫﺎي اﺻﻼح ﺟﻬﺶ زا در ﮔﯿﺎﻫﺎن ﻣﻌﻤﻮﻻً از ﻋﻮاﻣﻞ ﺟﻬﺶ زا ﺑﻪ ﻣﻨﻈﻮر اﻟﻘﺎي ﺟﻬﺶ اﺳﺘﻔﺎده ﺷﺪه اﺳﺖ ]2 [ . داوودي ﻣﺎﻧﻨﺪ ﻣﻬﻢ زﯾﻨﺘﯽ ﮔﯿﺎﻫﺎن از ﺑﺮﺧﯽ در )Chrysanthemum morifolium ( ﺗﻌﺪاد ﺑﺴﯿﺎر زﯾﺎدي رﻗﻢ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﺑﻪ ﺛﺒﺖ رﺳﯿﺪه اﺳﺖ ]3 ، 4 [. ﻫﻢ ﭼﻨﯿﻦ ﺗﻐﯿﯿﺮ ﺧﺼﻮﺻﯿﺎت ﺑﺮگ ﻫﺎ در اﺻﻼح ﺟﻬﺶ زاي ﮔﯿﺎﻫﺎن زﯾﻨﺘﯽ از ﺟﻤﻠﻪ ﻟﯿﻠﯿﻮم و داوودي ﺻﻮرت ﮔﺮﻓﺘﻪ اﺳﺖ ]5 ، 6 [ . در ﭘﮋوﻫﺸﯽ دﯾﮕﺮ ﺳﻮاﻧﺴﺮي و ﻫﻤﮑﺎران ]7 [ ﺑﺎ ﭘﺮﺗﻮﺗﺎﺑﯽ رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﮔﺮه ﮔﯿﺎه Torenia hybrida ﺑﻪ ﮔﯿﺎﻫﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ اي ﺑﺎ ﮔﻞ ﻫﺎي آﺑﯽ روﺷﻦ، ﺻﻮرﺗﯽ و ﺻﻮرﺗﯽ روﺷﻦ دﺳﺖ ﯾﺎﻓﺘﻨﺪ در ﺣﺎﻟﯽ ﮐﻪ ﮔﻞ ﻫﺎي ﻃﺒﯿﻌﯽ آﺑﯽ رﻧﮓ ﺑﻮدﻧﺪ. ﮐﺎرﺑﺮد ﺗﮑﻨﯿﮏ ﻫﺎي اﻟﻘﺎي ﺟﻬﺶ ﻫﻤﺮاه ﺑﺎ اﻧﺘﺨﺎب ﺑﺮ اﺳﺎس ﻧﺸﺎﻧﮕﺮﻫﺎ، ﺟﻬﺶ زا ﻫﺎ را ﮐﺎرﺑﺮدي ﺗﺮ ﺧﻮاﻫﺪ ﺳﺎﺧﺖ. در روش ﻫﺎي ﺟﻬﺶ زاﯾﯽ در ﺷﺮاﯾﻂ درون ﺷﯿﺸﻪ اي ﻣﯽ ﺗﻮان ﺑﺎ اﺳﺘﻔﺎده از ﻧﺸﺎﻧﮕﺮﻫﺎي ﻣﺨﺘﻠﻒ ﺑﻪ ﺑﺮرﺳﯽ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﭘﺮداﺧﺘﻪ و اﻧﻮاع ﺟﻬﺶ را ﺷﻨﺎﺳﺎﯾﯽ ﻧﻤﻮد ]8 [. ﻧﺸﺎﻧﮕﺮﻫﺎي ﻣﻮﻟﮑﻮﻟﯽ ﯾﮑﯽ از اﺑﺰارﻫﺎي ﺑﺴﯿﺎر ﻣﻬﻢ ﻗﻮي و در زﻣﯿﻨﻪ ارزﯾﺎﺑﯽ رواﺑﻂ ﺧﻮﯾﺸﺎوﻧﺪي ژﻧﺘﯿﮑﯽ، اﻧﺘﺨﺎب ﮔﯿﺎﻫﺎن ﺑﺮﺗﺮ و ﺑﺮرﺳﯽ ﺷﺒﺎﻫﺖ ﯾﺎ ﺗﻔﺎوت ﺑﯿﻦ ﻧﻤﻮﻧﻪ ﻫﺎي ﻣﺨﺘﻠﻒ ﻣﯽ ﺑﺎﺷﻨﺪ ]9 [ . 1 ISSR - ﻫﺎ ﻧﺸﺎﻧﮕﺮﻫﺎﯾﯽ ﻫﺴﺘﻨﺪ ﮐﻪ ﺗﻮﺳﻂPCR در ﺣﻀﻮر آﻏﺎزﮔﺮي ﮐﻪ ﻣﮑﻤﻞ رﯾﺰ ﻣﺎﻫﻮاره ﻫﺪف اﺳﺖ، ﺗﮑﺜﯿﺮ ﻣﯽ ﺷﻮﻧﺪ. ﻧﺘﺎﯾﺞ ﺗﺤﻘﯿﻘﺎت ﭘﯿﺸﯿﻦ ﻧﺸﺎن ﻣﯽ دﻫﺪ ﮐﻪ ﮐﺎرﺑﺮد ﻧﺸﺎﻧﮕﺮ ISSR ﻣﯽ ﺗﻮاﻧﺪ ﺑﻪ ﻋﻨﻮان ﯾﮏ روش ﻣﻔﯿﺪ ﺑﺮاي ارزﯾﺎﺑﯽ ﺳﺮﯾﻊ و آﺳﺎن ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﺑﯿﻦ وارﯾﺘﻪ ﻫﺎ در ﮔﯿﺎه داوودي ﻣﺪﻧﻈﺮ ﻗﺮار ﮔﯿﺮد. ﺻﻔﺎت ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ ارﻗﺎم ﺟﺪﯾﺪ، ﺗﻨﻮﻋﯽ را در ﻣﻘﺎﯾﺴﻪ ﺑﺎ واﻟﺪﯾﻦ ﻧﺸﺎن دادﻧﺪ، ﺑﻪ ﻃﻮري ﮐﻪ ﻇﻬﻮر اوﻟﯿﻦ ﻏﻨﭽﻪ ﮔﻞ و ارﺗﻔﺎع اوﻟﯿﻦ ﻏﻨﭽﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ در ﻣﻘﺎﯾﺴﻪ ﺑﺎ واﻟﺪﯾﻦ ﮐﻢ ﺗﺮ ﺑﻮد ]10 [ . Abstract Saintpaulia ionantha is an attractive ornamental plant. To induce diversity by creating distinct genotypes of African violet in vitro Gamma rays were applied as physical and chemical mutagens. Explants were cultured in MS medium containing 2 mg L-1 BA, 1 mg L-1 NAA, 30g L-1 sucrose, and 7g L-1 agar. After two days they were treated with radiation (0, 20, and 40Gy). The dose of 40Gy reduced explant regeneration and the number of regenerated plantlets and leaves. This caused enhanced variation and morphological changes. The leaves' color, shape, and size were modified. Afterward, the genetic diversity of the mutant plants was investigated with an ISSR marker. Eight primer pairs in PCR amplified different fragments of genomic DNA. Overall, 71 bands were scored. The dendrogram of cluster analysis using the Jaccard coefficient and UPGMA algorithm showed high variability. The mutants were divided into 4 groups with a similarity of 0.51%. The genetic similarity coefficient varied between 0.45-0.98 and 6 mutants were introduced based on morphological and molecular variations. The results showed that in vitro mutagenesis of African violets by gamma radiation is a suitable strategy to increase diversity. Keywords: Diversity, Mutagenesis, Tissue culture, ISSR Keywords: Diversity, Mutagenesis, Tissue culture, ISSR ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ 109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 Journal of Nuclear Science, Engineering and Technology Vol. 45 (4), Serial Number 109, 2024, P 66-74 اﺣﻤﺪ ﺷﺮ ﯾﻔﯽ ، زﻫﺮا ﺳﺮﮔﺰ ي ﻣﻘﺪم، ﻣﻬﺪ ﯾ ﻪ ﺧﺮاز ي ، . . . 67 ﮐﺸﻮر را اﯾﺠﺎب ﻣﯽ ﮐﻨﺪ. از اﯾﻦ رو ﺑﻪ ﻧﻈﺮ ﻣﯽ رﺳﺪ ﺑﺎ ﮐﺎرﺑﺮد ﻋﻮاﻣﻞ ﺟﻬﺶ زا در ﮐﺸﺖ ﺑﺎﻓﺖ آن ﺑﺘﻮان اﯾﻦ روش را ﺑﻪ ﻋﻨﻮان ﯾﮏ راﻫﺒﺮد ﮐﻮﺗﺎه ﻣﺪت ﺑﺮاي ﺗﻮﻟﯿﺪ ﺗﻨﻮع ژﻧﺘ ﯿﮑﯽ ﻣﻮرد ﺗﻮﺟﻪ ﻗﺮار داد و از آن در ﺑﺮﻧﺎﻣﻪ ﻫﺎي اﺻﻼﺣﯽ اﺳﺘﻔﺎده ﮐﺮد. ﻟﺬا اﯾﻦ ﺗﺤﻘﯿﻖ ﺑﺎ ﻫﺪف ﺑﺮرﺳﯽ اﻣﮑﺎن اﯾﺠﺎد ﺗﻨﻮع در ﺻﻔﺎت و دﺳﺖ ﯾﺎﺑﯽ ﺑﻪ ژﻧﻮﺗﯿﭗ ﻫﺎي ﺟﺪﯾﺪ ﺑﺎ ﻓﻨﻮﺗﯿﭗ ﻣﺘﻔﺎوت ﺑﺎ اﺳﺘﻔﺎده از ﺟﻬﺶ زا ﻫﺎي ﻓﯿﺰﯾﮑﯽ در ﮐﺸﺖ درون ﺷﯿﺸﻪ اي و ﮐﺎرﺑﺮد ﻧﺸﺎﻧﮕﺮﻫﺎي ﻣﻮﻟﮑﻮﻟﯽ، در ﮔﯿﺎه ﺑﻨﻔﺸ ﻪ آﻓﺮﯾﻘﺎﯾﯽ )Saintpaulia ionantha ( اﻧﺠﺎم ﮔﺮﻓﺖ. 1 . ﻣﻘﺪﻣﻪ ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ ﻣﻮارد ﮔﻔﺘﻪ ﺷﺪه و ﺑﺎزارﭘﺴﻨﺪي و ارزش ﺗﺠﺎري ﮔﯿﺎه ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺑﻪ ﻋﻨﻮان ﯾﮏ ﮔﯿﺎه زﯾﻨﺘﯽ ﮔﻠﺪاﻧﯽ، ﻧﯿﺎز ﺑﺮاي ﺗﺤﻘﯿﻖ در ﺧﺼﻮص ﺑﺮﻧﺎﻣﻪ ﻫﺎي اﺻﻼﺣﯽ ﻣﻨﺴﺠﻢ ﺑﺮاي ﻣﻌﺮﻓﯽ ارﻗﺎم ﺟﺪﺑﺪ ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎي در 1 Inter Simple Sequence Repeats 2. ﻣﻮاد و روش ﻫﺎ 2.1 ﺗﻬﯿﻪ رﯾﺰﻧﻤﻮﻧﻪ و اﻋﻤﺎل ﺟﻬﺶ در اﯾﻦ ﺗﺤﻘﯿﻖ از ﮔﯿﺎه ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ رﻗﻢRococo اﺳﺘﻔﺎده ﺷﺪ. رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺑﺮگ، دﻣﺒﺮگ و ﮔﯿﺎﻫﭽﻪ ﻫﺎي اﺳﺘﺮﯾﻞ در ﻣﺤﯿﻂ ﮐﺸﺖ MS ﺣﺎوي2 ﻣﯿﻠﯽ ﮔﺮم در ﻟﯿﺘﺮ BA ، 2 /0 ﻣﯿﻠﯽ ﮔﺮم در ﻟﯿﺘﺮ NAA ﮐﺸﺖ ﺷﺪه و دو روز ﭘﺲ از ﮐﺸﺖ اوﻟﯿﻪ ﺑﺎ دُزﻫﺎ ي ° ، 20 و40 ﮔﺮي اﺷﻌﻪ ﮔﺎﻣﺎ ﻧﺎﺷﯽ از ﮐﺒﺎﻟﺖ60 ﺗﯿﻤﺎر ﺷﺪﻧﺪ. ﭘﺲ از ﺗﯿﻤﺎر، رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎ ﺑﻪ اﺗﺎق رﺷﺪ ﺑﺎ ﺷﺮاﯾﻂ ﻧﻮري 16 ﺳﺎﻋﺖ ﻧﻮر ﺑﻪ ﻫﻤﺮاه8 ﺳﺎﻋﺖ ﺗﺎرﯾﮑﯽ ﺑﺎ ﺷﺪت ﻧﻮر 30 ﻣﯿﮑﺮوﻣﻮل ﺑﺮ ﻣﺘﺮ ﻣﺮﺑﻊ ﺑﺮ ﺛﺎﻧﯿﻪ و دﻣﺎي1 ± 25 درﺟﻪ ﺳﺎﻧﺘﯽ ﮔﺮاد ﻣﻨﺘﻘﻞ ﺷﺪﻧﺪ. ﺑﺮاي ﻫﺮ ﯾﮏ از ﺗﯿﻤﺎرﻫﺎ، 3 ﺗﮑﺮار و در ﻫﺮ ﺗﮑﺮار25 ﻧﻤﻮﻧﻪ در ﻧﻈﺮ ﮔﺮﻓﺘﻪ ﺷﺪ. واﮐﺸﺖ رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎ ﺑﻪ ﻓﺎﺻﻠﻪ ﻫﺮ 4 ﻫﻔﺘﻪ ﯾﮑﺒﺎر در ﻣﺤﯿﻂ ﮐﺸﺖ ﻣﺸﺎﺑﻪ اﻧﺠﺎم ﺷﺪ. ﺑﻌﺪ از ﮔﺬﺷﺖ 60 روز از اﻋﻤﺎل ﺗﯿﻤﺎر، ﺻﻔﺎت درﺻﺪ زﻧﺪه ﻣﺎﻧﯽ و ﺗﻌﺪاد ﮔﯿﺎﻫﭽﻪ ﺑﺎززا ﺷﺪه اﻧﺪ ازه ﮔﯿﺮي ﺷﺪ. ﺑﻪ ﻣﻨﻈﻮر ﺳﺎزﮔﺎري ﺑﺎ ﻣﺤﯿﻂ ﮔﻠﺨﺎﻧﻪ، ﮔﯿﺎﻫﭽﻪ ﻫﺎي رﯾﺸﻪ دار ﺷﺪه ﺑﻪ ﺑﺴﺘﺮ ﮐﺸﺖ ﮐﻮﮐﻮﭘﯿﺖ و ﭘﺮﻟﯿﺖ ﺑﻪ ﻧﺴﺒﺖ ﻣﺴﺎوي )1:1 ( اﻧﺘﻘﺎل داده ﺷﺪﻧﺪ و30 روز ﭘﺲ از ﺳﺎزﮔﺎري وزن ﺗﺮ ﮔﯿﺎﻫﭽﻪ ﻫﺎ و ﺗﻌﺪاد ﺑﺮگ ﻣﻮرد ارزﯾﺎﺑﯽ ﻗﺮار ﮔﺮﻓﺖ. 45 روز ﭘﺲ از زﻣﺎن ﮔﻞ دﻫﯽ ﻧﯿﺰ ﮔﯿﺎﻫﺎﻧﯽ ﮐﻪ ﻧﺴﺒﺖ ﺑﻪ ﺷﺎﻫﺪ ﺗﻐﯿﯿﺮات ﻓﻨﻮﺗﯿﭙﯽ ﻧﺸﺎن دادﻧﺪ اﻧﺘﺨﺎب ﺷﺪه و از ﻧﻈﺮ ﺻﻔﺎت ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ ﺷﺎﻣﻞ ﺗﻌﺪاد ﺑﺮگ، ﺳﻄﺢ ﺑﺮگ ﺑﺰرگ ﺗﺮﯾﻦ ﺑﺮگ، ﻃﻮل دﻣﺒﺮگ ﺑﺰرگ ﺗﺮﯾﻦ ﺑﺮگ، ﺗﻌﺪاد ﮔﻞ آذﯾﻦ، ﺗﻌﺪاد ﮔﻞ در ﻫﺮ ﮔﻞ آذﯾﻦ، ﺗﻌﺪاد ﮔﻠﺒﺮگ در ﻫﺮ ﮔﻞ، اﻧﺪازه ﺑﺰرگ ﺗﺮﯾﻦ ﮔﻞ، ﺗﻌﺪاد ﭘﺮﭼﻢ در ﮔﻞ ﻣﻮرد ارزﯾﺎﺑﯽ ﻗﺮار ﮔﺮﻓ ﺘﻨﺪ. Journal of Nuclear Science, Engineering and Technology Vol. 45 (4), Serial Number 109, 2024, P 66-74 2.2 ﺑﺎززاﯾﯽ ﻣﺠﺪد ﻓﺮم ﻫﺎي ﺟﻬﺶ ﯾﺎﻓﺘﻪ و ﻣﺸﺎﻫﺪه ﺗﻐﯿﯿﺮات ﻓﻨﻮﺗﯿﭙﯽ در آن ﻫﺎ ﺑﻪ ﻣﻨﻈﻮر ارزﯾﺎﺑﯽ ﻣﯿﺰان ﺛﺒﺎت ژﻧﺘﯿﮑﯽ ﻓﺮم ﻫﺎي ﺟﻬﺶ ﯾﺎﻓﺘﻪ اﻧﺘﺨﺎﺑﯽ، از آن ﻫﺎ ﻧﻤﻮﻧﻪ ﺑﺮﮔﯽ ﺗﻬﯿﻪ ﺷﺪ و ﻫﻤﺎﻧﻨﺪ آزﻣﺎﯾﺶ ﻗﺒﻞ ﻣﺮاﺣﻞ ﺿﺪﻋﻔﻮﻧﯽ ﺳﻄﺤﯽ، ﺑﺎززاﯾﯽ، ﺗﮑﺜﯿﺮ و ﺳﺎزﮔﺎري رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎ اﻧﺠﺎم ﺷﺪ. ﭘﺲ ا ز ﮔﺬﺷﺖ 60 روز از ﺳﺎزﮔﺎري، اﯾﻦ ﮔﯿﺎﻫﺎن از ﻧﻈﺮ 1. Inter Simple Sequence Repeats ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 Sequence Repeats ﺗﻮﻟﯿﺪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ )Saintpaulia ionantha ( . . . 68 ﭘﺎراﻣﺘﺮﻫﺎي ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ ﺷﺎﻣﻞ ﺗﻌﺪاد ﺑﺮگ، ﻣﯿﺎﻧﮕﯿﻦ ﻃﻮل دﻣﺒﺮگ ﻫﺎ، ﻣﺠﻤﻮع ﺳﻄﺢ ﺑﺮگ و ﻣﯿﺰان ﺳﻄﺢ ﻓﺘﻮﺳﻨﺘﺰﮐﻨﻨﺪه ﻣﻮﺟﻮد در ﺑﺮگ ﻫﺎ ﻣﻮرد ارزﯾﺎﺑﯽ ﻗﺮار ﮔﺮﻓﺘﻨﺪ. 2. ﻣﻮاد و روش ﻫﺎ ﺻﻔﺎت ﻣﺠﻤﻮع ﺳﻄﺢ ﺑﺮگ و ﻣﯿﺰان ﺳﻄﺢ ﻓﺘﻮﺳﻨﺘﺮﮐﻨﻨﺪه ﻣﻮﺟﻮد در ﺑﺮگ ﻫﺎ ﺑﺎ اﺳﺘﻔﺎده از ﺗﮑﻨﯿﮏ آﻧﺎﻟﯿﺰ ﺗﺼﻮﯾﺮ و ﺗﻮﺳﻂ ﻧﺮم اﻓﺰار آﻧﺎﻟﯿﺰ ﺗﺼﻮﯾﺮ ImageJ اﻧﺠﺎم ﺷﺪ ]11 [. اﻧﺪازه ﮔﯿﺮي ﻣﯿﺰان ﮐﻠﺮوﻓﯿﻞ ﻧﯿﺰ ﺑﺎ اﺳﺘﻔﺎده از روش ﻟﯿﭽﺘﻨﺴﺎﻟﺮ و ﺑﻮﺷﻤﻦ ]12 [ اﻧﺠﺎم ﺷﺪ. ﺑﺮاي ﻫﺮ ﻧﻤﻮﻧﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﭼﻬﺎر ﺗﮑﺮار در ﻧﻈﺮ ﮔﺮﻓﺘﻪ ﺷﺪ و ﻣﻘﺎﯾﺴﻪ ﻣﯿﺎﻧﮕﯿﻦ ﻫﺎ در ﻃﺮح آزﻣﺎﯾﺸﯽ ﻓﺎﮐﺘﻮرﯾﻞ ﺗﺼﺎدﻓﯽ ﺑﺮاﺳﺎس ﭼﻬﺎر ﺗﮑﺮار ﻣﻮرد ارزﯾﺎﺑﯽ ﻗﺮار ﮔﺮﻓﺖ. 3. ﻧﺘﺎﯾﺞ و ﺑﺤﺚ ﻃﺒﻖ ﻧﺘﺎﯾﺞ ﺑﻪ دﺳﺖ آﻣﺪه ﺣﺎﺻﻞ از ﺗﺠﺰﯾﻪ وارﯾﺎﻧﺲ، ارﺗﺒﺎط ﻣﺘﻘﺎﺑﻞ دُز اﺷﻌﻪ ﺑﺎ ﻧﻮع رﯾﺰﻧﻤﻮﻧﻪ ﺑﺮاي ﻋﺎﻣﻞ درﺻﺪ زﻧﺪه ﻣﺎﻧﯽ رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎ در ﺳﻄﺢ ﭘﻨﺞ درﺻﺪ ﻣﻌﻨﯽ دار ﺑﻮد و ﻧﺘﺎﯾﺞ ﻣﻘﺎﯾﺴﻪ ﻣﯿﺎﻧﮕﯿﻦ ﻧﺸﺎن داد ﮐﻪ ﺑﯿﺶ ﺗﺮﯾﻦ درﺻﺪ زﻧﺪه ﻣﺎﻧﯽ ﻣﺮﺑﻮط ﺑﻪ رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﮔﯿﺎﻫﭽﻪ و دُز ﺗﺎﺑﺶ اﺷﻌﻪ 20 ﮔﺮي و ﮐﻢ ﺗﺮﯾﻦ درﺻﺪ زﻧﺪه ﻣﺎﻧﯽ ﻣﺮﺑﻮط ﺑﻪ رﯾﺰﻧﻤﻮﻧﻪ دﻣﺒﺮگ و دُز ﺗﺎﺑﺶ 40 ﮔﺮي ﺑﻮد. ﻧﺘﺎﯾﺞ ﺣﺎﺻﻞ از ﺟﺪول ﺗﺠﺰﯾﻪ وا رﯾﺎﻧﺲ در ﻣﻮرد اﺛﺮات ﻣﺘﻘﺎﺑﻞ دُزﻫﺎي ﺗﺎﺑﺶ اﺷﻌﻪ و ﻧﻮع رﯾﺰﻧﻤﻮﻧﻪ ﺑﺮاي ﻋﺎﻣﻞ ﻣﯿﺎﻧﮕﯿﻦ ﺗﻌﺪاد ﮔﯿﺎﻫﭽﻪ در ﺳﻄﺢ ﭘﻨﺞ درﺻﺪ ﻣﻌﻨﯽ دار ﺑﻮد و ﻣﻘﺎﯾﺴﻪ ﻣﯿﺎﻧﮕﯿﻦ ﻧﺸﺎن داد ﮐﻪ ﺑﯿﺶ ﺗﺮﯾﻦ ﺗﻌﺪاد ﮔﯿﺎﻫﭽﻪ ﺗﺸﮑﯿﻞ ﺷﺪه ﻣﺘﻌﻠﻖ ﺑﻪ رﯾﺰﻧﻤﻮﻧﻪ ﮔﯿﺎﻫﭽﻪ و دُز ﺗﺎﺑﺶ اﺷﻌﻪ 20 ﮔﺮي و ﮐﻢ ﺗﺮﯾﻦ ﺗﻌﺪاد ﻣﺮﺑﻮط ﺑﻪ رﯾﺰ ﻧﻤﻮﻧﻪ دﻣﺒﺮگ در ﻫﺮ ﺳﻪ ﺗﯿﻤﺎر ﺗﺎﺑﺶ ﺑﻮده اﺳﺖ و ﺑﯿﻦ ﺑﯿﺶ ﺗﺮﯾﻦ و ﮐﻢ ﺗﺮﯾﻦ ﺗﻌﺪاد ﮔﯿﺎﻫﭽﻪ آن ﻫﺎ 66 / 64 درﺻﺪ ﺗﻔﺎوت ﻣﺸﺎﻫﺪه ﮔﺮدﯾﺪ. 2. 3 ارزﯾﺎﺑﯽ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﺑﺎ اﺳﺘﻔﺎده از ﻧﺸﺎﻧﮕﺮ ﻣﻮﻟﮑﻮﻟﯽISSR از ﺑﺎﻓﺖ ﺑﺮگ ﮔﯿﺎﻫﭽﻪ ﻫﺎﯾﯽ ﮐﻪ ﺗﻨﻮع ﻣﻮرﻓﻮﻟﻮژﯾﮏ در آن ﻫﺎ ﻣﺸﺎﻫﺪه ﺷﺪ و ﻫﻢ ﭼﻨﯿﻦ ﮔﯿﺎﻫﭽﻪ ﻫﺎي ﺷﺎﻫﺪ، اﺳﺘﺨﺮاج DNA ﺑﺎ اﺳﺘﻔﺎده از دﺳﺘﻮراﻟﻌﻤﻞ اراﺋﻪ ﺷﺪه ﺗﻮﺳﻂ اوﻧﺎت ﺳﺎﻧﭽﺰ و وﯾﺴﻨﺖ ﮐﺎرﺑﺎﺟﻮﺳﺎ ]13 [ اﻧﺠﺎم ﺷﺪ. در اﯾﻦ ﺗﺤﻘﯿﻖ8 آﻏﺎزﮔﺮISSR ﮐﻪ در ﺗﺤﻘﯿﻘﺎت ﻗﺒﻞ ﺗﻮﺳﻂ ﻓﺮﺟﺎدي ﺷﮑﯿﺐ و ﻫﻤﮑﺎران ]14 [ ﺑﺮ روي ﺳﺎﯾﺮ ﮔﯿﺎﻫﺎن زﯾﻨﺘﯽ از ﺧﺎﻧﻮاده ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ اﺳﺘﻔﺎده ﺷﺪه و ﻧﺘﺎﯾﺞ رﺿﺎﯾﺖ ﺑﺨﺸﯽ داﺷﺘﻪ اﻧﺪ اﻧﺘﺨﺎب و ﺗﻮﺳﻂ ﺷﺮﮐﺖ ﻣﺎﮐﺮوژن ﮐﺮه ﺳﻨﺘﺰ ﺷﺪ. ﺗﻐﯿﯿﺮات ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ ﻗﺎﺑﻞ ﺗﻮﺟﻬﯽ در ﮔﯿﺎﻫﭽﻪ ﻫﺎي ﺑﺎززا ﺷﺪه ﻣﺸﺎﻫﺪه ﺷﺪ ﮐﻪ در ﺑﺮگ ﮔﯿﺎﻫﭽﻪ ﻫﺎي ﺗﯿﻤﺎر ﺷﺪه ﺑﻪ آﺳﺎﻧﯽ ﻗﺎﺑﻞ ﺗﺸﺨﯿﺺ ﺑﻮد و ﺗﻨﻮع ﺧﻮﺑﯽ اﯾﺠﺎد ﮐ ﺮده ﺑﻮد )ﺷﮑﻞ 1( . اﯾﻦ ﺗﻐﯿﯿﺮات ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ ﺷﺎﻣﻞ ﺗﻐﯿﯿﺮ رﻧﮓ، ﺷﮑﻞ و ﭘﯿﭽﺶ ﺑﺮگ ﮔﯿﺎﻫﭽﻪ ﻫﺎي ﺗﯿﻤﺎر ﺷﺪه و در ﺑﺮﺧﯽ از ﮔﯿﺎﻫﺎن ﺗﻐﯿﯿﺮ در اﻧﺪازه دﻣﺒﺮگ ﻧﺴﺒﺖ ﺑﻪ اﻧﺪازه ﻃﺒﯿﻌﯽ آن ﻫﺎ ﺑﻮد. 2. ﻣﻮاد و روش ﻫﺎ ﺑﻪ ﮔﻮﻧﻪ اي ﮐﻪ ﺑﺎ اﻓﺰاﯾﺶ دُز اﺷﻌﻪ ﮔﺎﻣﺎ ﺗﺎ ﺳﻄﺢ 40 ﮔﺮي درﺻﺪ ﺑﺮگ ﻫﺎي ﺗﻐﯿﯿﺮ ﯾﺎﻓﺘﻪ اﻓﺰاﯾﺶ ﻗﺎﺑﻞ ﺗﻮﺟﻬﯽ داﺷﺖ. در ﺑﺮرﺳﯽ ﺗﺤﻘﯿﻘﺎت اﻧﺠﺎم ﺷﺪه ﺗﻮﺳﻂ ﺳﺎﯾﺮ ﻣﺤﻘﻘﺎن ﻣﺸﺨﺺ ﮔﺮدﯾﺪ ﮐﻪ ﺗﻐﯿﯿﺮ ﺧﺼﻮﺻﯿﺎت ﺑﺮگ ﻫﺎ در اﺻﻼح ﺟﻬﺸﯽ ﮔﯿﺎﻫﺎن زﯾﻨﺘﯽ ﻋﻤﺪﺗﺎً ﺑﺎ ﻫﺪف ﺗﻐﯿﯿﺮ اﻧﺪازه ﺑﺮگ ﻫﺎ و ﻧﯿﺰ اﻓﺰاﯾﺶ ﺗﻨﻮع رﻧﮓ ﺑﺮگ ﻫﺎ ﺑﻪ وﯾﮋه در ﮔﯿﺎﻫﺎن زﯾﻨﺘﯽ ﺻﻮرت ﮔﺮﻓﺘﻪ اﺳﺖ ]15 [. در دُزﻫﺎي ﺧﯿﻠﯽ ﺑﺎﻻ، ﺗﺎﺑﺶ ﻣﯽ ﺗﻮاﻧﺪ ﺑﺎ ﺑﺮوز ﺻﺪﻣﺎت اﺑﺘﺪاﯾﯽ، ﺗﻘﺴﯿﻢ ﺳﻠﻮﻟﯽ را ﺑﻪ ﺗﺄﺧﯿﺮ ﺑﯿﺎﻧﺪازد ﮐﻪ اﯾﻦ اﻣﺮ اﻟﻘﺎي ﺗﻐﯿﯿﺮاﺗﯽ در ﻣﻮرﻓﻮﻟﻮژي ﮔﯿﺎه را ﺑﻪ ﻫﻤﺮاه ﺧﻮاﻫﺪ داﺷﺖ ] 16 [ ،. ﺑﻪ ﻋﻨﻮان ﻣﺜﺎل وﻧﮓ ﭘﯿﺎﺳﺎﺗﯿﺪ و ﻫﻤﮑﺎران ] 17 [ در ﺑﺮرﺳﯽ اﺛﺮ اﺷﻌﻪ ﮔﺎﻣﺎ ﺑﺮ ﺗﺸﮑﯿﻞ ﮔﯿﺎﻫﭽﻪ ﺣﺎﺻﻞ از ﺑﺮگ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﻧﺸﺎن دادﻧﺪ ﮐﻪ اﺷﻌﻪ ﮔﺎﻣﺎ ﺳﺒﺐ اﻓﺰاﯾﺶ ﺑﺮگ ﻫﺎي ﺗﻐﯿﯿﺮ ﺷﮑﻞ ﯾﺎﻓﺘﻪ ﻣﯽ ﺷﻮد. ﻣﺎﻧﺪال و ﻫﻤﮑﺎران ]5 [ ﺑﻪ وﺳﯿﻠﻪ ﮐﺎرﺑﺮد دُزﻫﺎي ﻣﺨﺘﻠﻒ ﺗﺎﺑﺶ ﮔﺎﻣﺎ )5 /2 - 5 /1 ﮔﺮي(، ﻣﻮﻓﻖ ﺑ ﻪ ﺗﻮﻟﯿﺪ ﺑﻮﺗﻪ ﻫﺎي اﺑﻠﻖ داوودي )C. morifolium( ﺷﺪﻧﺪ. ﻫﻢ ﭼﻨﯿﻦ ﮔﺰارش ﺷﺪه ﮐﻪ ﺗﻮﻟﯿﺪ ﮔﯿﺎﻫﺎن داراي ﺑﺮگ ﻫﺎي ﺑﺎ ﺷﮑﻞ ﺗﻐﯿﯿﺮﯾﺎﻓﺘﻪ و ﻓﺎﻗﺪ رﻧﮓ داﻧﻪ ﻫﺎي ﻓﺘﻮﺳﻨﺘﺰي از ﺟﻤﻠﻪ ﻧﺘﺎﯾﺞ اﻟﻘﺎي ﺟﻬﺶ ﺑﺮ اﺛﺮ ﺗﺎﺑﺶ ﭘﺮﺗﻮي ﯾﻮﻧﯿﺰه ﮐﺮﺑﻨﯽ، اﺷﻌﻪ اﯾﮑﺲ و ﮔﺎﻣﺎ ﺑﻪ رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺑﺮگ ﮔﯿﺎه ﺑﻨﻔﺸﻪ واﮐﻨﺶ زﻧﺠﯿﺮه اي ﭘﻠﯿﻤﺮاز ﺑﺎ اﺳﺘﻔﺎده از 8 آﻏﺎزﮔﺮISSR و ﮐﯿﺖ Master Mix PCR ﺳﺎﺧﺖ ﺷﺮﮐﺖAmpliqon اﻧﺠﺎم ﮔﺮﻓﺖ. ﺗﺮﮐﯿﺒﺎت، ﻣﻘﺎدﯾﺮ و ﭼﺮﺧﻪ دﻣﺎﯾﯽ ﻣﻮرد ﻧﯿﺎز PCR ﺑﺎ اﺳﺘﻔﺎده از دﺳﺘﻮراﻟﻌﻤﻞ ﻓﺮﺟﺎدي ﺷﮑﯿﺐ و ﻫﻤﮑﺎران ]14 [ اﻧﺠﺎ م ﮔﺮﻓﺖ. ﻣﺤﺼﻮﻻت PCR ﺑﺎ اﺳﺘﻔﺎده از ژل آﮔﺎرز1 درﺻﺪ در ﺑﺎﻓﺮ X 5 /0 TBE ﺑﻪ ﻣﺪت5 /1 ﺳﺎﻋﺖ و ﺑﺎ وﻟﺘﺎژ100 وﻟﺖ ﺑﺎ رﻧﮓ ﮔﺮﯾﻦ وﯾﻮور )ﺳﺎﺧﺖ ﺷﺮﮐﺖ GenetBio ( اﻟﮑﺘﺮوﻓﻮرز ﺷﺪ و در ﺗﺮاﻧﺴﻠﻮﻣﯿﻨﺎﺗﻮر دﺳﺘﮕﺎه در ﺑﻨﻔﺶ ﻣﺎوراء ﻧﻮر زﯾﺮ M 20 Vilber lourmat TCR- ﻋﮑﺲ ﺑﺮداري ﺷﺪ. ﺗﺠﺰﯾﻪ ﺧﻮﺷﻪ ﺑﺮاي اي ﺗﻌﯿﯿﻦ ﺗﺸﺎﺑﻪ ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﻣﻮرد ﻣﻄﺎﻟﻌﻪ ﺑﺎ اﻟﮕﻮرﯾﺘﻢ UPGMA ﻣﺒﺘﻨﯽ ﺑﺮ ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ﺟﺎﮐﺎرد و ﺗﻌﯿﯿﻦ ﻫﻤﺒﺴﺘﮕﯽ ﺑﯿﻦ ﻣﺎﺗﺮﯾﺲ ﻫﺎي ﺗﺸﺎﺑﻪ ﺣﺎﺻﻞ از ﺗﺠﺰﯾﻪ داده ﻫﺎ، ﺑﺎ اﺳﺘﻔﺎده ﻧﺮم از اﻓﺰار NTSYS-pc ﻧﺴﺨﻪ 02 .2 اﻧﺠﺎم ﮔﺮﻓﺖ. ﺗﺠﺰﯾﻪ وارﯾﺎﻧﺲ ﻣﻮﻟﮑﻮﻟﯽ )AMOVA ( ﺑﻪ ﻣﻨﻈﻮر ﺗﻔﮑﯿﮏ وارﯾﺎﻧﺲ ﻣﻮﻟﮑﻮﻟﯽ ﺑﻪ ﮐﻞ وارﯾﺎﻧﺲ ژﻧﺘﯿﮑﯽ درون و ﺑﯿﻦ ﮔﺮوه و ﻫﺎ ﻣﺤﺎﺳﺒﻪ ﻓﺎﺻﻠﻪ ژﻧﺘﯿﮑﯽ ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻧﺮم ﺑﺎ ﻫﺎ اﻓﺰار Excel اﻧﺠﺎم ﮔﺮدﯾﺪ . 2. ﻣﻮاد و روش ﻫﺎ اﺛﺮ ﺟﻬﺶ اﻟﻘﺎﯾﯽ ﺑﺮ ﮐﺎﻫﺶ ﻣﯿﺰان رﻧﮓ داﻧﻪ ﻫﺎي ﻓﺘﻮﺳﻨﺘﺰي ﺑﻪ دﻟﯿﻞ اﯾﺠﺎد ﻧﻘﺺ در ژن ﻫﺎي ﻣﺴﺆل ﺑﯿﻮﺳﻨﺘﺰ رﻧﮓ داﻧﻪ ﻫﺎ، ﺗﺨﺮﯾﺐ ﻣﺴﯿﺮﻫﺎي ﺑﯿﻮﺳﻨﺘﺰي ﺗﻮﻟﯿﺪ رﻧﮓ داﻧﻪ ﻫﺎ، ﺗﺨﺮﯾﺐ ﮐﻠﺮوﭘﻼﺳﺖ و ﻧﯿﺰ ﺗﺠﺰﯾﻪ رﻧﮓ داﻧﻪ ﻫﺎي ﻓﺘﻮﺳﻨﺘﺰي ﻣﯽ ﺑﺎﺷﺪ ]19 [. ﻣﻨﻈﻮر اﻟﻘﺎي ﺟﻬﺶ در رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﮔﯿﺎه ﻟﯿﻠﯿﻮم، ﺗﻨﻮع در ﺷﮑﻞ و ﻣﻮرﻓﻮﻟﻮژي ﮔﻞ ﻫﺎي ﺟﻬﺶ ﯾﺎﻓﺘﻪ را در ﭘﯽ داﺷﺖ ]6 [ . رﻧﮓ داﻧﻪ ﻫﺎ، ﺗﺨﺮﯾﺐ ﻣﺴﯿﺮﻫﺎي ﺑﯿﻮﺳﻨﺘﺰي ﺗﻮﻟﯿﺪ رﻧﮓ داﻧﻪ ﻫﺎ، ﺗﺨﺮﯾﺐ ﮐﻠﺮوﭘﻼﺳﺖ و ﻧﯿﺰ ﺗﺠﺰﯾﻪ رﻧﮓ داﻧﻪ ﻫﺎي ﻓﺘﻮﺳﻨﺘﺰي ﻣﯽ ﺑﺎﺷﺪ ]19 [. 30 روز ﭘﺲ از ﺳﺎزﮔﺎري ﮔﯿﺎﻫﭽﻪ ﻫﺎ و اﻧﺘﻘﺎل ﺑﻪ ﮔﻞ ﺧﺎﻧﻪ ﺧﺼﻮﺻﯿﺎت رﺷﺪي ﺷﺎﻣﻞ وزن ﺗﺮ و ﺗﻌﺪاد ﺑﺮگ در ﻫﺮ ﮔﯿﺎﻫﭽﻪ اﻧﺪازه ﮔﯿﺮي ﺷﺪ. ﭘﺲ از اﻧﺠﺎم ﺗﺠﺰﯾﻪ و ﺗﺤﻠﯿﻞ آﻣﺎري، ﺗﻔﺎوت ﻣﻌﻨﯽ داري ﻣﯿﺎن وزن ﺗﺮ ﻧﻤﻮﻧﻪ ﻫﺎ در ﺗﯿﻤﺎرﻫﺎ ﻣﺸﺎﻫﺪه ﻧﺸﺪ و ﻣﯿﺎﻧﮕﯿﻦ وزن ﺗﺮ در ﻧﻤﻮﻧﻪ ﻫﺎي ﻣﻮرد ﺑﺮرﺳﯽ 65 /1 ﮔﺮم ﺑﻪ دﺳﺖ آﻣﺪ. در ﺑﺮرﺳﯽ ﻋﺎﻣﻞ ﺗﻌﺪا د ﺑﺮگ، اﺛﺮ ﻣﻌﻨﯽ داري در ﺟﺪول ﺗﺠﺰﯾﻪ وارﯾﺎﻧﺲ ﻣﺸﺎﻫﺪه ﮔﺮدﯾﺪ و ﻧﺘﺎﯾﺞ ﺣﺎﺻﻞ از ﻣﻘﺎﯾﺴﻪ ﻣﯿﺎﻧﮕﯿﻦ داده ﻫﺎ ﻧﺸﺎن داد ﮐﻪ ﺑﯿﺶ ﺗﺮﯾﻦ ﺗﻌﺪاد ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه )33 / 10 ﻋﺪد( در رﯾﺰﻧﻤﻮﻧﻪ ﺑﺮگ ﺑﺎ دُز ﺗﺎﺑﺶ اﺷﻌﻪ20 ﮔﺮي و ﮐﻢ ﺗﺮﯾﻦ ﺗﻌﺪاد ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه ﻣﺘﻌﻠﻖ ﺑﻪ رﯾﺰﻧﻤﻮﻧﻪ ﮔﯿﺎﻫﭽﻪ و ﺷﺎﻫﺪ ﺑﺎ ﻣﯿﺎﻧﮕﯿﻦ 16 /7 ﺑﺮگ ﺑﻮده اﺳﺖ )ﺷﮑﻞ2 (. در ﻣﻄﺎﻟﻌﻪ ﺣﺎﺿﺮ ﻣﯿﺰان ﺑﺮگ در ﺷﺎﻫﺪ ﺗﻌﺪاد ﮐﻢ ﺗﺮي داﺷﺘﻪ اﺳﺖ وﻟﯽ ﺑﺎ اﻓﺰاﯾﺶ دُز ﺑﻪ 20 ﮔﺮي اﯾﻦ ﺗﻌﺪاد اﻓﺰاﯾﺶ داﺷﺘﻪ و در دُز40 ﮔﺮي روﻧﺪ ﮐﺎﻫﺸﯽ را ﻃﯽ ﮐﺮده اﺳﺖ. اﯾﻦ ﮐﺎﻫﺶ ﻣﯽ ﺗﻮاﻧﺪ ﺑﻪ دﻟﯿﻞ ﮐﺎﻫﺶ ﺗﻘﺴﯿﻢ ﺳﻠﻮﻟﯽ در دُزﻫﺎي ﺑﺎﻻﺗﺮ اﺷﻌﻪ ﮔﺎﻣﺎ ﺑ ﺎﺷﺪ. ﺗﻘﺴﯿﻢ ﺳﻠﻮﻟﯽ ﺑﻬﯿﻨﻪ، ﺑﻪ دﻟﯿﻞ ﻋﻤﻠﮑﺮد ﺑﺎﻻي اﻧﺪاﻣﮏ ﻫﺎ و ﻓﺮاﯾﻨﺪﻫﺎي آﻧﺰﯾﻤﯽ اﺳﺖ. ﻫﻢ ﭼﻨﯿﻦ ﮔﺰارش ﺷﺪه ﮐﻪ ﺗﺠﻤﻊ ﻧﺸﺎﺳﺘﻪ در ﮐﻠﺮوﭘﻼﺳﺖ و ﻫﻢ ﭼﻨﯿﻦ آﺳﯿﺐ و ﺧﺴﺎرت ﺑﻪ ﮔﺮاﻧﺎﻫﺎ و ﺗﯿﻼﮐﻮﺋﯿﺪﻫﺎ ﺳﺒﺐ ﺑﺎزدارﻧﺪﮔﯽ در اﻧﺘﻘﺎل ﮐﺮﺑﻮﻫﯿﺪرات ﻫﺎ ﺧﻮاﻫﺪ ﺷﺪ ]20 [ .در ﺑﺮرﺳﯽ ﻫﺎﯾﯽ ﮐﻪ ﺑﻌﺪ از ﺑﻪ ﮔﻞ رﻓﺘﻦ ﮔﯿﺎﻫﺎن ﺗﯿﻤﺎر ﺷﺪه ﺑﺎ دُز اﺷﻌﻪ ﮔﺎﻣﺎ در ﮔﻠﺨﺎﻧﻪ اﻧﺠﺎم ﺷﺪ ﺗﻨﻮع ﮐﺎﻣﻼً ﺑﺎرزي در ﺗﻌﺪادي از ﮔﻞ ﻫﺎ ﻣﺸﺎﻫﺪه ﺷﺪ ﮐﻪ ﻧﺸﺎن دﻫﻨﺪه اﺛﺮ ﻣﻌﻨﯽ دار ﮐﺎرﺑﺮد ﭘﺮﺗﻮدﻫﯽ ﺑﺮ ﺗﻨﻮع رﻧﮓ و ﺷﮑﻞ ﮔﻞ در اﯾﻦ ﮔﯿﺎه ﺑﻮد، ﺑﻪ ﻃﻮري ﮐﻪ در ﺑﺮﺧﯽ از ﮔﻞ ﻫﺎ اﻓﺰاﯾﺶ اﻧﺪ ازه ﮔﻞ، ﮐﺎﻫﺶ ﺗﻌﺪاد ﮔﻠﺒﺮگ، ﻣﻀﺮس ﺷﺪن ﺣﺎﺷﯿﻪ ﮔﻞ و ﺗﻐﯿﯿﺮ در اﻧﺪازه ﮔﻠﺒﺮگ ﻫﺎ ﻣﺸﺎﻫﺪه ﺷﺪ )ﺷﮑﻞ 3(. 2. ﻣﻮاد و روش ﻫﺎ ﺑ ﺮاي ﺗﻌﯿﯿﻦ ﻗﺪرت ﺗﻤﺎﯾﺰ ﻧﺸﺎﻧﮕﺮﻫﺎ در ﺟﻬﺶ ﯾﺎﻓﺘﻪ از ، ﻫﺎ ﭘﺎراﻣﺘﺮﻫﺎي ﻣﯿﺰان اﻃﻼﻋﺎت ﭼﻨﺪﺷﮑﻞ PIC اﺳﺘﻔﺎده ﮔﺮدﯾﺪ ﮐﻪ از ﻓﺮﻣﻮل 2 q - 2 P - 1 PIC= .ﻣﺤﺎﺳﺒﻪ ﺷﺪ ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 اﺣﻤﺪ ﺷﺮ ﯾﻔﯽ ، زﻫﺮا ﺳﺮﮔﺰ ي ﻣﻘﺪم، ﻣﻬﺪ ﯾ ﻪ ﺧﺮاز ي ، . . . ﻣﻨﻈﻮر اﻟﻘﺎي ﺟﻬﺶ در رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﮔﯿﺎه ﻟﯿﻠﯿﻮم، ﺗﻨﻮع در ﺷﮑﻞ و ﻣﻮرﻓﻮﻟﻮژي ﮔﻞ ﻫﺎي ﺟﻬﺶ ﯾﺎﻓﺘﻪ را در ﭘﯽ داﺷﺖ ]6 [ . ﺷﮑﻞ1 . ﺗﻨﻮع ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ در ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه در ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﭘﺮﺗﻮدﯾﺪه اﺷﻌﻪ ﮔﺎﻣﺎ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ. اﻟﻒ( ﺷﺎﻫﺪ، ب( ﻣﻀﺮس ﺷﺪن ﺑﺮگ، پ( ﭘﯿﭽﯿﺪﮔﯽ ﺑﺮگ ﺑﻪ داﺧﻞ، ت( ﺗﻐﯿﯿﺮ ﺷﮑﻞ ﺑﺮگ، ث( اﺑﻠﻖ ﺷﺪن ﺟﺰﯾﯽ در ﺑﺮگ، ج( ﮐﺎﻫﺶ ﺳﺎﯾﺰ ﺑﺮگ )ﮐﻮﺗﻮﻟﮕﯽ ﺑﺮگ(، چ( ﮐﻮﺗﻮﻟﮕﯽ ﻫﻤﺮاه ﺑﺎ ﻣﻀﺮس ﺷﺪن، ح( ﺷﮑﺎف در ﻣﺤﻞ اﺗﺼﺎل دﻣﺒﺮگ ﺑﻪ ﺑﺮگ، خ( ﮐﺸﯿﺪه ﺷﺪن دﻣﺒﺮگ و ﺑﺮگ و ﺻﺎف ﺷﺪن ﺑﺮگ و ﻋﺪم ﻣﻀﺮس ﺑﻮدن، د( ﺗﻐﯿﯿﺮ رﻧﮓ ﺑﺮگ ﻫﺎ، ذ( زرد ﺷﺪن ﮐﺎﻣﻞ ﺑﺮگ و ر( اﺑﻠﻖ ﺷﺪن ﺑﺨﺸﯽ از ﺑﺮگ. ﺷﮑﻞ2 .ﺗﻌﺪاد ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه در ﻫﺮ ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺑﻪ ﺗﻔﮑﯿﮏ دز ﺗﺎﺑﺶ ) در ﻫﺮ ﺳﺘﻮن ﻣﯿﺎﻧﮕﯿﻦ ﻫﺎﯾﯽ ﺑﺎ ﺣﺪاﻗﻞ ﯾﮏ ﺣﺮف ﻣ ﺸﺘﺮك، در ﺳﻄﺢ اﺣﺘﻤﺎل ﭘﻨﺞ درﺻﺪ ﺗﻔﺎوت ﻣﻌﻨﯽ داري ﻧﺪارﻧﺪ (. ﺷﮑﻞ3 . ﺗﻨﻮع در ﮔﻞ ﺗﺸﮑﯿﻞ ﺷﺪه ﺑﺮ روي ﻫﺮ ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺗﺎﺑﺶ دﯾﺪه اﺷﻌﻪ ﮔﺎﻣﺎ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ. اﻟﻒ( ﺷﺎﻫﺪ، ب( اﻓﺰاﯾﺶ ﺳﺎﯾﺰ ﮔﻞ، پ و ت( ﮐﺎﻫﺶ ﺗﻌﺪاد و ﻧﺎﻣﺘﻘﺎرن ﺑﻮدن ﺳﺎﯾﺰ ﮔﻠﺒﺮگ، ث( ﻣﻀﺮس ﺷﺪنﺣﺎﺷﯿﻪﮔﻠﺒﺮگ، ج(ﮐﺎﻫﺶﺗﻌﺪادﮔﻠﺒﺮگ وﻣﺘﻘﺎرنﺑﻮدنﺳﺎﯾﺰ و 69 اﺣﻤﺪ ﺷﺮ ﯾﻔﯽ ، زﻫﺮا ﺳﺮﮔﺰ ي ﻣﻘﺪم، ﻣﻬﺪ ﯾ ﻪ ﺧﺮاز ي ، . . . آﻓﺮﯾ ﻘﺎﯾﯽ )S. ionantha( در ﺷﺮاﯾﻂ درون ﺷﯿﺸﻪ اي ﺑﻮده اﺳﺖ ]14 ، 18 [. اﺛﺮ ﺟﻬﺶ اﻟﻘﺎﯾﯽ ﺑﺮ ﮐﺎﻫﺶ ﻣﯿﺰان رﻧﮓ داﻧﻪ ﻫﺎي ﻓﺘﻮﺳﻨﺘﺰي ﺑﻪ دﻟﯿﻞ اﯾﺠﺎد ﻧﻘﺺ در ژن ﻫﺎي ﻣﺴﺆل ﺑﯿﻮﺳﻨﺘﺰ رﻧﮓ داﻧﻪ ﻫﺎ، ﺗﺨﺮﯾﺐ ﻣﺴﯿﺮﻫﺎي ﺑﯿﻮﺳﻨﺘﺰي ﺗﻮﻟﯿﺪ رﻧﮓ داﻧﻪ ﻫﺎ، ﺗﺨﺮﯾﺐ ﮐﻠﺮوﭘﻼﺳﺖ و ﻧﯿﺰ ﺗﺠﺰﯾﻪ رﻧﮓ داﻧﻪ ﻫﺎي ﻓﺘﻮﺳﻨﺘﺰي ﻣﯽ ﺑﺎﺷﺪ ]19 [. آﻓﺮﯾ ﻘﺎﯾﯽ )S. ionantha( در ﺷﺮاﯾﻂ درون ﺷﯿﺸﻪ اي ﺑﻮده اﺳﺖ ]14 ، 18 [. 2. ﻣﻮاد و روش ﻫﺎ ﺑﺮﺧﯽ از ﮔﯿﺎﻫﭽﻪ ﻫﺎي داراي ﺗﻐﯿﯿﺮ ﻓﻨﻮﺗﯿﭙﯽ ﺳﺮﻋﺖ رﺷﺪ ﮐﻢ ﺗﺮي ﻧﺴﺒﺖ ﺑﻪ ﮔﯿﺎﻫﭽﻪ ﻫﺎي دﯾﮕﺮ داﺷﺘﻨﺪ ﺑﻪ ﻃﻮري ﮐﻪ اﯾﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ در زﻣﺎن داده ﺑﺮداري در ﻓﺎز ﮔﻞ دﻫﯽ ﻧﺒﻮدﻧﺪ )ﺟﺪول 1 .( در ﺗﺤﻘﯿﻘﯽ ﮐﻪ داﺗﺎ و ﻫﻤﮑﺎران ]3 [ ﺑﻪ ﻣﻨﻈﻮر ﺗﻮﻟﯿﺪ ﮔﯿﺎﻫﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﭘﺎﯾﺪار ﮔﯿﺎه داوودي اﻧﺠﺎم دادﻧﺪ، در ﮔﯿﺎﻫﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﺣﺎﺻﻞ ﻋﻼوه ﺑﺮ رﻧﮓ، ﺷﮑﻞ ﮔﻞ ﻫﺎ ﻧﯿﺰ ﺗﻐﯿﯿﺮ ﯾﺎﻓﺖ، ﺑﻪ ﻃﻮري ﮐﻪ ﮔﻞ ﻫﺎي زﺑﺎﻧﻪ اي ﮔﯿﺎه ﺑﻪ ﺻﻮرت ﻟﻮﻟﻪ اي ﺗﻐﯿﯿﺮﺷﮑﻞ ﭘﯿﺪا ﮐﺮدﻧﺪ. ﻫﻢ ﭼﻨﯿﻦ ﺗﺎﺑﺶ ﭘﺮﺗﻮي ﮔﺎﻣﺎ ﺑﻪ Journal of Nuclear Science, Engineering and Technology Vol 45 (4) Serial Number 109 2024 P 66-74 ﺷﮑﻞ1 . ﺗﻨﻮع ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ در ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه در ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﭘﺮﺗﻮدﯾﺪه اﺷﻌﻪ ﮔﺎﻣﺎ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ. اﻟﻒ( ﺷﺎﻫﺪ، ب( ﻣﻀﺮس ﺷﺪن ﺑﺮگ، پ( ﭘﯿﭽﯿﺪﮔﯽ ﺑﺮگ ﺑﻪ داﺧﻞ، ت( ﺗﻐﯿﯿﺮ ﺷﮑﻞ ﺑﺮگ، ث( اﺑﻠﻖ ﺷﺪن ﺟﺰﯾﯽ در ﺑﺮگ، ج( ﮐﺎﻫﺶ ﺳﺎﯾﺰ ﺑﺮگ )ﮐﻮﺗﻮﻟﮕﯽ ﺑﺮگ(، چ( ﮐﻮﺗﻮﻟﮕﯽ ﻫﻤﺮاه ﺑﺎ ﻣﻀﺮس ﺷﺪن، ح( ﺷﮑﺎف در ﻣﺤﻞ اﺗﺼﺎل دﻣﺒﺮگ ﺑﻪ ﺑﺮگ، خ( ﮐﺸﯿﺪه ﺷﺪن دﻣﺒﺮگ و ﺑﺮگ و ﺻﺎف ﺷﺪن ﺑﺮگ و ﻋﺪم ﻣﻀﺮس ﺑﻮدن، د( ﺗﻐﯿﯿﺮ رﻧﮓ ﺑﺮگ ﻫﺎ، ذ( زرد ﺷﺪن ﮐﺎﻣﻞ ﺑﺮگ و ر( اﺑﻠﻖ ﺷﺪن ﺑﺨﺸﯽ از ﺑﺮگ. ﺷﮑﻞ2 .ﺗﻌﺪاد ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه در ﻫﺮ ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺑﻪ ﺗﻔﮑﯿﮏ دز ﺗﺎﺑﺶ ) در ﻫﺮ ﺳﺘﻮن ﻣﯿﺎﻧﮕﯿﻦ ﻫﺎﯾﯽ ﺑﺎ ﺣﺪاﻗﻞ ﯾﮏ ﺣﺮف ﻣ ﺸﺘﺮك، در ﺳﻄﺢ اﺣﺘﻤﺎل ﭘﻨﺞ درﺻﺪ ﺗﻔﺎوت ﻣﻌﻨﯽ داري ﻧﺪارﻧﺪ (. ﺷﮑﻞ3 . ﺗﻨﻮع در ﮔﻞ ﺗﺸﮑﯿﻞ ﺷﺪه ﺑﺮ روي ﻫﺮ ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺗﺎﺑﺶ دﯾﺪه اﺷﻌﻪ ﮔﺎﻣﺎ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ. اﻟﻒ( ﺷﺎﻫﺪ، ب( اﻓﺰاﯾﺶ ﺳﺎﯾﺰ ﮔﻞ، پ و ت( ﮐﺎﻫﺶ ﺗﻌﺪاد و ﻧﺎﻣﺘﻘﺎرن ﺑﻮدن ﺳﺎﯾﺰ ﮔﻠﺒﺮگ، ث( ﻣﻀﺮس ﺷﺪن ﺣﺎﺷﯿﻪ ﮔﻠﺒﺮگ، ج( ﮐﺎﻫﺶ ﺗﻌﺪاد ﮔﻠﺒﺮگ و ﻣﺘﻘﺎرن ﺑﻮدن ﺳﺎﯾﺰ و چ( ﺟﺎﻣﯽ ﺷﮑﻞ ﺷﺪن ﮔﻞ. ﺷﮑﻞ1 . ﺗﻨﻮع ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ در ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه در ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﭘﺮﺗﻮدﯾﺪه اﺷﻌﻪ ﮔﺎﻣﺎ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ. اﻟﻒ( ﺷﺎﻫﺪ، ب( ﻣﻀﺮس ﺷﺪن ﺑﺮگ، پ( ﭘﯿﭽﯿﺪﮔﯽ ﺑﺮگ ﺑﻪ داﺧﻞ، ت( ﺗﻐﯿﯿﺮ ﺷﮑﻞ ﺑﺮگ، ث( اﺑﻠﻖ ﺷﺪن ﺟﺰﯾﯽ در ﺑﺮگ، ج( ﮐﺎﻫﺶ ﺳﺎﯾﺰ ﺑﺮگ )ﮐﻮﺗﻮﻟﮕﯽ ﺑﺮگ(، چ( ﮐﻮﺗﻮﻟﮕﯽ ﻫﻤﺮاه ﺑﺎ ﻣﻀﺮس ﺷﺪن، ح( ﺷﮑﺎف در ﻣﺤﻞ اﺗﺼﺎل دﻣﺒﺮگ ﺑﻪ ﺑﺮگ، خ( ﮐﺸﯿﺪه ﺷﺪن دﻣﺒﺮگ و ﺑﺮگ و ﺻﺎف ﺷﺪن ﺑﺮگ و ﻋﺪم ﻣﻀﺮس ﺑﻮدن، د( ﺗﻐﯿﯿﺮ رﻧﮓ ﺑﺮگ ﻫﺎ، ذ( زرد ﺷﺪن ﮐﺎﻣﻞ ﺑﺮگ و ر( اﺑﻠﻖ ﺷﺪن ﺑﺨﺸﯽ از ﺑﺮگ. ﺷﮑﻞ1 . 2. ﻣﻮاد و روش ﻫﺎ ﺗﻨﻮع ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ در ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه در ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﭘﺮﺗﻮدﯾﺪه اﺷﻌﻪ ﮔﺎﻣﺎ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ. اﻟﻒ( ﺷﺎﻫﺪ، ب( ﻣﻀﺮس ﺷﺪن ﺑﺮگ، پ( ﭘﯿﭽﯿﺪﮔﯽ ﺑﺮگ ﺑﻪ داﺧﻞ، ت( ﺗﻐﯿﯿﺮ ﺷﮑﻞ ﺑﺮگ، ث( اﺑﻠﻖ ﺷﺪن ﺟﺰﯾﯽ در ﺑﺮگ، ج( ﮐﺎﻫﺶ ﺳﺎﯾﺰ ﺑﺮگ )ﮐﻮﺗﻮﻟﮕﯽ ﺑﺮگ(، چ( ﮐﻮﺗﻮﻟﮕﯽ ﻫﻤﺮاه ﺑﺎ ﻣﻀﺮس ﺷﺪن، ح( ﺷﮑﺎف در ﻣﺤﻞ اﺗﺼﺎل دﻣﺒﺮگ ﺑﻪ ﺑﺮگ، خ( ﮐﺸﯿﺪه ﺷﺪن دﻣﺒﺮگ و ﺑﺮگ و ﺻﺎف ﺷﺪن ﺑﺮگ و ﻋﺪم ﻣﻀﺮس ﺑﻮدن، د( ﺗﻐﯿﯿﺮ رﻧﮓ ﺑﺮگ ﻫﺎ، ذ( زرد ﺷﺪن ﮐﺎﻣﻞ ﺑﺮگ و ر( اﺑﻠﻖ ﺷﺪن ﺑﺨﺸﯽ از ﺑﺮگ. ﺷﮑﻞ2 .ﺗﻌﺪاد ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه در ﻫﺮ ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺑﻪ ﺗﻔﮑﯿﮏ دز ﺗﺎﺑﺶ ) در ﻫﺮ ﺳﺘﻮن ﻣﯿﺎﻧﮕﯿﻦ ﻫﺎﯾﯽ ﺑﺎ ﺣﺪاﻗﻞ ﯾﮏ ﺣﺮف ﻣ ﺸﺘﺮك، در ﺳﻄﺢ اﺣﺘﻤﺎل ﭘﻨﺞ درﺻﺪ ﺗﻔﺎوت ﻣﻌﻨﯽ داري ﻧﺪارﻧﺪ (. ﺷﮑﻞ2 .ﺗﻌﺪاد ﺑﺮگ ﺗﺸﮑﯿﻞ ﺷﺪه در ﻫﺮ ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺑﻪ ﺗﻔﮑﯿﮏ دز ﺗﺎﺑﺶ ) در ﻫﺮ ﺳﺘﻮن ﻣﯿﺎﻧﮕﯿﻦ ﻫﺎﯾﯽ ﺑﺎ ﺣﺪاﻗﻞ ﯾﮏ ﺣﺮف ﻣ ﺸﺘﺮك، در ﺳﻄﺢ اﺣﺘﻤﺎل ﭘﻨﺞ درﺻﺪ ﺗﻔﺎوت ﻣﻌﻨﯽ داري ﻧﺪارﻧﺪ (. ﺷﮑﻞ3 . ﺗﻨﻮع در ﮔﻞ ﺗﺸﮑﯿﻞ ﺷﺪه ﺑﺮ روي ﻫﺮ ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺗﺎﺑﺶ دﯾﺪه اﺷﻌﻪ ﮔﺎﻣﺎ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ. اﻟﻒ( ﺷﺎﻫﺪ، ب( اﻓﺰاﯾﺶ ﺳﺎﯾﺰ ﮔﻞ، پ و ت( ﮐﺎﻫﺶ ﺗﻌﺪاد و ﻧﺎﻣﺘﻘﺎرن ﺑﻮدن ﺳﺎﯾﺰ ﮔﻠﺒﺮگ، ث( ﻣﻀﺮس ﺷﺪن ﺣﺎﺷﯿﻪ ﮔﻠﺒﺮگ، ج( ﮐﺎﻫﺶ ﺗﻌﺪاد ﮔﻠﺒﺮگ و ﻣﺘﻘﺎرن ﺑﻮدن ﺳﺎﯾﺰ و چ( ﺟﺎﻣﯽ ﺷﮑﻞ ﺷﺪن ﮔﻞ. ﺷﮑﻞ3 . ﺗﻨﻮع در ﮔﻞ ﺗﺸﮑﯿﻞ ﺷﺪه ﺑﺮ روي ﻫﺮ ﮔﯿﺎﻫﭽﻪ ﺳﺎزﮔﺎر ﺷﺪه از رﯾﺰﻧﻤﻮﻧﻪ ﻫﺎي ﺗﺎﺑﺶ دﯾﺪه اﺷﻌﻪ ﮔﺎﻣﺎ در ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ. اﻟﻒ( ﺷﺎﻫﺪ، ب( اﻓﺰاﯾﺶ ﺳﺎﯾﺰ ﮔﻞ، پ و ت( ﮐﺎﻫﺶ ﺗﻌﺪاد و ﻧﺎﻣﺘﻘﺎرن ﺑﻮدن ﺳﺎﯾﺰ ﮔﻠﺒﺮگ، ث( ﻣﻀﺮس ﺷﺪن ﺣﺎﺷﯿﻪ ﮔﻠﺒﺮگ، ج( ﮐﺎﻫﺶ ﺗﻌﺪاد ﮔﻠﺒﺮگ و ﻣﺘﻘﺎرن ﺑﻮدن ﺳﺎﯾﺰ و چ( ﺟﺎﻣﯽ ﺷﮑﻞ ﺷﺪن ﮔﻞ. Journal of Nuclear Science, Engineering and Tech Vol. 45 (4), Serial Number 109, 2024, P 66-74 دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 ﺗﻮﻟﯿﺪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ )Saintpaulia ionantha ( . . . 70 70 ﺟﺪول1. 2. ﻣﻮاد و روش ﻫﺎ در اﯾﻦ ﻣﯿﺎن آﻏﺎزﮔﺮ808 UBC ﮐﻢ ﺗﺮﯾﻦ ﻣﮑﺎن ژﻧﯽ را ﺑﺎ 7 ﺑﺎﻧﺪ و آﻏﺎزﮔﺮﻫﺎي840 UBC ، 842 UBC و873 UBC ﺑﯿﺶ ﺗﺮﯾﻦ ﺗﻌﺪاد ﻣﮑﺎن ژﻧﯽ را ﺑﺎ 10 ﺑﺎﻧﺪ ﺗﻮﻟﯿﺪ ﮐﺮدﻧﺪ ﮐﻪ ﺑﯿﺎﻧ ﮕﺮ ﻗﺪرت اﯾﻦ آﻏﺎزﮔﺮﻫﺎ در ﺗﻔﮑﯿﮏ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﻣﯽ ﺑﺎﺷﺪ. درﺻﺪ ﭼﻨﺪ ﺷﮑﻠﯽ از 61 ﺗﺎ85 درﺻﺪ ﻣﺘﻐﯿﺮ ﺑﻮد و ﻣﯿﺎﻧﮕﯿﻦ ﻣﯿﺰان ﭼﻨﺪﺷﮑﻠﯽ 42 / 70 درﺻﺪ ﻣﺤﺎﺳﺒﻪ .ﺷﺪ ﺳﭙﺲ ﻣﯿﺰان اﻃﻼﻋﺎت ﭼﻨﺪﺷﮑﻠﯽ ) PIC ( ﺑﺎ اﺳﺘﻔﺎده از ﻓﺮاواﻧﯽ آﻟﻠﯽ ﻫﺮ آﻏﺎزﮔﺮ ﻣﺤﺎﺳﺒﻪ ﮔﺮدﯾﺪ ﮐﻪ در واﻗﻊ ﺑﯿﺎﻧﮕﺮ ارزش ﯾﮏ ﻧﺸﺎﻧﮕﺮ ﺑﺮ اي ﺗﺸﺨﯿﺺ ﭼﻨﺪ ﺷﮑﻠﯽ در ﯾﮏ ﺟﻤﻌﯿﺖ ﻣﯽ ﺑﺎﺷﺪ. ﻣﯿﺎﻧﮕﯿﻦ PIC آﻏﺎزﮔﺮﻫﺎ در اﯾﻦ ﭘﮋوﻫﺶ37 /0 ﺑﻮد ﮐﻪ ﺑﯿﻦ43 /0 ﺗﺎ32 /0 ﻣﺘﻐﯿﺮ ﺑﻮد و اﯾﻦ ﻣﯿﺰان ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﯾﻦ ﮐﻪ ﺣﺪاﮐﺜﺮ PIC در ﻧﺸﺎﻧﮕﺮﻫﺎي ﻏﺎﻟﺐ5 /0 ﻣﯽ ﺑﺎﺷﺪ، ﻧﺸﺎن دﻫﻨﺪه اﯾﻦ اﺳﺖ ﮐﻪ آﻏﺎزﮔﺮﻫﺎي اﻧﺘﺨﺎب ﺷﺪه ﭘﺮاﮐﻨﺪﮔﯽ ﻣﻨﺎﺳﺒﯽ در ژﻧﻮم ﺟﻤﻌﯿﺖ ﻣﻮرد ﺑﺮرﺳﯽ داﺷﺘﻪ اﻧﺪ. ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ ﺟﺪول 2، ﺑﯿﺶ ﺗﺮﯾﻦ ﻣﯿﺰان PIC ﻣﺮﺑﻮط ﺑﻪ آﻏﺎزﮔﺮﻫﺎي840 UBC ) ﺑﺎ43 /0 ( و857 UBC ﺑﺎ )42 /0 ( ﺑﻮد. ﺑﺪﯾﻦ ﻣﻔﻬﻮم ﮐﻪ آﻏﺎزﮔﺮﻫﺎي840 UBC و 857 UBC ﺑﺎ دارا ﺑﻮدن ﺣﺪاﮐﺜﺮ ﭘﺮاﮐﻨﺪﮔﯽ آﻟﻠﯽ ﺑﯿﻦ ﻧﻤﻮﻧﻪ ﻫﺎ ﺑﻬﺘﺮ از ﺳﺎﯾﺮ آﻏﺎزﮔﺮﻫﺎ ﻓﺎﺻﻠﻪ ژﻧﺘﯿﮑﯽ را ﻣﻌﻠﻮم ﮐﺮده و ﻣﯽ ﺗﻮاﻧﻨﺪ ﺑﻪ ﻋﻨﻮان آﻏﺎزﮔﺮﻫﺎﯾﯽ ﺑﺎ ﺗﻮان ﺑﺎﻻي ﺗﻌﯿﯿﻦ ﺗﻨﻮع ﻣﻮرد اﺳﺘﻔﺎده ﻗﺮار ﮔﯿﺮﻧﺪ. 3 .2 ﺗﺠﺰﯾﻪ ﺧﻮﺷﻪ اي ﺑﺮ اﺳﺎس ﺗﻤﺎم آﻏﺎزﮔﺮﻫﺎ ﺗﺮﺳﯿﻢ دﻧﺪروﮔﺮا م ﺑﺎ اﺳﺘﻔﺎده از ﺑﺎﻧﺪﻫﺎي ﭼﻨﺪ ﺷﮑﻞ اﻧﺠﺎم و ﺷﺒﺎﻫﺖ ژﻧﺘﯿﮑﯽ ﺑﯿﻦ ژﻧﻮﺗﯿﭗ ﻫﺎ و ﺷﺎﻫﺪ ﺑﺎ اﺳﺘﻔﺎده از ﺿﺮﯾﺐ ﺟﺎﮐﺎرد1 ﻣﺤﺎﺳﺒﻪ و آﻧﺎﻟﯿﺰ ﺧﻮﺷﻪ ﺑﻨﺪي UPGMA .ﺻﻮرت ﮔﺮﻓﺖ ﻧﺘﺎﯾﺞ ﺑﻪ دﺳﺖ آﻣﺪه از دﻧﺪروﮔﺮام، ﺗﻤﺎﯾﺰ ژﻧﺘﯿﮑﯽ ژﻧﻮﺗﯿﭗ ﻫﺎي ﻣﻮرد ﻣﻄﺎﻟﻌﻪ را ﻧﺸﺎن داد. ﻫﻤﺒﺴﺘﮕﯽ )ﺿﺮﯾﺐ( ﮐﻮﻓﻨﺘﯿﮏ ﮐﻪ ﯾﮏ ﻫﻤﺒﺴﺘﮕﯽ ﺑﯿﻦ ﺷﺒﺎﻫ ﺖ ﻫﺎي ﻧﺸﺎن داده ﺷﺪه روي دﻧﺪروﮔﺮام و درﺟﻪ واﻗﻌﯽ ﺷﺒﺎﻫﺖ آن ﻫﺎﺳﺖ و ﺑﯿﺎﻧﮕﺮ اﻋﺘﺒﺎر و ﻫﻢ ﺧﻮاﻧﯽ ﺧﻮﺷﻪ اي ﺣﺎﺻﻞ ﺑﺎ ﻣﺎﺗﺮﯾﺲ داده ورودي ﻣﯽ ﺑﺎﺷﺪ، ﻣﺤﺎﺳﺒﻪ ﺷﺪ. ﺿﺮﯾﺐ ﮐﻮﻓﻨﺘﯿﮏ ﺑﯿﻦ ﻣﺎﺗﺮﯾﺲ ﺗﺸﺎﺑﻪ و دﻧﺪروﮔﺮام ﺣﺎﺻﻞ 992 /0 =r ﺑﻪ دﺳﺖ آﻣﺪ. ﺿﺮﯾﺐ ﮐﻮﻓﻨﺘﯿﮏ ﺑﺎﻻي ﺑﻪ دﺳﺖ آﻣﺪه ﺑﻪ ﮐﻤﮏ روش ﺗﺠﺰﯾﻪ ﺧﻮﺷﻪ اي ﺑﺮ اﺳﺎس ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ﺟﺎﮐﺎرد )85 /0( ﻧﺸﺎن دﻫﻨﺪه ﮐﺎراﯾﯽ اﯾﻦ روش ﺑﺮاي ﮔﺮوه ﺑﻨﺪي ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﻓﺮﺿﯽ اﺳﺖ. در دﻧﺪروﮔﺮام 11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻓﺮﺿﯽ و 3 ﺷﺎﻫﺪ ﺑﻪ4 ﮔﺮوه و دو ﻓﺮد ﻣﺠﺰا در ﺳﻄﺢ ﺗﺸﺎﺑﻪ ژﻧﺘﯿﮑﯽ 49 /0 ﮔﺮوه ﺑﻨﺪي ﺷﺪﻧﺪ )ﺷﮑﻞ 4 (. 2. ﻣﻮاد و روش ﻫﺎ ﺑﺮرﺳﯽ ﻓﻨﻮﺗﯿﭙﯽ ﮔﯿﺎﻫﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻧﺎﺷﯽ از ﺗﺎﺑﺶ اﺷﻌﻪ ﮔﺎﻣﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﺗﻌﺪاد ﺑﺮگ ﻗﻄﺮ ﺑﺮگ ) mm ( ﻃﻮل دﻣﺒﺮگ (mm) ﺗﻌﺪاد ﮔﻞ آذﯾﻦ ﺗﻌﺪاد ﮔﻞ روي ﮔﻞ آذﯾﻦ ﺗﻌﺪاد ﮔﻠﺒﺮگ در ﻫﺮ ﮔﻞ ﻗﻄﺮ ﺑﺰرگ ﺗﺮﯾﻦ ﮔﻞ(mm) ﺗﻌﺪاد ﭘﺮﭼﻢ در ﻫﺮ ﮔﻞ ﺷﺎﻫﺪ 12 14 / 43 45 / 60 3 5 10 65 / 32 ° 1 13 40 / 62 60 / 35 2 4 10 40 / 24 3 2 12 17 / 47 97 / 18 4 5 5 46 / 22 2 3 15 92 / 43 98 / 29 3 5 9 59 / 39 3 4 13 42 / 42 95 / 29 4 5 5 19 / 31 2 5 8 63 / 30 84 / 18 ° ° ° ° ° 6 14 24 / 35 23 / 23 3 5 7 47 / 35 ° 7 * 4 28 / 22 89 /6 ° ° ° ° ° 8 * 9 81 / 18 13 / 13 ° ° ° ° ° 9 17 34 / 46 50 / 45 6 5 10 31 / 33 2 10 18 43 / 54 68 / 64 9 7 12 92 / 32 2 11 15 94 / 50 95 / 38 4 8 5 20 / 43 ° 12 17 60 / 45 28 / 49 6 6 13 19 / 31 3 13 16 38 / 40 92 / 38 7 7 13 43 / 35 3 14 16 53 / 50 05 / 41 5 6 11 89 / 31 3 *داده ﺑﺮداري ﺑﻪ دﻟﯿﻞ ﯾﮑﺴﺎن ﺑﻮدن ﺳﻦ رﺷﺪي ﮔﯿﺎﻫﺎن در ﯾﮏ زﻣﺎن واﺣﺪ اﻧﺠﺎم ﺷﺪ و اﯾﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ در زﻣﺎن داده ﺑﺮداري در ﻓﺎز ﮔﻞ .دﻫﯽ ﻧﺒﻮدﻧﺪ 3 .1 آﻧﺎﻟﯿﺰ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﻧﻤﻮﻧﻪ ﻫﺎي ﺗﯿﻤﺎر ﺷﺪه ﺑﺎ ﭘﺮﺗﻮ ﮔﺎﻣﺎ ﺑﺎ اﺳﺘﻔﺎده از ﻧﺸﺎﻧﮕﺮ ﻣﻮﻟﮑﻮﻟﯽ ISSR ﺗﻐﯿﯿﺮﭘﺬﯾﺮي ژﻧﺘﯿﮑﯽ11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻓﺮﺿﯽ و3 ﺷﺎﻫﺪ ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺑﺎ اﺳﺘﻔﺎده از 8 آﻏﺎزﮔﺮISSR آﻧﺎﻟﯿﺰ ﺷﺪﻧﺪ ﮐﻪ در ﻣﺠﻤﻮع 71 ﻣﮑﺎن )ﺑﺎﻧﺪ( ﻗﺎﺑﻞ اﻣﺘﯿﺎزدﻫﯽ اﯾﺠﺎد ﮐﺮدﻧﺪ ﮐﻪ در اﯾﻦ ﻣﯿﺎن 50 ) ﻣﮑﺎن70 ( ﭼﻨﺪﺷﮑﻠﯽ را% ﻧﺸﺎن دادﻧﺪ. ﻣﯿﺎﻧﮕﯿﻦ ﺗﻌﺪاد ﺑﺎﻧﺪﻫﺎي ﺗﮑﺜﯿﺮ ﺷﺪه و ﺑﺎﻧﺪﻫﺎي ﭼﻨﺪ ﺷﮑﻞ ﺑﻪ ازاي ﻫﺮ آﻏﺎزﮔﺮ ﺑﻪ ﺗﺮﺗﯿﺐ 87 /8 و25 /6 ﺑﻮد. Journal of Nuclear Science, Engineering and Technology Vol. 45 (4), Serial Number 109, 2024, P 66-74 1. Jaccard Coefficient 2. ﻣﻮاد و روش ﻫﺎ ﺗﻮاﻟﯽ آﻏﺎزﮔﺮ ﻣﻮرد اﺳﺘﻔﺎده و اﻃﻼﻋﺎت ﭼﻨﺪﺷﮑﻠﯽ آن ﭘﺮاﯾﻤﺮ ﺗﻮاﻟﯽ Ta )دﻣﺎي اﺗﺼﺎل ﭘﺮاﯾﻤﺮ ( ﺗﻌﺪاد ﺑﺎﻧﺪﻫﺎ ﺗﻌﺪاد ﺑﺎﻧﺪﻫﺎي ﭼﻨﺪﺷﮑﻞ درﺻﺪ ﻓﺮاواﻧﯽ ﺑﺎﻧﺪﻫﺎي ﭼﻨﺪﺷﮑﻞ ﻣﺤﺘﻮاي اﻃﻼﻋﺎﺗﯽ ﭼﻨﺪ ﺷﮑﻠﯽ 808 UBC 5'-AGA GAG AGA GAG AGA GC-3' 4 / 52 7 4 83 % 35 /0 810 UBC 5'-GAG AGA GAG AGA GAG AT-3' 0 / 50 8 6 75 % 32 /0 834 UBC 5'-AGA GAG AGA GAG AGA GYT-3' 6 / 51 9 6 63 % 35 /0 836 UBC 5'-AGA GAG AGA GAG AGA GYA-3' 6 / 51 8 4 76 % 40 /0 840 UBC 5'-GAG AGA GAG AGA GAG AYT-3' 6 / 51 10 9 81 % 43 /0 842 UBC 5'-GAG AGA GAG AGA GAG AYG-3' 9 / 53 10 6 61 % 36 /0 857 UBC 5'-ACA CAC ACA CAC ACA CYG-3' 9 / 53 9 8 81 % 42 /0 873 UBC 5'-GAC AGA CAG ACA GAC A-3' 2 / 48 10 7 85 % 40 /0 All 71 50 - - ﺷﮑﻞ4. دﻧﺪروﮔﺮام ﺑﻪ دﺳﺖ آﻣﺪه ﺑﺎ اﺳﺘﻔﺎده از 8 آﻏﺎزﮔﺮISSR ﺑﺮ اﺳﺎس روش UPGMA و ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ﺟﺎﮐﺎرد ﺑﺮاي11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻓﺮﺿﯽ )1 - 11 ( و3 ) ﺷﺎﻫﺪ1 ، 2 و3 .( در ﮔﯿﺎه ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺷﮑﻞ5 . ﻧﻤﺎﯾﺶ دوﺑﻌﺪي آﻧﺎﻟﯿﺰPCoA ﻧﻤﺎﯾﺎﻧﮕﺮ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ و ﺳﻪ ﺷﺎﻫﺪ. ﺟﺪول2 . ﺗﻮاﻟﯽ آﻏﺎزﮔﺮ ﻣﻮرد اﺳﺘﻔﺎده و اﻃﻼﻋﺎت ﭼﻨﺪﺷﮑﻠﯽ آن ﺷﮑﻞ5 . ﻧﻤﺎﯾﺶ دوﺑﻌﺪي آﻧﺎﻟﯿﺰPCoA ﻧﻤﺎﯾﺎﻧﮕﺮ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ و ﺳﻪ ﺷﺎﻫﺪ. ﺷﮑﻞ5 . ﻧﻤﺎﯾﺶ دوﺑﻌﺪي آﻧﺎﻟﯿﺰPCoA ﻧﻤﺎﯾﺎﻧﮕﺮ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ و ﺳﻪ ﺷﺎﻫﺪ. ﺟﻬﺶ ﯾﺎﻓﺘﻪ و ﺳﻪ ﺷﺎﻫﺪ. 3 . 3 ﻓﺎﺻﻠﻪ ژﻧﺘﯿﮑﯽ ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﺟﻬﺶ ﯾﺎﻓﺘﻪ 1 ﺗﺎ4 ﺑﺎ ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ژﻧﺘﯿﮑﯽ986 /0 ﺑﯿﺶ ﺗﺮﯾﻦ ﺗﺸﺎﺑﻪ و ﺟﻬﺶ ﯾﺎﻓﺘﻪ 8 ﺑﺎ ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ژﻧﺘﯿﮑﯽ51 /0 ﮐﻢ ﺗﺮﯾﻦ ﺗﺸﺎﺑﻪ را ﻧﺴﺒﺖ ﺑﻪ ﺷﺎﻫﺪ داﺷﺘﻨﺪ. آﻧﺎﻟﯿﺰ UPGMA )ﺷﮑﻞ4 ( و PCA )ﺷﮑﻞ5( ﺑﺎ ﻫﻢ ﻫﻢ ﺧﻮاﻧﯽ داﺷﺘﻨﺪ و ﭘﻼت دوﺑﻌﺪي PCoA ﻧﯿﺰ ﭘﺮاﮐﻨﺪﮔﯽ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ را ﺑﻪ ﺧﻮﺑﯽ ﻧﺸﺎن ﻣﯽ دﻫﺪ ﮐﻪ ﺑﯿﺎﻧﮕﺮ وﺟﻮد ﺗﻨﻮع ژﻧﺘﯿﮑﯽ اﯾﺠﺎد ﺷﺪه در ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﻣﯽ ﺑﺎﺷﺪ. ﻧﺘﺎﯾﺞ ﺣﺎﺻﻞ از ﺗﺠﺰﯾﻪ وارﯾﺎﻧﺲ ﻣﻮ ﻟﮑﻮﻟﯽ )AMOVA ( ﻧﺸﺎن داد ﮐﻪ38 درﺻﺪ ﺗﻨﻮع در ﺑﯿﻦ ﺟﻤﻌﯿﺖ ﻫﺎ و 62 درﺻﺪ ﺗﻨﻮع در داﺧﻞ ﺟﻤﻌﯿﺖ اﺳﺖ و ﻣﯿﺰان ﺗﻨﻮع ﺑﯿﻦ ﺟﻤﻌﯿﺖ ﻫﺎ در ﻣﻘﺎﯾﺴﻪ ﺑﺎ ﺗﻨﻮع درون ﺟﻤﻌﯿﺖ ﻧﺸﺎن دﻫﻨﺪه ﻣﻌﻨﯽ داري ﺗﻔﺎوت ﻫﺎي اﯾﺠﺎد ﺷﺪه اﺳﺖ. 2. ﻣﻮاد و روش ﻫﺎ آﻧﺎﻟﯿﺰ UPGMA )ﺷﮑﻞ4 ( و PCA )ﺷﮑﻞ5( ﺑﺎ ﻫﻢ ﻫﻢ ﺧﻮاﻧﯽ داﺷﺘﻨﺪ و ﭘﻼت دوﺑﻌﺪي PCoA ﻧﯿﺰ ﭘﺮاﮐﻨﺪﮔﯽ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ را ﺑﻪ ﺧﻮﺑﯽ ﻧﺸﺎن ﻣﯽ دﻫﺪ ﮐﻪ ﺑﯿﺎﻧﮕﺮ وﺟﻮد ﺗﻨﻮع ژﻧﺘﯿﮑﯽ اﯾﺠﺎد ﺷﺪه در ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﻣﯽ ﺑﺎﺷﺪ. ﻧﺘﺎﯾﺞ ﺣﺎﺻﻞ از ﺗﺠﺰﯾﻪ وارﯾﺎﻧﺲ ﻣﻮ ﻟﮑﻮﻟﯽ )AMOVA ( ﻧﺸﺎن داد ﮐﻪ38 درﺻﺪ ﺗﻨﻮع در ﺑﯿﻦ ﺟﻤﻌﯿﺖ ﻫﺎ و 62 درﺻﺪ ﺗﻨﻮع در داﺧﻞ ﺟﻤﻌﯿﺖ اﺳﺖ و ﻣﯿﺰان ﺗﻨﻮع ﺑﯿﻦ ﺟﻤﻌﯿﺖ ﻫﺎ در ﻣﻘﺎﯾﺴﻪ ﺑﺎ ﺗﻨﻮع درون ﺟﻤﻌﯿﺖ ﻧﺸﺎن دﻫﻨﺪه ﻣﻌﻨﯽ داري ﺗﻔﺎوت ﻫﺎي اﯾﺠﺎد ﺷﺪه اﺳﺖ. ﻧﺘﺎﯾﺞ ﺑﻪ دﺳﺖ آﻣﺪه از اﯾﻦ ﺗﺤﻘﯿﻖ ﻧﺸﺎن ﻣﯽ دﻫﺪ ﮐﻪ در ﺳﻄﺢ ﻣﻮﻟﮑﻮﻟﯽ ﺑﯿﻦ ﺟﻬ ﺶ ﯾﺎﻓﺘﻪ ﻫﺎي اﯾﺠﺎد ﺷﺪه ﺗﻔﺎوت ﻣﻌﻨﯽ داري وﺟﻮد دارد. ﻫﻢ ﭼﻨﯿﻦ ﻧﺘﺎﯾﺞ ﺑﯿﺎﻧﮕﺮ اﯾﻦ اﺳﺖ ﮐﻪ ISSR، ﻧﺸﺎﻧﮕﺮ ﻣﻔﯿﺪي ﺑﺮاي اﻧﺪازه ﮔﯿﺮي ﺳﺮﯾﻊ و آﺳﺎن ﺗﻨﻮع ژﻧﺘﯿﮑﯽ در ﻣﯿﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ اﺳﺖ. ﺷﮑﻞ5 . ﻧﻤﺎﯾﺶ دوﺑﻌﺪي آﻧﺎﻟﯿﺰPCoA ﻧﻤﺎﯾﺎﻧﮕﺮ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ و ﺳﻪ ﺷﺎﻫﺪ. ﺑﺮرﺳﯽ ﺗﻐﯿﯿﺮات ﻣﻮﻟﮑﻮﻟﯽ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي اﯾﺠﺎد ﺷﺪه ﺑﺎ ﻧﺸﺎﻧﮕﺮISSR ﻧﺸﺎن داد ﮐﻪ ﺗﺎﺑﺶ در ﺳﻄﺢ40 ﮔﺮي ﻣﯽ ﺗﻮاﻧﺪ ﭼﻨﺪ ﺷﮑﻠﯽ ﺑﺴﯿﺎر ﺑﺎﻻﯾﯽ در آن ﻫﺎ اﯾﺠﺎد ﮐﻨﺪ ﺑﻪ . ﻃﻮري ﮐﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﻓﺮﺿﯽ ﺑﺎ ﯾﮏ ﺳﻄﺢ ﺗﯿﻤﺎر در ﺳﻄﺢ ﺗﺸﺎﺑﻪ 51 /0 درﺻﺪ ﺑﻪ4 ﮔﺮوه ﺗﻘﺴﯿﻢ ﺑﻨﺪي ﺷﺪﻧﺪ و داﻣﻨﻪ ﺗﺸﺎﺑﻪ ژﻧﺘﯿ ﮑﯽ از 98 /0 ﺗﺎ45 /0 ﻣﺘﻐﯿﺮ ﺑﻮد. اﯾﻦ ﻧﺘﺎﯾﺞ ﻧﺸﺎن دﻫﻨﺪه ﻗﺪرت و ﮐﺎراﯾﯽ ﺗﺎﺑﺶ ﮔﺎﻣﺎ در اﯾﺠﺎد ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﻣﯽ ﺑﺎﺷﺪ ﮐﻪ ﺑﻪ اﺻﻼح ﮐﻨﻨﺪﮔﺎن ﮔﯿﺎﻫﯽ در اﻧﺘﺨﺎب وارﯾﺘﻪ ﻫﺎي ﮔﯿﺎﻫﯽ ﺑﺎ ﺧﺼﻮﺻﯿﺎت ﻣﻄﻠﻮب ﮐﻤﮏ ﻣﯽ ﮐﻨﺪ. ﻧﺘﺎﯾﺞ اﯾﻦ آزﻣﺎﯾﺶ در ﺑﻪ وﺟﻮد آﻣﺪن ﺗﻐﯿﯿﺮات ژﻧﺘﯿﮑﯽ ﺣﺎﺻﻞ از ﺗﺎﺑﺶ، ﻣﺸﺎﺑﻪ ﺑ ﺎ ﻧﺘﺎﯾﺞ اﻟﻘﺎي ﺟﻬﺶ در ﮔﯿﺎﻫﺎن ﻣﺨﺘﻠﻒ ﺑﻮد ] 21 [. در ﻃﯽ ﭘﮋوﻫﺶ ﺑﺮﮐﺎت و اﻟﺴﺎﻣﺎك ]22 [ ﮔﻠﭽﻪ ﻫﺎي ﮔﯿﺎه داوودي رﻗﻢ Delistar White ﺑﺎ دو دُز اﺷﻌﻪ ﮔﺎﻣﺎ ﺗﯿﻤﺎر و از ﻧﺸﺎﻧﮕﺮ ISSR ﺑﺮاي ﺗﺸﺨﯿﺺ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ و واﻟﺪﯾﻦ اﺳﺘﻔﺎده ﮐﺮدﻧﺪ و از 5 آﻏﺎزﮔﺮ ﺑﺮاي ﺗﮑﺜﯿﺮ ﻗﻄﻌﺎتDNA ژﻧﻮﻣﯽ اﺳﺘﻔﺎده ﺷﺪ و13 ﮐﻠﻮن ﺳﻮﻣﺎﺗﯿﮑﯽ ﺑﺎ اﺳﺘﻔﺎده از اﯾﻦ روش ﺟﺪا ﮔﺮدﯾﺪ. ﺷﺒﺎﻫﺖ ژﻧﺘﯿﮑﯽ در ﺑﯿﻦ 40 ژﻧﻮﺗﯿﭗ ﻣﻮرد ﺑﺮرﺳﯽ اﺣﻤﺪ ﺷﺮ ﯾﻔﯽ ، زﻫﺮا ﺳﺮﮔﺰ ي ﻣﻘﺪم، ﻣﻬﺪ ﯾ ﻪ ﺧﺮاز ي ، . . . اﺣﻤﺪ ﺷﺮ ﯾﻔﯽ ، زﻫﺮا ﺳﺮﮔﺰ ي ﻣﻘﺪم، ﻣﻬﺪ ﯾ ﻪ ﺧﺮاز ي ، . . . 71 ﺟﺪول2 . 2. ﻣﻮاد و روش ﻫﺎ ﺷﺎﻫﺪ و ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي 1 ﺗﺎ7 در درون ﮔﺮو ه I ﺗﻘﺴﯿﻢ ﺑﻨﺪي ﺷﺪﻧﺪ اﯾﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﻧﺴﺒﺖ ﺑﻪ ﺑﻘﯿﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ داراي ﺗﺸﺎﺑﻪ ژﻧﺘﯿﮑﯽ ﺑﯿﺶ ﺗﺮي ﻫﺴﺘﻨﺪ و ﺗﻐﯿﯿﺮ ژﻧﺘﯿﮑﯽ زﯾﺎدي ﻧﺴﺒﺖ ﺑﻪ ﺷﺎﻫﺪ ﻧﺸﺎن ﻧﻤﯽ دﻫﻨﺪ. ﮔﺮو ه II ﺑﻪ دو زﯾﺮﮔﺮوه9 و10 ﺗﻘﺴﯿﻢ ﺑﻨﺪي ﺷﺪ. ﮔﺮو ه III ﺑﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ11 ﺗﻔﮑﯿﮏ ﺷﺪ. ﮔﺮوهVI ﺑﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ 8 اﺧﺘﺼﺎص داده ﺷﺪ. اﯾﻦ دو ﮔﺮوه ﺳﻪ و ﭼﻬﺎر ﻧﺴﺒﺖ ﺑﻪ ﺑﻘﯿﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ داراي ﺗﺸﺎﺑﻪ ژﻧﺘﯿﮑﯽ ﮐﻢ ﺗﺮي ﺑﻮدﻧﺪ و ﺗﻐﯿﯿﺮ ژﻧﺘﯿﮑﯽ ﺑﯿﺶ ﺗﺮي را ﻧﺸﺎن دادﻧﺪ. ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 71 اﺣﻤﺪ ﺷﺮ ﯾﻔﯽ ، زﻫﺮا ﺳﺮﮔﺰ ي ﻣﻘﺪم، ﻣﻬﺪ ﯾ ﻪ ﺧﺮاز ي ، . . . ﺟﺪول2 . ﺗﻮاﻟﯽ آﻏﺎزﮔﺮ ﻣﻮرد اﺳﺘﻔﺎده و اﻃﻼﻋﺎت ﭼﻨﺪﺷﮑﻠﯽ آن ﭘﺮاﯾﻤﺮ ﺗﻮاﻟﯽ Ta )دﻣﺎي اﺗﺼﺎل ﭘﺮاﯾﻤﺮ ( ﺗﻌﺪاد ﺑﺎﻧﺪﻫﺎ ﺗﻌﺪاد ﺑﺎﻧﺪﻫﺎي ﭼﻨﺪﺷﮑﻞ درﺻﺪ ﻓﺮاواﻧﯽ ﺑﺎﻧﺪﻫﺎي ﭼﻨﺪﺷﮑﻞ ﻣﺤﺘﻮاي اﻃﻼﻋﺎﺗﯽ ﭼﻨﺪ ﺷﮑﻠﯽ 808 UBC 5'-AGA GAG AGA GAG AGA GC-3' 4 / 52 7 4 83 % 35 /0 810 UBC 5'-GAG AGA GAG AGA GAG AT-3' 0 / 50 8 6 75 % 32 /0 834 UBC 5'-AGA GAG AGA GAG AGA GYT-3' 6 / 51 9 6 63 % 35 /0 836 UBC 5'-AGA GAG AGA GAG AGA GYA-3' 6 / 51 8 4 76 % 40 /0 840 UBC 5'-GAG AGA GAG AGA GAG AYT-3' 6 / 51 10 9 81 % 43 /0 842 UBC 5'-GAG AGA GAG AGA GAG AYG-3' 9 / 53 10 6 61 % 36 /0 857 UBC 5'-ACA CAC ACA CAC ACA CYG-3' 9 / 53 9 8 81 % 42 /0 873 UBC 5'-GAC AGA CAG ACA GAC A-3' 2 / 48 10 7 85 % 40 /0 All 71 50 - - ﺷﮑﻞ4. دﻧﺪروﮔﺮام ﺑﻪ دﺳﺖ آﻣﺪه ﺑﺎ اﺳﺘﻔﺎده از 8 آﻏﺎزﮔﺮISSR ﺑﺮ اﺳﺎس روش UPGMA و ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ﺟﺎﮐﺎرد ﺑﺮاي11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻓﺮﺿﯽ )1 - 11 ( و3 ) ﺷﺎﻫﺪ1 ، 2 و3 .( در ﮔﯿﺎه ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ 3 . 3 ﻓﺎﺻﻠﻪ ژﻧﺘﯿﮑﯽ ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﺟﻬﺶ ﯾﺎﻓﺘﻪ 1 ﺗﺎ4 ﺑﺎ ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ژﻧﺘﯿﮑﯽ986 /0 ﺑﯿﺶ ﺗﺮﯾﻦ ﺗﺸﺎﺑﻪ و ﺟﻬﺶ ﯾﺎﻓﺘﻪ 8 ﺑﺎ ﺿﺮﯾﺐ ﺗﺸﺎﺑﻪ ژﻧﺘﯿﮑﯽ51 /0 ﮐﻢ ﺗﺮﯾﻦ ﺗﺸﺎﺑﻪ را ﻧﺴﺒﺖ ﺑﻪ ﺷﺎﻫﺪ داﺷﺘﻨﺪ. 2. ﻣﻮاد و روش ﻫﺎ ﻧﺘﺎﯾﺞ ﺑﻪ دﺳﺖ آﻣﺪه از اﯾﻦ ﺗﺤﻘﯿﻖ ﻧﺸﺎن ﻣﯽ دﻫﺪ ﮐﻪ در ﺳﻄﺢ ﻣﻮﻟﮑﻮﻟﯽ ﺑﯿﻦ ﺟﻬ ﺶ ﯾﺎﻓﺘﻪ ﻫﺎي اﯾﺠﺎد ﺷﺪه ﺗﻔﺎوت ﻣﻌﻨﯽ داري وﺟﻮد دارد. ﻫﻢ ﭼﻨﯿﻦ ﻧﺘﺎﯾﺞ ﺑﯿﺎﻧﮕﺮ اﯾﻦ اﺳﺖ ﮐﻪ ISSR، ﻧﺸﺎﻧﮕﺮ ﻣﻔﯿﺪي ﺑﺮاي اﻧﺪازه ﮔﯿﺮي ﺳﺮﯾﻊ و آﺳﺎن ﺗﻨﻮع ژﻧﺘﯿﮑﯽ در ﻣﯿﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ اﺳﺖ. Journal of Nuclear Science, Engineering and Technology Vol. 45 (4), Serial Number 109, 2024, P 66-74 ﺑﺮرﺳﯽ ﺗﻐﯿﯿﺮات ﻣﻮﻟﮑﻮﻟﯽ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي اﯾﺠﺎد ﺷﺪه ﺑﺎ ﻧﺸﺎﻧﮕﺮISSR ﻧﺸﺎن داد ﮐﻪ ﺗﺎﺑﺶ در ﺳﻄﺢ40 ﮔﺮي ﻣﯽ ﺗﻮاﻧﺪ ﭼﻨﺪ ﺷﮑﻠﯽ ﺑﺴﯿﺎر ﺑﺎﻻﯾﯽ در آن ﻫﺎ اﯾﺠﺎد ﮐﻨﺪ ﺑﻪ . ﻃﻮري ﮐﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﻓﺮﺿﯽ ﺑﺎ ﯾﮏ ﺳﻄﺢ ﺗﯿﻤﺎر در ﺳﻄﺢ ﺗﺸﺎﺑﻪ 51 /0 درﺻﺪ ﺑﻪ4 ﮔﺮوه ﺗﻘﺴﯿﻢ ﺑﻨﺪي ﺷﺪﻧﺪ و داﻣﻨﻪ ﺗﺸﺎﺑﻪ ژﻧﺘﯿ ﮑﯽ از 98 /0 ﺗﺎ45 /0 ﻣﺘﻐﯿﺮ ﺑﻮد. اﯾﻦ ﻧﺘﺎﯾﺞ ﻧﺸﺎن دﻫﻨﺪه ﻗﺪرت و ﮐﺎراﯾﯽ ﺗﺎﺑﺶ ﮔﺎﻣﺎ در اﯾﺠﺎد ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﻣﯽ ﺑﺎﺷﺪ ﮐﻪ ﺑﻪ اﺻﻼح ﮐﻨﻨﺪﮔﺎن ﮔﯿﺎﻫﯽ در اﻧﺘﺨﺎب وارﯾﺘﻪ ﻫﺎي ﮔﯿﺎﻫﯽ ﺑﺎ ﺧﺼﻮﺻﯿﺎت ﻣﻄﻠﻮب ﮐﻤﮏ ﻣﯽ ﮐﻨﺪ. ﻧﺘﺎﯾﺞ اﯾﻦ آزﻣﺎﯾﺶ در ﺑﻪ وﺟﻮد آﻣﺪن ﺗﻐﯿﯿﺮات ژﻧﺘﯿﮑﯽ ﺣﺎﺻﻞ از ﺗﺎﺑﺶ، ﻣﺸﺎﺑﻪ ﺑ ﺎ ﻧﺘﺎﯾﺞ اﻟﻘﺎي ﺟﻬﺶ در ﮔﯿﺎﻫﺎن ﻣﺨﺘﻠﻒ ﺑﻮد ] 21 [. در ﻃﯽ ﭘﮋوﻫﺶ ﺑﺮﮐﺎت و اﻟﺴﺎﻣﺎك ]22 [ ﮔﻠﭽﻪ ﻫﺎي ﮔﯿﺎه داوودي رﻗﻢ Delistar White ﺑﺎ دو دُز اﺷﻌﻪ ﮔﺎﻣﺎ ﺗﯿﻤﺎر و از ﻧﺸﺎﻧﮕﺮ ISSR ﺑﺮاي ﺗﺸﺨﯿﺺ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ و واﻟﺪﯾﻦ اﺳﺘﻔﺎده ﮐﺮدﻧﺪ و از 5 آﻏﺎزﮔﺮ ﺑﺮاي ﺗﮑﺜﯿﺮ ﻗﻄﻌﺎتDNA ژﻧﻮﻣﯽ اﺳﺘﻔﺎده ﺷﺪ و13 ﮐﻠﻮن ﺳﻮﻣﺎﺗﯿﮑﯽ ﺑﺎ اﺳﺘﻔﺎده از اﯾﻦ روش ﺟﺪا ﮔﺮدﯾﺪ. ﺷﺒﺎﻫﺖ ژﻧﺘﯿﮑﯽ در ﺑﯿﻦ 40 ژﻧﻮﺗﯿﭗ ﻣﻮرد ﺑﺮرﺳﯽ ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 ﺗﻮﻟﯿﺪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ )Saintpaulia ionantha ( . . . 72 95 /0 ﺗﺎ43 /0 ﺑﻪ دﺳﺖ آﻣﺪ و درﺧﺖ ﻓﯿﻠﻮژﻧﯽ در اﯾﻦ آزﻣﺎﯾﺶ در 5 ﺷﺎﺧﻪ ﻃﺒﻘﻪ ﺑﻨﺪي ﺷﺪ. در آزﻣﺎﯾﺶ دﯾﮕﺮي ﮐﻪ ﺑﺮ روي اﯾﻦ ﮔﯿﺎه اﻧﺠﺎم ﮔﺮﻓﺖ، از 60 آﻏﺎزﮔﺮISSR ﺑﺮاي ﺗﺸﺨﯿﺺ وارﯾﺘﻪ ﻫﺎي ﺟﺪﯾﺪ اﺳﺘﻔﺎده ﺷﺪ. ﻧﺘﺎﯾﺞ ﻧﺸﺎن داد ﮐﻪ ﭘﻠﯽ ﻣﻮرﻓﯿﺴﻢ ﺗﻔﺎوت ﻫﺎي ﻣﻌﻨﯽ داري را در ﻣﻘﺎﯾﺴﻪ ﺑﺎ وارﯾﺘﻪ ﻫﺎي ﻣﻮ ﺟﻮد دارد. اﯾﻦ آزﻣﺎﯾﺶ ﻧﺸﺎن داد ﮐﻪ ﮐﺎرﺑﺮد ﻧﺸﺎﻧﮕﺮ ISSR ﻣﯽ ﺗﻮاﻧﺪ ﺑﻪ ﻋﻨﻮان ﯾﮏ روش ﻣﻔﯿﺪ ﺑﺮاي ارزﯾﺎﺑﯽ ﺳﺮﯾﻊ و آﺳﺎن ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﺑﯿﻦ وارﯾﺘﻪ ﻫﺎ ﻣﺪﻧﻈﺮ ﻗﺮار ﮔﯿﺮد. 2. ﻣﻮاد و روش ﻫﺎ ز ا ي و ﻫﻤﮑﺎران ] 6 [ ﯾﮏ ﭘﺮوﺗﮑﻞ ﮐﺎرآﻣﺪ را ﺟﻬﺖ اﻟﻘﺎي ﺟﻬﺶ در ﮔﯿﺎه ﻟﯿﻠﯿﻮم اراﺋﻪ ﮐﺮدﻧﺪ. آن ﻫﺎ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ در ﺑﯿﻦ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ را ﺑﻪ وﺳﯿﻠﻪ ﻧﺸﺎﻧﮕﺮ ﻣﻮﻟﮑﻮﻟﯽ ISSR ﺗﺄﯾﯿﺪ ﮐﺮدﻧﺪ و ﻓﺮاواﻧﯽ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ06 / 36 درﺻﺪ ﺑﺎ اﺳﺘﻔﺎده از7 ﭘﺮاﯾﻤﺮISSR را ﮔﺰارش ﮐﺮدﻧﺪ. از اﯾﻦ9 ﻻﯾﻦ ﺑﺮرﺳﯽ ﺷﺪه ﺑﺎ ﻧﺸﺎﻧﮕﺮ ISSR ، 4 ﻻﯾﻦ ﻫﯿﭻ ﺗﻔﺎوﺗﯽ از ﻧﻈﺮ ﻣﻮﻟﮑﻮﻟﯽ ﻧﺸﺎن ﻧﺪادﻧﺪ اﻣﺎ 5 ﻻﯾﻦ دﯾﮕﺮ ﺗﻔﺎوت ﻫﺎﯾﯽ را ﻧﺸﺎن دادﻧﺪ ﮐﻪ ﺗﻔﺎوت ﻫﺎي ﻓﻨﻮﺗﯿﭙﯽ را در اﯾﻦ ﮔﯿﺎه اﯾﺠﺎد ﮐﺮدﻧﺪ. ﻧﺘﺎﯾﺞ ﺑﺮ روي ﻟﯿﻠﯿﻮم ﻧﺸﺎن داد ﮐﻪ ﻧﺸﺎﻧﮕﺮ ISSR ﻗﺎدر ﺑﻪ ﻧﺸﺎن دادن ﺗﻔﺎوت ﻫﺎ در ﺳﻄﺢ ﻣﻮﻟﮑﻮﻟﯽ اﺳﺖ. ﻣﯿﺰان ﮐﻞ ﮐﻠﺮوﻓﯿﻞ در ﺳﻄﺢ 5 درﺻﺪ ﻣﻌﻨﯽ دار ﺑﻮد وﻟﯽ ﭘﺎراﻣﺘﺮ ﺗﻌﺪاد ﺑﺮگ و ﻣﯿﺰان ﮐﺎروﺗﻨﻮﺋﯿﺪ از ﻧﻈﺮ آﻣﺎري در ﺳﻄﺢ 5 درﺻﺪ ﻣﻌﻨﯽ دار ﻧﺒﻮد، ﺑﻨﺎﺑﺮاﯾﻦ ﻣﻘﺎﯾﺴﻪ ﻣﯿﺎﻧﮕﯿﻦ ﻫﺎ ﺑﺮاي آن ﻫﺎ ﺻﻮرت ﻧﮕﺮﻓﺖ )ﺟﺪول 3 (. ﺑﺎ ﺑﺮرﺳﯽ اﯾﻦ داده ﻫﺎ ﻣﯽ ﺗﻮان ﮔﻔﺖ ﺟﻬﺶ ﯾﺎﻓﺘﻪ 11 M ﮐﻪ داراي رﻧﮓ ﺗﯿﺮه ﺗﺮ در ﺑﺮگ ﻫﺎ ﻣﯽ ﺑﺎﺷﺪ، ﺑﺎ داده ﻫﺎي اﻓﺰاﯾﺶ ﺷﺪت رﻧﮓ ﺳﺒﺰ )ﺑﻪ دﺳﺖ آﻣﺪه از آﻧﺎﻟﯿﺰ ﺗﺼﺎوﯾﺮ( ﻣﻄﺎﺑﻘﺖ دارد، و ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ داده ﻫﺎي اﺳﺘﺨﺮاج ﮐﻠﺮوﻓﯿﻞ )ﺟﺪول 3( ﻣﯽ ﺗﻮاﻧﺪ ﺑﻪ دﻟﯿﻞ اﻓﺰاﯾﺶ ﻣﯿﺰان ﮐﻠﺮوﻓﯿﻞ ﮐﻞ در اﯾﻦ ﻓﺮم ﺑﺎﺷﺪ. در اﯾﻦ ﻓﺮم ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﻢ ﭼﻨﯿﻦ ﻣﯿﺰان ﮐﻠﺮوﻓﯿﻞ b اﻓﺰاﯾﺶ ﭘﯿﺪا ﮐﺮده ﮐﻪ اﯾﻦ ﻣﯿﺰان در ﻓﺮم ﻫﺎي ﺟﻬﺶ ﯾﺎﻓﺘﻪ دﯾﮕﺮ ﺗﻘﺮﯾﺒﺎً ﺑﺮاﺑﺮ ﺑﺎ ﻣﯿﺰان ﮐﻠﺮوﻓﯿﻞ b در ﻧﻤﻮﻧﻪ ﺷﺎﻫﺪ ﻣﯽ ﺑﺎﺷﺪ. اﯾﻦ ﺗﻐﯿﯿﺮات در ﻣﯿﺰان ﮐﻠﺮوﻓﯿﻞ در اﯾﻦ ﮔﯿﺎه ﻣﯽ ﺗﻮاﻧﺪ ﺑﻪ ﮔﯿﺎه اﻣﮑﺎن رﺷﺪ ﺑﻬﺘﺮ ﻧﺴﺒﺖ ﺑﻪ ﻓﺮم اﺑﻠﻖ در ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي دﯾﮕﺮ را ﺑﺪﻫﺪ ﺑﻨﺎﺑﺮاﯾﻦ ﺳﻄﺢ ﺑ ﺮگ و ﺳﻄﺢ ﻓﺘﻮﺳﻨﺘﺰﮐﻨﻨﺪه در اﯾﻦ ﻓﺮم ﻧﺴﺒﺖ ﺑﻪ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي دﯾﮕﺮ ﺑﯿﺶ ﺗﺮ اﺳﺖ )ﺟﺪول 3 و ﺷﮑﻞ6 .( ﺷﮑﻞ6. ﭘﻨﺞ ﮔﯿﺎه ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﮐﻪ ﺑﯿﺶ ﺗﺮﯾﻦ ﻓﺎﺻﻠﻪ ژﻧﺘﯿﮑﯽ را از ﮔﯿﺎه ﺷﺎﻫﺪ داﺷﺘﻨﺪ. 3 .4 ﺑﺮرﺳﯽ ﭘﺎﯾﺪاري ﺗﻐﯿﯿﺮات ﻓﻨﻮﺗﯿﭙﯽ در ﻓﺮم ﻫﺎي ﺟﻬﺶ ﯾﺎﻓﺘﻪ از ﻣﯿﺎن11 ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻣﻮرد آﻧﺎﻟﯿﺰ ISSR ﮐﻪ در ﺑﺎﻻ ﺑﻪ آن اﺷﺎره ﮔﺮدﯾﺪ، 5 ﮔﯿﺎه ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﮐﻪ ﺑﯿﺶ ﺗﺮﯾﻦ ﻓﺎﺻﻠﻪ ژﻧﺘﯿﮑﯽ را از ﮔﯿﺎه ﺷﺎﻫﺪ داﺷﺘﻨﺪ و ﻫﻢ ﭼﻨﯿﻦ ﺑﯿﺶ ﺗﺮﯾﻦ ﺗﻔﺎوت ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ را ﻧﺸﺎن ﻣﯽ دادﻧﺪ، اﻧﺘﺨﺎب ﺷﺪﻧﺪ و ﭘﺲ از ﭼﻨﺪ ﯾﻦ واﮐﺸﺖ، ﮔﯿﺎﻫﭽﻪ ﻫﺎي ﺑﺎززا ﺷﺪه از ﻧﻈﺮ ﭘﺎراﻣﺘﺮﻫﺎي ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ و ﻓﯿﺰﯾﻮﻟﻮژﯾﮑﯽ ﻣﻮرد ﺑﺮرﺳﯽ ﻗﺮار ﮔﺮﻓﺘﻨﺪ. 2. ﻣﻮاد و روش ﻫﺎ از ﻣﯿﺎن ﻧﺘﺎﯾﺞ ﺣﺎﺻﻞ از ﺟﺪول ﺗﺠﺰﯾﻪ وارﯾﺎﻧﺲ، ﭘﺎراﻣﺘﺮﻫﺎي ﺳﻄﺢ ﺑﺮگ، ﻃﻮل دﻣﺒﺮگ، ﺳﻄﺢ ﻓﺘﻮﺳﻨﺘﺰﮐﻨﻨﺪه، ﺷﺪت رﻧﮓ ﺳﺒﺰ در ﻫﺮ ﻣﯿﻠﯽ ﻣﺘﺮ ﻣﺮﺑﻊ ﺑﺮگ و ﻣﯿﺰان ﮐﻠﺮوﻓﯿﻞ b در ﺳﻄﺢ ﯾﮏ در ﺻﺪ و ﻣﯿﺰان ﮐﻠﺮوﻓﯿﻞ a و ﺟﺪول3. ﺑﺮرﺳﯽ ﻓﻨﻮﺗﯿﭙﯽ ﮔﯿﺎﻫﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻧﺎﺷﯽ از ﺗﺎﺑﺶ اﺷﻌﻪ ﮔﺎﻣﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺳﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي urnal of Nuclear Science, Engineering and Technology ﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 5 (4), Serial Number 109, 2024, P 66-74 ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي Journal of Nuclear Science, Engineering and Technology ﺟﺪول3. ﺑﺮرﺳﯽ ﻓﻨﻮﺗﯿﭙﯽ ﮔﯿﺎﻫﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻧﺎﺷﯽ از ﺗﺎﺑﺶ اﺷﻌﻪ ﮔﺎﻣﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﻣﺠﻠﻪ ﻋﻠﻮم، ﻣﻬﻨﺪﺳ ﯽ و ﻓﻨﺎور ي ﻫﺴﺘﻪ اي Journal of Nuclear Science, Engineering and Technology دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 Vol. 45 (4), Serial Number 109, 2024, P 66-74 ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﺗﻌﺪاد ﺑﺮگ ﺳﻄﺢ ﺑﺮگ )2 mm ( ﻃﻮل دﻣﺒﺮگ (mm) ﺳﻄﺢ ﻓﺘﻮﺳﻨﺘﺰي )2 mm ( ﺷﺪت ﺳﺒﺰﯾﻨﮕﯽ در ﻫﺮ ﻣﯿﻠﯽ ﻣﺘﺮ ﻣﺮﺑﻊ ﺑﺮگ ﻣﺤﺘﻮا ي ﮐﻠﺮوﻓﯿﻞ a ﻣﺤﺘﻮاي ﮐﻠﺮوﻓﯿﻞ b ﻣﺤﺘﻮاي ﮐﻠﺮوﻓﯿﻞ ﮐﻞ ﻣﺤﺘﻮاي ﮐﺎروﺗﻨﻮﺋﯿﺪ ﺷﺎﻫﺪ a7 /8 a 62 / 239 a 83 /0 a 62 / 239 b 59 /4 ab 061 /0 b 032 /0 b 093 /0 a 029 /0 7 M a8 /8 c 78 / 49 d 39 /0 c 19 / 31 bc 71 /2 ab 069 /0 b 039 /0 ab 109 /0 a 036 /0 8 M a6 /8 b 03 / 110 cd 47 /0 c 06 / 34 c 03 /2 b 047 /0 b 035 /0 b 083 /0 a 029 /0 9 M a2 /7 bc 42 / 93 d 43 /0 c 87 / 54 c 23 /2 a 080 /0 b 043 /0 ab 123 /0 a 040 /0 10 M a5 /8 b 81 / 131 d 66 /0 b 81 / 131 bc 72 /3 b 047 /0 b 053 /0 b 101 /0 a 027 /0 11 M a5 /7 a 81 / 244 bc 60 /0 a 02 / 238 a 22 /7 ab 053 /0 a 10 /0 a 162 /0 a 031 /0 دوره45 ، ﺷﻤﺎره3 ، ﺟﻠﺪ109 ، ﭘﺎﯾﯿﺰ1403 ، ص66 - 74 اﺣﻤﺪ ﺷﺮ ﯾﻔﯽ ، زﻫﺮا ﺳﺮﮔﺰ ي ﻣﻘﺪم، ﻣﻬﺪ ﯾ ﻪ ﺧﺮاز ي ، . . . 73 4. ﻧﺘﯿﺠﻪ ﮔﯿﺮي ﺑﻪ ﻃﻮر ﮐﻠﯽ ﻧﺘﺎﯾﺞ اﯾﻦ ﺗﺤﻘﯿﻖ ﻧﺸﺎن داد وﺟﻮد ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ و ژﻧﺘﯿﮑﯽ در ﮔﯿﺎه ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﻧﺸﺎن دﻫﻨﺪه اﺛﺮ ﺗﺎﺑﺶ ﮔﺎﻣﺎ در اﻓﺰاﯾﺶ ﺗﻨﻮع اﺳﺖ و در ﺣﻘﯿﻘﺖ ﻧﺘﯿﺠﻪ ﺗﻐﯿﯿﺮاﺗﯽ اﺳﺖ ﮐﻪ در ﺳﺎﺧﺘﺎر ﮔﯿﺎه از ﻃﺮﯾﻖ ﺗﺎﺑﺶ روي داده اﺳﺖ. ﺑﻪ ﻋﺒﺎرﺗﯽ ﺗﺎﺑﺶ ﻣﯽ ﺗﻮاﻧﺪ از ﻃﺮﯾﻖ اﺛﺮ ﺑﺮ ژن ﻫﺎ، اﻓﺰاﯾﺶ ﻓﻌﺎﻟﯿﺖ آن ﻫﺎ را ﺳﺒﺐ ﺷﻮد ﯾﺎ ﺣﺘﯽ از ﻃﺮﯾﻖ اﯾﺠﺎد ﺗﻐﯿﯿﺮ در ﺑﯿﺎن ژن ﻫﺎ ﺑﺎﻋﺚ ﻓﻌﺎل ﺷﺪن ژن ﻫﺎي ﺟﺪﯾﺪ و ﯾﺎ ﺑﺎﻋﺚ ﺧﺎﻣﻮش ﺷﺪن ﺑﻌﻀﯽ از ژن ﻫﺎ ﺷﻮد. ﺟﺪول3. ﺑﺮرﺳﯽ ﻓﻨﻮﺗﯿﭙﯽ ﮔﯿﺎﻫﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻧﺎﺷﯽ از ﺗﺎﺑﺶ اﺷﻌﻪ ﮔﺎﻣﺎي ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ ﺗﺎﺑﺶ ﻗﺎدر اﺳﺖ ﺑﺎ اﺛﺮ ﺑﺮ ﻗﺴﻤﺖ ﻫﺎي ﻣﺨﺘﻠﻒ ژﻧﻮم، ﺑﺎﻋﺚ ﺗﻐﯿﯿﺮ در ﻓﻌﺎﻟﯿﺖ ﻫﺎي ﺳﺎﺧﺘﺎري، ﺑﯿﻮﺷﯿﻤﯿﺎﯾﯽ و ﮐﺎرﮐﺮدي ﮔﯿﺎه ﺷﺪه و از اﯾﻦ ﻃﺮﯾﻖ ﻓﺮم ﻫﺎي ﺟﺪﯾﺪي از ﮔﯿﺎه ﺑﺎ ﺻﻔﺎت ﺗﻐﯿﯿﺮ ﯾﺎﻓﺘﻪ ﺗﻮﻟﯿﺪ ﺷﻮد. ﻧﺘﯿﺠﻪ ﺗﺤﻘﯿﻖ ﺣﺎﺿﺮ از ﻣﯿﺎن 1227 رﯾﺰﻧﻤﻮﻧﻪ اوﻟﯿﻪ ﺗﺎﺑﺶ دﯾﺪه ﮐﻪ ﭘﺲ از ﺗﮑﺜﯿﺮ اوﻟﯿﻪ ﺑﻪ 3600 ،ﮔﯿﺎﻫﭽﻪ رﺳﯿﺪ در ﺑﺮآورد اوﻟﯿﻪ ﻣﻨﺠﺮ ﺑﻪ اﯾﺠﺎد 11 ﻓﺮم ﺗﻐﯿﯿﺮ ﯾﺎﻓﺘﻪ ﺷﺪ ﮐﻪ ﭘﺲ از ﺑﺮرﺳﯽ ﻫﺎي ﻣﻮﻟﮑﻮﻟﯽ 6 ﮔﯿﺎه ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﺗﺄﯾﯿﺪ ﺷﺪ ﮐﻪ ﻧﺸﺎن ﻣﯽ دﻫﺪ درﺻﺪ ﺟﻬﺶ در اﯾ ﻦ ﺗﺤﻘﯿﻖ 004 /0 ﻣﯽ ﺑﺎﺷﺪ. ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ داراي ﺑﺮگ ﻫﺎي اﺑﻠﻖ ﯾﺎ ﺳﺒﺰ ﺗﯿﺮه و ﯾﺎ داراي ﻓﺮم ﺑﻮﺗﻪ ﻣﺘﻔﺎوت ﺑﻮدﻧﺪ. ﻧﺸﺎﻧﮕﺮ ISSR ﺑﺮاي ارزﯾﺎﺑﯽ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﻣﻮﺟﻮد در ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي اﻟﻘﺎ ﺷﺪه ﺑﻪ وﺳﯿﻠﻪ ﺗﺎﺑﺶ ﮔﺎﻣﺎ در ﮔﯿﺎه ﺑﻨﻔﺸﻪ آﻓﺮﯾﻘﺎﯾﯽ، ﺗﻮاﻧﺎﯾﯽ ﺑﺎﻻ و ﻗﺪرت ﺗﻤﺎﯾﺰ ﺧﻮﺑﯽ ﻧﺸﺎن داد و ﮐ ﺎراﯾﯽ اﯾﻦ ﻧﺸﺎﻧﮕﺮ را ﺑﺮاي ﺷﻨﺎﺳﺎﯾﯽ ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎي ﺑﻪ دﺳﺖ آﻣﺪه از ﺗﺎﺑﺶ ﻧﺸﺎن ﻣﯽ دﻫﺪ، از اﯾﻦ رو ﺑﺎ اﺳﺘﻔﺎده از آن ﻣﯽ ﺗﻮان ﮔﺰﯾﻨﺶ زود ﻫﻨﮕﺎم و ﮐﺎﻫﺶ اﻧﺪازه ي ﺟﻤﻌﯿﺖ ﺟﻬﺶ ﯾﺎﻓﺘﻪ را اﻣﮑﺎن ﭘﺬﯾﺮ ﺳﺎﺧﺖ. اﯾﻦ ﺗﺤﻘﯿﻖ ﻧﺸﺎن داد ﮐﻪ ﭘﺎﯾﺪاري ژﻧﺘﯿﮑﯽ در ﺑﺮﺧﯽ ﮔﯿﺎﻫﺎن ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﺷﻨﺎﺳﺎﯾﯽ ﺷﺪه ﺑﺮاي ﺻﻔﺖ ﻣﻮرد ﻧﻈﺮ، در ﻧﺴﻞ ﺑﻌﺪي ﺑﺎﻗﯽ ﻣﯽ ﻣﺎﻧﺪ، اﻣﺎ در ﺑﺮﺧﯽ دﯾﮕﺮ از ﺟﻬﺶ ﯾﺎﻓﺘﻪ ﻫﺎ ﺗﻐﯿﯿﺮات ﺑﻪ ﺷﮑﻞ ﺷﯿﻤﺮ ﺑﻮده و ﺣﺘﯽ در ﻣﺮاﺣﻞ ﻣﺨﺘﻠﻒ رﺷﺪي ﮔﯿﺎه ﻧﯿﺰ ﭘﺎﯾﺪار ﻧﻤﯽ ﻣﺎﻧﺪ . ﻣﺮاﺟﻊ ﻣﺮاﺟﻊ 1. 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https://openalex.org/W3205378385	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0259006&type=printable	English	null	Active pulmonary tuberculosis and coronavirus disease 2019: A systematic review and meta-analysis	PloS one	2,021	cc-by	10,285	PLOS ONE RESEARCH ARTICLE Methods We queried the PubMed and Embase databases for studies providing data on (a) proportion of COVID-19 patients with active pulmonary tuberculosis or (b) severe disease, hospitaliza- tion, or mortality among COVID-19 patients with and without active pulmonary tuberculosis. We calculated the proportion of tuberculosis patients, and the relative risk (RR) for each reported outcome of interest. We used random-effects models to summarize our data. Editor: Girish Chandra Bhatt, All India Institute of Medical Science - Bhopal, INDIA Received: July 19, 2021 Accepted: October 8, 2021 Published: October 21, 2021 Editor: Girish Chandra Bhatt, All India Institute of Medical Science - Bhopal, INDIA Received: July 19, 2021 Accepted: October 8, 2021 Published: October 21, 2021 Results Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0259006 We retrieved 3,375 citations, and included 43 studies, in our review. The pooled estimate for proportion of active pulmonary tuberculosis was 1.07% (95% CI 0.81%-1.36%). COVID-19 patients with tuberculosis had a higher risk of mortality (summary RR 1.93, 95% CI 1.56– 2.39, from 17 studies) and for severe COVID-19 disease (summary RR 1.46, 95% CI 1.05– 2.02, from 20 studies), but not for hospitalization (summary RR 1.86, 95% CI 0.91–3.81, from four studies), as compared to COVID-19 patients without tuberculosis. Copyright: © 2021 Aggarwal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS OPEN ACCESS Citation: Aggarwal AN, Agarwal R, Dhooria S, Prasad KT, Sehgal IS, Muthu V (2021) Active pulmonary tuberculosis and coronavirus disease 2019: A systematic review and meta-analysis. PLoS ONE 16(10): e0259006. https://doi.org/ 10.1371/journal.pone.0259006 Active pulmonary tuberculosis and coronavirus disease 2019: A systematic review and meta-analysis Ashutosh Nath AggarwalID, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu Ashutosh Nath AggarwalID, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu Department of Pulmonary Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India * aggarwal.ashutosh@outlook.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Objective The proportion of COVID-19 patients having active pulmonary tuberculosis, and its impact on COVID-19 related patient outcomes, is not clear. We conducted this systematic review to evaluate the proportion of patients with active pulmonary tuberculosis among COVID-19 patients, and to assess if comorbid pulmonary tuberculosis worsens clinical outcomes in these patients. * aggarwal.ashutosh@outlook.com PLOS ONE PLOS ONE Search strategy We initially looked up the PubMed and EMBASE databases for publications indexed till March 31, 2021, and further updated our search on June 30, 2021. We queried the PubMed database using the following search string: (Tuberculosis OR Tubercular OR Tuberculous OR TB OR Mycobacterium OR Mycobacterial) AND (COVID-19 OR “COVID 19” OR COVID19 OR nCoV OR 2019nCoV OR 2019-nCoV OR CoV-2 OR “CoV 2” OR SARS-CoV-2 OR SARSCoV2). The Embase database was similarly searched. We further scanned the WHO compendium of tuberculosis/COVID-19 studies for any additional published studies [23]. We also examined the bibliographies of selected articles and recent reviews. Introduction The ongoing coronavirus disease 2019 (COVID-19) pandemic is spreading relentlessly, and has affected more than 230 million people worldwide. COVID-19 is associated with worse out- comes in the elderly population, and those with comorbid health conditions such as obesity, diabetes mellitus, hypertension, and cardiovascular disorders [1–9]. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. y Tuberculosis is a destructive pulmonary disease and therefore widely perceived to be associ- ated with increased susceptibility to acquiring COVID-19, and poorer prognosis in patients having both diseases concurrently, especially among people living with human immunodefi- ciency virus infection (PLHIV). A note from World Health organization (WHO) also antici- pated poorer outcomes in these patients [10]. However, the actual impact of tuberculosis on occurrence and clinical outcomes of COVID-19 is not clear. A case series from Italy reported a benign clinical course for patients having both infections [11]. An early meta-analysis of six Chinese studies found no association between tuberculosis and COVID-19 severity or mortal- ity [12]. Population based data from South Korea also did not suggest tuberculosis to be signif- icantly associated with COVID-19-related mortality [13]. However, other investigators describe a disproportionately higher rate of adverse clinical outcomes among patients with tuberculosis and COVID-19 [14–16]. Tuberculosis was identified as the commonest comor- bidity on verbal autopsy among 70 COVID-19 deaths, and in 10% of whole-body autopsies, in Zambia [17, 18]. Two meta-analyses suggest higher odds of underlying tuberculosis among patients with severe COVID-19 and those dying from COVID-19 [19, 20]. Due to these incon- sistencies, we felt a need to perform a detailed analysis of the available evidence till date. Herein, we evaluate the frequency of concurrent active pulmonary tuberculosis among COVID-19 patients. We also assess if comorbid pulmonary tuberculosis increases the risk of severe disease, hospitalization, or mortality in COVID-19 patients. Methods We registered our systematic review protocol with the PROSPERO database (registration number CRD42021245835). We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and the Meta-analysis of Observational Studies in Epi- demiology (MOOSE) recommendations for reporting our review [21, 22]. An approval from our Institutional Review Board was not necessary as we extracted only summary information from previously published articles. PLOS ONE PLOS ONE Tuberculosis and COVID-19 Funding: The authors received no specific funding for this work. Conclusion Active pulmonary tuberculosis is relatively common among COVID-19 patients and increases the risk of severe COVID-19 and COVID-19-related mortality. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. 1 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 Selection of studies After removing duplicate citations, two reviewers (ANA and RA) screened all the titles and abstracts. We omitted publications not reporting on COVID-19 or tuberculosis. We also excluded experimental, radiological or autopsy studies, case reports, letters to editor not 2 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 PLOS ONE Tuberculosis and COVID-19 describing original observations, reviews, guidelines, conference abstracts, editorials, and study protocols. Full texts of citations considered potentially suitable by either reviewer were assessed further. describing original observations, reviews, guidelines, conference abstracts, editorials, and study protocols. Full texts of citations considered potentially suitable by either reviewer were assessed further. We included a publication for data synthesis if it (a) included patients with COVID-19 con- firmed by detection of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens, or strongly suspected on clinical or radiological assessment if a confirmatory test was not available, (b) either described the frequency of patients having con- current active pulmonary tuberculosis among COVID-19 patients, or reported on any of the following outcomes in COVID-19 patients with and without tuberculosis—severe COVID-19, hospital admission, or mortality. Severe COVID-19 was defined based on institutional or national guidelines, or as per the prevalent guidance from international professional bodies or the World Health Organization. If the same (or substantially overlapping) patient cohort was reported in two or more publications, we included the one describing the largest patient popu- lation. In case of any disagreement, consensus between the two reviewers determined study inclusion. Data extraction and study quality We obtained information on study design, location and healthcare setting, participant inclu- sion and exclusion criteria, period of patient enrollment, the source of patient information, and the outcomes reported, from all eligible studies. We used the Newcastle-Ottawa Scale (NOS) to assess methodological quality of studies [24]. We considered a study to be of good quality if its NOS score was seven or more (out of a maximum possible score of nine). Statistical analysis There were 30 (69.8%) publications from Asia, and 10 (23.3%) from Afric with maximum contribution from China (22 studies) (Table 1). One (2.3%) study analyzed data from m ltiple co ntries [43] All st dies e al ated data from retrospecti e patient Fig 1. Flow chart for study selection. https://doi.org/10.1371/journal.pone.0259006.g001 Results We identified 3,375 publications from our literature search (Fig 1). We finally selected 43 stud- ies, describing 236,863 patients with COVID-19, for data synthesis [35–77]. Thirty-three (76.7%) of them provided information on one or more of the adverse clinical outcomes of interest (Table 1). There were 30 (69.8%) publications from Asia, and 10 (23.3%) from Africa, with maximum contribution from China (22 studies) (Table 1). One (2.3%) study analyzed data from multiple countries [43]. All studies evaluated data from retrospective patient cohorts, except for four (9.3%) that collected the information prospectively [39, 40, 56, 69]. Six (14.0%) studies reported population-based data [35, 36, 42, 52, 59, 62], while the others were conducted in a hospital setting. Two (4.7%) studies also included COVID-19 patients based on Fig 1. Flow chart for study selection. https://doi.org/10.1371/journal.pone.0259006.g001 https://doi.org/10.1371/journal.pone.0259006.g001 Statistical analysis We estimated the percentage of active tuberculosis patients among those with COVID-19 dis- ease in each study and calculated the corresponding 95% confidence interval (95% CI) by Clopper-Pearson exact method [25]. We also computed the relative risk (RR), and the corre- sponding 95% CI, for each predefined outcome from each study [26]. We employed a continu- ity correction of 0.5 for studies having ‘zero’ cell frequencies prior to these calculations. We pooled our data using the DerSimonian-Laird random effects model to generate sum- mary estimates [27]. Freeman-Tukey double arcsine transformation was used to summarize data on proportions [28]. We assessed between-study heterogeneity through the Higgins’ inconsistency index (I2), which was considered high for values greater than 0.75 [29]. The con- tribution of each study to overall heterogeneity, and its influence on the summary estimate, were assessed through the Baujat’s plot [30]. For searching the reasons for heterogeneity, we performed subgroup analyses for predefined covariates that included study location and set- ting, study design, COVID-19 diagnostic standards, description of criteria used to define active tuberculosis, national burden of tuberculosis, and the overall study quality. World Health Organization standards were used to refer to countries as high burden, and to extract country incidence estimates, for tuberculosis [31]. In a sensitivity analysis, the influence of each study on the summary estimate was also assessed by repeating meta-analysis after iteratively omitting one study at a time. Further, any influential study was identified using a battery of diagnostic tests using Studentized Residuals, Difference in Fits (DFFITS), Cooks Distance, Covariance Ratio, Tau Square, and the contribution of each study in the Q, H2 test statistics value and the weights assigned to these studies [32]. Publication bias was assessed through Eggers’ test and by visualizing contour-enhanced trim-and-fill funnel plots [33, 34]. We utilized the statistical softwares Stata (Intercooled edition 12.0, Stata Corp, USA) and R (version 4.1.1, R Foundation for Statistical Computing, Austria) for analyzing our data. 3 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 Fig 1. Flow chart for study selection. https://doi.org/10.1371/journal.pone.0259006.g001 Tuberculosis and COVID- PLOS ONE Tuberculosis and COVID-19 Results We identified 3,375 publications from our literature search (Fig 1). We finally selected 43 stu ies, describing 236,863 patients with COVID-19, for data synthesis [35–77]. Thirty-three (76.7%) of them provided information on one or more of the adverse clinical outcomes of interest (Table 1). Results We identified 3,375 publications from our literature search (Fig 1). We finally selected 43 stud- ies, describing 236,863 patients with COVID-19, for data synthesis [35–77]. Thirty-three (76.7%) of them provided information on one or more of the adverse clinical outcomes of interest (Table 1). There were 30 (69.8%) publications from Asia, and 10 (23.3%) from Africa, with maximum contribution from China (22 studies) (Table 1). One (2.3%) study analyzed data from multiple countries [43]. All studies evaluated data from retrospective patient cohorts, except for four (9.3%) that collected the information prospectively [39, 40, 56, 69]. Six (14.0%) studies reported population-based data [35, 36, 42, 52, 59, 62], while the others were conducted in a hospital setting. Two (4.7%) studies also included COVID-19 patients based on We identified 3,375 publications from our literature search (Fig 1). We finally selected 43 stud- ies, describing 236,863 patients with COVID-19, for data synthesis [35–77]. Thirty-three (76.7%) of them provided information on one or more of the adverse clinical outcomes of interest (Table 1). There were 30 (69.8%) publications from Asia, and 10 (23.3%) from Africa, with maximum contribution from China (22 studies) (Table 1). One (2.3%) study analyzed data from multiple countries [43]. All studies evaluated data from retrospective patient cohorts, except for four (9.3%) that collected the information prospectively [39, 40, 56, 69]. Six (14.0%) studies reported population-based data [35, 36, 42, 52, 59, 62], while the others were conducted in a hospital setting. Two (4.7%) studies also included COVID-19 patients based on PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 4 / 21 PLOS ONE Tuberculosis and COVID-19 Author, year Location Study design Setting Inclusion criteria for COVID-19 patients Exclusion criteria Study period Source of data Tuberculosis definition No. Results of COVID- 19 patients PLHIV Information reported NOS score Al Kuwari HM, 2020 [35] Qatar Retrospective General population Confirmed disease NS Feb 28 —Apr 18, 2020 National COVID database ICD codes 5,685 NS Proportion, severity 6 Boulle A, 2020 [36] Western Cape, South Africa Retrospective General population Confirmed disease in adult patients (> = 20 years) NS Mar 1 —Jun 9, 2020 Health records database Microbiological confirmation, anti- tubercular treatment, admission to tuberculosis hospital 22,308 17.8% Proportion, hospitalization, mortality 8 Chen T, 2020 [37] Wuhan, China Retrospective Inpatients Confirmed disease NS Jan 1— Feb 10, 2020 Medical records Medical records 203 1.0% Proportion, mortality 6 Dai M, 2020 [38] China Retrospective Inpatients Confirmed disease in patients with at least two CT examinations and discharged by study end date Poor quality CT scan images Feb 5 —Mar 8, 2020 Medical records NS 73 NS Proportion, severity 5 Du RH, 2020 [39] Wuhan, China Prospective Inpatients Confirmed or highly probable disease NS Dec 25, 2019— Feb 7, 2020 Medical records NS 179 NS Proportion, mortality 6 Gupta N, 2020 [40] New Delhi, India Prospective Inpatients Confirmed disease NS Mar 20 —May 8, 2020 Medical records NS 200 NS Proportion 5 Ibrahim OR, 2020 [41] Katsina, Nigeria Retrospective Inpatients Confirmed disease in adult patients (> = 18 years) NS Apr 10 —Jun 10, 2020 Medical records Confirmation during hospital stay 45 NS Proportion, mortality 6 Lee SG, 2020 [42] South Korea Retrospective General population Confirmed disease in adult patients (> = 18 years) NS Mar 26 —May 15, 2020 Health insurance database Diagnostic codes 7,339 0.1% Proportion, severity, mortality 7 Li G, 2020 [43] 59 countries in China, North America, and Europe Retrospective Inpatients Confirmed disease Patients receiving remdesivir or dexamethasone, lack of treatment records, data from countries with <5 records Jan 1— Apr 30, 2020 Medical records NS 598 NS Proportion, mortality 7 (Continued) LOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 5 / 21 PLOS ONE Tuberculosis and COVID-19 Author, year Location Study design Setting Inclusion criteria for COVID-19 patients Exclusion criteria Study period Source of data Tuberculosis definition No. Results of COVID- 19 patients PLHIV Information reported NOS score Li X, 2020 [44] Wuhan, China Ambispective Inpatients Confirmed or highly probable disease NS Jan 26 —Feb 5, 2020 Personal/ telephonic interviews, medical records ICD-10 diagnostic codes 548 NS Proportion, severity 6 Liu J, 2020 [45] Wuhan, China Retrospective Outpatients and inpatients Confirmed disease in adult patients (> = 18 years) NS Dec 29, 2019— Feb 28, 2020 Medical records Medical records 1,190 NS Proportion, hospitalization, mortality 6 Liu SJ, 2020 [46] Ezhou, China Retrospective Inpatients NS NS Jan 23 —Feb 12, 2020 Medical records NS 342 NS Proportion, severity 5 Ma Y, 2020 [47] 9 Chinese provinces Retrospective Inpatients Confirmed disease in adult patients (> = 18 years) NS Jan 13 —Apr 13, 2020 Medical records Self-reported or diagnosed at admission 1,160 NS Proportion, severity, mortality 6 Maciel EL, 2020 [48] Espirito Santo, Brazil Retrospective Inpatients Confirmed disease in patients with definite outcomes (discharge or death) NS Feb 26 —May 14, 2020 Regional epidemiologic studies database NS 440 1.0% Proportion, mortality 5 Nachega JB, 2020 [49] Kinshasa, DR Congo Retrospective Inpatients Confirmed disease Incomplete information Mar 10 —Jul 31, 2020 Medical records NS 766 1.6% Proportion, severity 6 Parker A, 2020 [50] Cape Town, South Africa Retrospective Inpatients Confirmed disease in adult patients (> = 18 years) NS Mar 24–11 May, 2020 Medical records NS 113 21.2% Proportion, hospitalization 5 Sun Y, 2020 [51] Beijing, China Retrospective Inpatients Confirmed disease NS NS Medical records NS 63 NS Proportion 5 Sy KTL, 2020 [52] Philippines Retrospective General population Confirmed disease NS Up to May 17, 2020 National COVID-19 surveillance registry History or current diagnosis of tuberculosis 12,513 NS Proportion, hospitalization, mortality 8 Xiao KH, 2020 [53] Chongqing, China Retrospective Inpatients Confirmed disease NS Jan 23 —Feb 8, 2021 Medical records NS 143 NS Proportion, severity 5 Yu HH, 2020 [54] Wuhan, China Retrospective Inpatients Confirmed disease NS Jan 27 —Mar 5, 2020 Medical records NS 1561 NS Proportion, severity 7 ( d) LOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 6 / 21 PLOS ONE Tuberculosis and COVID-19 Author, year Location Study design Setting Inclusion criteria for COVID-19 patients Exclusion criteria Study period Source of data Tuberculosis definition No. PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 Results of COVID- 19 patients PLHIV Information reported NOS score Zeng JH, 2020 [55] Shenzhen, China Retrospective Inpatients Confirmed disease NS Jan 11 —Apr 1, 2020 Medical records NS 416 NS Proportion, severity 5 Zhang JJ, 2020 [56] Wuhan, China Prospective Inpatients Confirmed disease NS Jan 16 —Feb 3, 2020 Medical records NS 140 NS Proportion, severity 6 Zhang YT, 2020 [57] Guangdong, China Retrospective Inpatients Confirmed disease NS Jan 15 —Mar 4, 2020 Disease surveillance database NS 1,350 NS Proportion, severity 5 Abraha HE, 2021 [58] Mekelle, Ethiopia Retrospective Inpatients Confirmed disease NS May 10 —Oct 16, 2020 Medical records NS 2,617 0.9% Proportion, severity 7 Dave JA, 2021 [59] Western Cape, South Africa Retrospective General population Confirmed disease NS Mar 4 —Jul 15, 2020 Regional health information database Database records 64,476 12.3% Proportion 6 du Bruyn, 2021 [60] Cape Town, South Africa w Retrospective Inpatients Confirmed disease NS Jun 11 —Aug 28, 2020 Medical records Microbiologically proven or clinically diagnosed 104 29.8% Proportion, severity 6 Gajbhiye RK, 2021 [61] Mumbai, India Retrospective Inpatients Confirmed disease in pregnant/ postpartum women NS Apr— Sep, 2020 Medical records NS 879 NS Proportion 4 Hesse R, 2021 [62] South Africa Retrospective General population Confirmed disease in adult patients (> = 18 years) Indeterminate COVID-19 test results Mar 1 —Jul 7, 2020 Health records GeneXpert positivity within six months before COVID diagnosis 98,335 6.3% Proportion 6 Kapoor D, 2021 [63] New Delhi, India Retrospective Inpatients Confirmed disease in children <18 years age NS Mar 1 —Dec 31, 2020 Medical records NS 120 NS Proportion, mortality 4 Lagrutta L, 2021 [64] Buenos Aires, Argentina Retrospective Outpatients and inpatients Confirmed disease NS Jul 5— Oct 17, 2020 Hospital registry Bacteriological confirmation or recent clinical diagnosis 5,447 7.2% Proportion 6 Li S, 2021 [65] Wuhan, China Retrospective Inpatients Confirmed disease in patients with definite outcomes (discharge or death) Patients still hospitalized at study end date, death within 24 hours a fter admission, loss to follow up Jan 18 —Mar 29, 2020 Medical records NS 2,924 NS Proportion, mortality 4 (Continued) LOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 7 7 / 21 PLOS ONE Tuberculosis and COVID-19 Author, year Location Study design Setting Inclusion criteria for COVID-19 patients Exclusion criteria Study period Source of data Tuberculosis definition No. Results of COVID- 19 patients PLHIV Information reported NOS score Lu Y, 2021 [66] Wuhan, China Retrospective Inpatients Confirmed severe disease in adult patients (<65 years) NS Jan 25 —Feb 15, 2020 Medical records NS 77 NS Proportion, mortality 5 Meng M, 2021 [67] Wuhan, China Retrospective Inpatients Confirmed severe disease NS Jan 2— Mar 28, 2020 Medical records NS 415 None Proportion, mortality 5 Mithal A, 2021 [68] New Delhi, India Retrospective Inpatients Confirmed disease in adult patients (> = 18 years) NS Jul 9— Aug 8, 2020 Medical records NS 401 NS Proportion 5 Moolla MS, 2021 [69] Cape Town, South Africa Prospective Inpatients Confirmed disease NS Mar 26 —Aug 31, 2020 Medical records NS 363 14.6% Proportion 6 Song J, 2021 [70] Wuhan, China Retrospective Inpatients Confirmed disease in patients with definite outcomes (discharge or death) NS Feb 1 —Mar 6, 2020 Medical records Self-report on admission 961 NS Proportion, severity 6 van der Zalm MM, 2021 [71] Cape Town, South Africa Retrospective Inpatients Confirmed disease in children (< = 13 years) Infants born diagnosed in the neonatal service, multisystem inflammatory syndrome Apr 17 —Jul 24, 2020 Medical records Medical records 159 1.3% Proportion, mortality 5 Verma R, 2021 [72] Firozabad, India Retrospective Inpatients Confirmed disease among critically ill patients in ICU Patients referred to other centres Jul 1— Dec 31, 2020 Medical records NS 120 NS Proportion 4 Yan B, 2021 [73] Jilin, China Retrospective Inpatients Confirmed disease NS Jan 28 —Mar 25, 2020 Medical records NS 190 NS Proportion, severity 5 Yang C, 2021 [74] Taiyuan, China Retrospective Inpatients Confirmed disease NS Jan 24 —Apr 25, 2020 Medical records NS 104 NS Proportion, severity 6 Yitao Z, 2021 [75] Guangzhou, China Retrospective Inpatients Confirmed disease NS Jan 21 —Mar 23, 2020 Medical records NS 257 NS Proportion, severity 5 (Continued) LOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 8 / 21 PLOS ONE Tuberculosis and COVID-19 ( ) Author, year Location Study design Setting Inclusion criteria for COVID-19 patients Exclusion criteria Study period Source of data Tuberculosis definition No. of COVID- 19 patients PLHIV Information reported NOS score Zhang W, 2021 [76] Taiyuan, China Retrospective Inpatients Confirmed disease Patients with malignant tumors, hypertension, heart disease, diabetes, etc. Results Jan 1— May 31, 2020 Medical records NS 500 NS Proportion 5 Zheng B, 2021 [77] Honghu, China Retrospective Inpatients Confirmed disease Laboratory and radiology workup at other hospitals, no pulmonary lesion of chest CT scan Jan 1— Mar 27, 2020 Medical records NS 198 NS Proportion, severity 5 COVID-19 Coronavirus disease 2019, ICD International Classification of Diseases, NOS Newcastle-Ottawa Scale for study quality, NS Not specified, PLHIV People living with human immunodeficiency virus infection. https://doi.org/10.1371/journal.pone.0259006.t001 Author, year Location Study design Setting Inclusion criteria for COVID-19 patients Exclusion criteria Study period Source of data Tuberculosis definition No. of COVID- 19 patients PLHIV Information reported NOS score Zhang W, 2021 [76] Taiyuan, China Retrospective Inpatients Confirmed disease Patients with malignant tumors, hypertension, heart disease, diabetes, etc. Jan 1— May 31, 2020 Medical records NS 500 NS Proportion 5 Zheng B, 2021 [77] Honghu, China Retrospective Inpatients Confirmed disease Laboratory and radiology workup at other hospitals, no pulmonary lesion of chest CT scan Jan 1— Mar 27, 2020 Medical records NS 198 NS Proportion, severity 5 COVID-19 Coronavirus disease 2019, ICD International Classification of Diseases, NOS Newcastle-Ottawa Scale for study quality, NS Not specified, PLHIV People living with human i d fi i i i f ti LOS ONE \| https://doi.org/10.1371/journal.pone.0259 9 / 21 PLOS ONE Tuberculosis and COVID-19 high clinical or radiological suspicion [39, 44]. All others only studied patients with disease confirmed by the detection of SARS-CoV2 RNA in respiratory specimens. One (2.3%) study did not specify the inclusion criteria [46]. Only two (4.7%) studies specifically evaluated chil- dren [63, 71]; others included only adults or described a mixed population. Patient informa- tion was retrieved mainly from medical records at participating healthcare facilities, or from surveillance registries or insurance databases (Table 1). Most investigators reviewed patient records or used tuberculosis-related diagnostic codes in databases to identify patients having active tuberculosis (Table 1). Fourteen (32.6%) studies reported human immunodeficiency virus (HIV) seroprevalence in their patient cohorts [36, 37, 42, 48–50, 58–60, 62, 64, 67, 69, 71]. Of these, a single study from South Africa provided tuberculosis prevalence and outcome data based on HIV status [36]. Only six (14.0%) studies were considered high quality (S1 Table) [36, 42, 43, 52, 54, 58]. Proportion of patients with active tuberculosis The proportion of patients having active pulmonary tuberculosis among COVID-19 patients could be computed from all 43 studies. It ranged from 0.18% to 14.42% (Fig 2). The highest occurrence was noted in a study conducted in a high HIV prevalence South African setting [60]. All other studies described figures below 6%. Almost all studies reported proportion esti- mates of comorbid pulmonary tuberculosis among COVID-19 patients that were higher than their corresponding WHO country estimates for annual tuberculosis incidence (Fig 2). The pooled proportion estimate from all 43 studies was 1.07% (95% CI 0.81%-1.36%). p p p ( ) There was substantial heterogeneity between the studies (I2 94.7%). Baujat’s plot suggested that three studies unduly influenced heterogeneity as well as pooled estimates (S1 Fig) [36, 59, 62]. Omitting these three studies from meta-analysis resulted in a slightly higher summary esti- mate of proportion (1.15%, 95% CI 0.84%-1.50%) with only a minor reduction in heterogene- ity (I2 86.9%). On influence analysis, a single study with the highest reported proportion of active tuberculosis patients was associated with large values of Studentized residuals, Cook’s distance and DFFITS (S2 Fig), and was considered potentially influential [60]. After removing this study, the pooled proportion estimate from remaining 42 studies was lower at 1.00% (95% CI 0.75%-1.28%) with hardly any reduction in heterogeneity (I2 94.5%). On sensitivity analysis, omitting other studies one at a time also did not appreciably influence summary estimates or heterogeneity (S3 Fig). On subgroup analysis, studies conducted in low tuberculosis burden or multiple countries showed lesser heterogeneity (Table 2). Overall, the pooled estimates on pro- portion were much lower from studies conducted in countries not having high tuberculosis burden, as well as from population-based and high-quality studies (Table 2). Severe COVID-19 Twenty studies with 24,371 COVID-19 patients, of whom 161 (0.7%) had tuberculosis, pro- vided information on severe COVID-19 [35, 38, 42, 44, 46, 47, 49, 51, 53–58, 70, 73–77]. All, except four (20.0%), of these publications were from China [35, 42, 49, 58]. Severe COVID-19 was defined based on World Health Organization guidance in three (15.0%) studies [35, 49, 58], recommendations from international professional bodies in two (10.0%) studies [44, 70], national guidelines in 11 (55.0%) studies [38, 42, 46, 47, 51, 53, 56, 57, 73, 74, 76], and institu- tional policy in four (20.0%) studies [54, 55, 75, 77]. Only three (15.0%) studies were consid- ered high quality [42, 54, 58]. All studies, except two, included patients with laboratory confirmed COVID-19 [44, 46]. Only one (12.5%) had a prospective study design [56]. Of the 3431 patients with severe disease in the included cohorts, 36 (1.0%) had underlying tuberculo- sis. Only four (20.0%) studies reported a RR for severe COVID-19 that significantly exceeded PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 10 / 21 PLOS ONE Tuberculosis and COVID-19 Fig 2. Proportion of COVID-19 patients also having tuberculosis and corresponding 95% confidence intervals (CI). Individual proportion estimates are depicted by solid squares, and the corresponding country estimate of annual tuberculosis incidence by hollow circles. https://doi.org/10.1371/journal.pone.0259006.g002 Fig 2. Proportion of COVID-19 patients also having tuberculosis and corresponding 95% confidence intervals (CI). Individual proportion estimates are depicted by solid squares, and the corresponding country estimate of annual tuberculosis incidence by hollow circles. https://doi.org/10.1371/journal.pone.0259006.g002 Fig 2. Proportion of COVID-19 patients also having tuberculosis and corresponding 95% confidence intervals (CI). Individual proportion estimates are depicted by solid squares, and the corresponding country estimate of annual tuberculosis incidence by hollow circles. https://doi.org/10.1371/journal.pone.0259006.g002 https://doi.org/10.1371/journal.pone.0259006.g002 1.0 (Fig 3) [42, 46, 56, 77]. COVID-19 patients who also had tuberculosis were 1.46 (95% CI 1.05–2.02) times more likely to develop severe COVID-19 as compared to COVID-19 patients without tuberculosis (Fig 3). 2 There was moderate heterogeneity between the studies (I2 42.9%). Baujat’s plot indicated that one Korean study unduly influenced heterogeneity as well as pooled estimates [42]. No additional influential study was identified on formal influence analysis. Omitting this single study from analysis resulted in a lower summary RR estimate (1.35, 95% CI 0.98–1.87) and lesser heterogeneity (I2 29.5%). On sensitivity analysis, omitting other studies one at a time did not significantly affect heterogeneity (S4 Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 Severe COVID-19 On subgroup analysis, studies conducted in Africa or in low tuberculosis burden or multiple countries, as well as population-based studies, showed negligible heterogeneity (Table 3). Overall, the pooled RR estimates were much higher from studies conducted in countries not having high tuberculosis burden, as well as from pop- ulation-based studies (Table 3). There was no significant publication bias (S5 Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 11 / 21 PLOS ONE Tuberculosis and COVID-19 Table 2. Subgroup analysis for summary estimates for proportion of COVID-19 patients with active pulmonary tuberculosis. Criteria and subgroups No. of studies Summary proportion, % (95% CI) I2, % Overall 43 0.99 (0.74–1.27) 94.7 Continent: Africa 10 1.31 (0.73–2.04) 98.4 Asia 30 1.13 (0.81–1.49) 83.5 Other/Multiple countries 3 0.37 (0.02–1.02) - Study design: Prospective 4 1.49 (0.47–2.98) 68.1 Not prospective 39 1.04 (0.78–1.34) 95.1 Study setting: Hospital-based 37 1.34 (0.94–1.80) 84.5 Population-based 6 0.71 (0.35–1.19) 99.1 Patient inclusion: Confirmed cases only 40 1.03 (0.77–1.33) 94.9 Probable cases also 3 1.80 (0.41–4.01) - Tuberculosis definition: Criteria specified 15 0.95 (0.60–1.38) 97.9 Criteria not specified 28 1.21 (0.82–1.65) 75.1 Tuberculosis burden: High burden countries 39 1.24 (0.94–1.57) 94.5 Other/multiple countries 4 0.30 (0.15–0.49) 72.6 Study quality: NOS score > = 7 6 0.83 (0.43–1.35) 95.7 NOS score <7 37 1.19 (0.87–1.56) 94.2 95% CI 95% confidence interval, I2 Higgins’ inconsistency index, NOS Newcastle-Ottawa Scale for study quality. https://doi org/10 1371/journal pone 0259006 t002 Table 2. Subgroup analysis for summary estimates for proportion of COVID-19 patients with active pulmonary tuberculosis. s for summary estimates for proportion of COVID-19 patients with active pulmonary 95% CI 95% confidence interval, I2 Higgins’ inconsistency index, NOS Newcastle-Ottawa Scale for study quality. Need for hospitalization Four publications with 28,438 COVID-19 patients, of whom 479 (1.7%) had tuberculosis, pro- vided data on hospitalization due to COVID-19 [36, 45, 52, 71]. All these studies were from high tuberculosis burden countries (two from South Africa, and one each from China and Philippines), had a retrospective study design, and included patients with laboratory con- firmed COVID-19. Two (50.0%) studies were of high quality [36, 52]. Overall, 20.6% of patients were hospitalized. Of the 5853 patients who required hospitalization in the included cohorts, 227 (3.9%) had underlying tuberculosis. Two studies reported a RR for hospitalization that statistically significantly exceeded 1.0 (Fig 3) [36, 71]. COVID-19 patients who also had tuberculosis were 1.86 (95% CI 0.91–3.81) times more likely require hospitalization as com- pared to COVID-19 patients without tuberculosis (Fig 3). This pointed to the absence of any statistically significant risk of hospitalization among COVID-19 patients with tuberculosis. There was considerable heterogeneity between the studies (I2 97.5%). A subgroup analysis was not undertaken due to small number of studies. There was no significant publication bias (S5 Fig). https://doi.org/10.1371/journal.pone.0259006.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 Mortality Seventeen studies with 42,321 COVID-19 patients, of whom 632 (1.5%) had tuberculosis, reported on deaths due to COVID-19 [36, 39, 41–43, 45, 47–50, 52, 57, 60, 63, 65–67]. All stud- ies were conducted in high tuberculosis burden countries, except one from South Korea and another that combined data from multiple nations [42, 43]. All studies, except one, included patients with laboratory confirmed COVID-19 [39]. Only one publication had a prospective study design [39]. Four (23.5%) studies were considered as high quality [36, 42, 43, 52]. Of the 2822 patients who died in the included cohorts, 97 (3.4%) had underlying tuberculosis. Only four (23.5%) studies reported RR for mortality that clearly exceeded 1.0 (Fig 3) [36, 42, 45, 52]. The confidence limits for all other studies were wide (Fig 3). COVID-19 patients who also had PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 12 / 21 PLOS ONE Tuberculosis and COVID-19 Fig 3. Relative risk, and corresponding 95% confidence intervals (CI), of adverse clinical outcomes among COVID-19 patients having tuberculosis. Fig 3. Relative risk, and corresponding 95% confidence intervals (CI), of adverse clinical outcomes among COVID-19 patients having tuberculosis. https://doi.org/10.1371/journal.pone.0259006.g003 clinical outcomes among COVID-19 patients having https://doi.org/10.1371/journal.pone.0259006.g003 tuberculosis were 1.93 (95% CI 1.56–2.39) times more likely to die as compared to COVID-19 patients without tuberculosis (Fig 3). 2 There was only mild heterogeneity between the studies (I2 21.5%). Inspection of Baujat’s plot indicated that four studies unduly affected heterogeneity as well as pooled estimates [36, 42, 52, 67]. Two of these were also considered to be influential on formal influence analysis [52, 67]. Omitting these four studies from analysis lowered the summary RR estimate (1.65%, 95% CI 1.25–2.18) and resulted in negligible heterogeneity (I2 0.0%). On sensitivity analysis, heterogeneity could also be further decreased by individually omitting three of these studies one at a time (S4 Fig) [36, 42, 67]. Stratification by whether criteria for tuberculosis definition were specified in the studies resulted in homogeneity in either group (Table 3). On subgroup analysis, studies conducted outside Asia or Africa or in low tuberculosis burden or multiple PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 13 / 21 PLOS ONE Tuberculosis and COVID-19 Table 3. Subgroup analysis for summary relative risk of adverse outcomes among COVID-19 patients with active pulmonary tuberculosis. Criteria and subgroups COVID-19 severity Mortality No. of studies Summary relative risk (95% CI) I2, % No. Mortality of studies Summary relative risk (95% CI) I2, % Overall 20 1.46 (1.05–2.02) 42.9 17 1.93 (1.56–2.39) 21.5 Continent: Africa 2 0.87 (0.38–2.01) 0.0 5 2.10 (1.38–3.18) 24.7 Asia 18 1.52 (1.08–2.15) 42.9 10 1.94 (1.47–2.56) 23.1 Other/Multiple countries - - - 2 1.14 (0.53–2.44) 0.0 Study design: Prospective 1 2.05 (1.19–3.53) - 1 0.44 (0.03–6.76) - Not prospective 19 1.37 (0.95–1.98) 45.3 16 1.93 (1.57–2.41) 21.5 Study setting: Hospital-based 18 1.32 (0.95–1.82) 30.9 14 1.66 (1.25–2.22) 14.4 Population- based 2 3.17 (2.02–4.97) 0.0 3 2.37 (1.90–2.96) 0.0 Patient inclusion: Confirmed cases only 18 1.44 (0.98–2.11) 43.1 16 1.93 (1.56–2.39) 21.5 Probable cases also 2 1.39 (0.65–2.97) 65.0 1 0.44 (0.03–6.76) 0.0 Tuberculosis definition: Criteria specified 5 1.46 (0.64–3.33) 72.3 6 2.58 (2.08–3.20) 0.0 Criteria not specified 15 1.50 (1.06–2.11) 23.7 11 1.41 (1.08–1.84) 0.0 Tuberculosis burden: High burden countries 18 1.32 (0.95–1.82) 30.9 15 1.94 (1.59–2.38) 11.4 Other/multiple countries 2 3.17 (2.02–4.97) 0.0 2 2.38 (0.65–8.66) 77.7 Study quality: NOS score > = 7 3 1.31 (0.29–5.97) 75.3 4 2.44 (1.75–3.41) 42.3 NOS score <7 17 1.45 (1.05–1.99) 27.0 13 1.58 (1.22–2.04) 0.0 95% CI 95% confidence interval, I2 Higgins’ inconsistency index, NOS Newcastle-Ottawa Scale for study quality. htt //d i /10 1371/j l 0259006 t003 Table 3. Subgroup analysis for summary relative risk of adverse outcomes among COVID-19 patients with active pulmonary tuberculosis. 95% CI 95% confidence interval, I2 Higgins’ inconsistency index, NOS Newcastle-Ottawa Scale for study quality. https://doi.org/10.1371/journal.pone.0259006.t003 countries, population-based studies, those including patients with a clinico-radiological diag- nosis of COVID-19, and low-quality publications showed negligible heterogeneity (Table 3). There was no significant publication bias (S5 Fig). countries, population-based studies, those including patients with a clinico-radiological diag- nosis of COVID-19, and low-quality publications showed negligible heterogeneity (Table 3). There was no significant publication bias (S5 Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 Discussion We found that 0.99% of the COVID-19 patients had active pulmonary tuberculosis. These patients showed higher risk for mortality, but not for severe disease or hospitalization, than COVID-19 patients without tuberculosis. Our data synthesis summarizes far greater number of studies than previous meta-analyses and provides information both on tuberculosis fre- quency and COVID-19 outcome estimates [12, 19, 20]. Unlike previous meta-analyses that reported summary odds ratios, we present summary RR estimates for adverse clinical out- comes, which are much easier to interpret and understand in a clinical setting. The summary proportion of those with active pulmonary tuberculosis among COVID-19 patients appears higher than the recent WHO estimates for annual incidence of tuberculosis in some of the high tuberculosis burden countries where most of the studies were conducted (China 0.06%, India 0.19%, Nigeria 0.22%, Philippines 0.55%, and South Africa 0.61%) [31]. However, this proportion of active tuberculosis is lower than the generally reported proportion of other comorbid conditions, like diabetes or hypertension [5, 8]. Whether the lower PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 14 / 21 PLOS ONE Tuberculosis and COVID-19 tuberculosis proportion is due to under-reporting or under-recognition of active tuberculosis among COVID-19 patients, or to safeguarding strategies commonly employed by people with respiratory disorders, is not certain. However, when patients with active pulmonary tuberculo- sis do acquire COVID-19, there is a significantly greater risk (about two-fold higher) of COVID-19 mortality. Our summary estimate for relative risk of mortality in COVID-19 patients having tuberculosis is quite similar to the relative risk estimates of mortality for COVID-19 patients having other comorbid conditions (like diabetes, hypertension, or cardio- vascular diseases) widely known to adversely affect prognosis in COVID-19 patients [1–3]. It is likely that superadded COVID-19 pneumonia in a lung that is already structurally damaged by tuberculosis may manifest as more severe disease. Importantly, local alterations in lung immunity resulting from active pulmonary tuberculosis can also adversely influence host response to SARS-CoV-2 virus. Recent in-vitro data from COVID-19 patients with active pul- monary tuberculosis has shown an attenuated interferon-gamma response after stimulation of whole blood with peptides derived from SARS-CoV-2 spike protein, in contrast to a normal response to Mycobacterium tuberculosis-specific antigens [78]. There are several similarities between COVID-19 and pulmonary tuberculosis. In several countries, COVID-19 too is a stigmatizing disorder, much like tuberculosis. Both diseases show airborne transmission when people are in close contact. Discussion Both present with similar symp- toms like fever and cough. This can complicate decision-making, especially is nations with high tuberculosis burden. Although several countries have proposed bidirectional screening of both COVID-19 and pulmonary tuberculosis patients, such policy remains difficult to imple- ment in resource-constrained settings. This might contribute to underdiagnosis of tuberculo- sis in COVID-19 patients. As it is, under-reporting of tuberculosis is a problem that is globally recognized. This is further compounded by reduced access to tuberculosis diagnosis and treat- ment as a result of COVID-19 related restrictions. It is therefore possible that our calculations regarding pulmonary tuberculosis among COVID-19 patients might be an underestimate. Our systematic review has a few limitations. Due to the dynamic nature of the pandemic, and the lag between data collection and publication of results, most studies provide information from the initial months of 2020 and from regions that were severely afflicted earlier. Thus, the figures may not truly represent the patient data from all the geographic locations. Also, most of the included studies had a retrospective design, and collated data from medical records that were likely completed in an overwhelmed health system. This could have resulted in both underreporting as well as misclassification of comorbid health conditions. Several studies reported only on inpatients who have a higher probability of adverse outcomes compared to patients in the community. Only 15.6% of the included studies were of sufficiently high quality. There were differences in healthcare strategies regarding SARS-CoV-2 testing and admission/transfer criteria, variability in institutional practices in the timing of investigations and other evaluations, and the level and extent of medical intervention available to patients. Such heterogeneity can restrict the generalizability of our results. We cannot rule out an overestimation from lack of adjustment for potential confounders (like age, HIV status, other comorbid health conditions, or other patient characteristics) as we focused on univariate estimates. In particular, only one South African study reported on tuberculosis fre- quency data and outcome parameters stratified by HIV status, and there is need to gather more information on the impact of HIV on COVID-19 and tuberculosis associations. Conceptualization: Ashutosh Nath Aggarwal. Data curation: Ashutosh Nath Aggarwal, Ritesh Agarwal. Data curation: Ashutosh Nath Aggarwal, Ritesh Agarwal. Formal analysis: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Methodology: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Project administration: Ashutosh Nath Aggarwal. Supervision: Ashutosh Nath Aggarwal, Ritesh Agarwal. Supervision: Ashutosh Nath Aggarwal, Ritesh Agarwal. Validation: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Visualization: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Writing – original draft: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kurus- wamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Writing – review & editing: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kur- uswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Conclusion Factors associated with disease severity and mortality among patients with COVID-19: A systematic review and meta- Author Contributions Conceptualization: Ashutosh Nath Aggarwal. Conclusion In summary, the available evidence suggests that COVID-19 patients show relatively higher proportion of concurrent active pulmonary tuberculosis. Active pulmonary tuberculosis sig- nificantly increases the risk of severe COVID-19 and COVID-19-related mortality. PLOS ONE \| https://doi.org/10.1371/journal.pone.0259006 October 21, 2021 15 / 21 PLOS ONE Tuberculosis and COVID-19 Supporting information S1 Table. Details of Newcastle-Ottawa Scale scoring for study quality. (PDF) S1 Fig. Baujat’s plot for studies reporting prevalence of tuberculosis among COVID-19 patients. (PDF) S2 Fig. Influence statistics for studies reporting prevalence of tuberculosis among COVID- 19 patients. (PDF) S3 Fig. Sensitivity analysis for studies reporting prevalence of tuberculosis among COVID-19 patients. (PDF) S4 Fig. Sensitivity analysis for studies reporting outcomes of patients COVID-19 patients with comorbid tuberculosis. (PDF) S5 Fig. Contour-enhanced trim-and-fill funnel plots. (PDF) Supporting information S1 Table. Details of Newcastle-Ottawa Scale scoring for study quality. (PDF) S1 Fig. Baujat’s plot for studies reporting prevalence of tuberculosis among COVID-19 patients. (PDF) S2 Fig. Influence statistics for studies reporting prevalence of tuberculosis among COVID- 19 patients. (PDF) S3 Fig. Sensitivity analysis for studies reporting prevalence of tuberculosis among COVID-19 patients. (PDF) S4 Fig. Sensitivity analysis for studies reporting outcomes of patients COVID-19 patients with comorbid tuberculosis. (PDF) S5 Fig. Contour-enhanced trim-and-fill funnel plots. (PDF) Author Contributions Conceptualization: Ashutosh Nath Aggarwal. Data curation: Ashutosh Nath Aggarwal, Ritesh Agarwal. Formal analysis: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Methodology: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Project administration: Ashutosh Nath Aggarwal. Supervision: Ashutosh Nath Aggarwal, Ritesh Agarwal. Validation: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Visualization: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kuruswamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. Writing – original draft: Ashutosh Nath Aggarwal, Ritesh Agarwal, Sahajal Dhooria, Kurus- wamy Thurai Prasad, Inderpaul Singh Sehgal, Valliappan Muthu. 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https://openalex.org/W4295661675	https://findresearcher.sdu.dk/ws/files/211037313/Larsen_2022_IOP_Conf._Ser._Earth_Environ._Sci._1078_012078.pdf	English	null	Filling the gaps Circular transition of affordable housing in Denmark	IOP conference series. Earth and environmental science	2,022	cc-by	6,137	Download date: 24. Oct. 2024 Filling the gaps - Circular transition of affordable housing in Denmark Citation for pulished version (APA): Larsen, V. G., Tollin, N., Antoniucci, V., Birkved, M., Sattrup, P. A., Holmboe, T., & Marella, G. (2022). Filling the gaps - Circular transition of affordable housing in Denmark. IOP Conference Series: Earth and Environmental Science, 1078(1), Article 012078. https://doi.org/10.1088/1755-1315/1078/1/012078 Citation for pulished version (APA): Larsen, V. G., Tollin, N., Antoniucci, V., Birkved, M., Sattrup, P. A., Holmboe, T., & Marella, G. (2022). Filling the gaps - Circular transition of affordable housing in Denmark. 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Oct. 2024 IOP Conference Series: Earth and Environmental Science You may also like Review of Spatial Model to Assess Suitability of Affordable Housing Based on Demand Criteria Zur’ain Zamri, Zakri Tarmidi and Nurul Hana Adi Maimun - Sea level rise and coastal flooding threaten affordable housing Maya K Buchanan, Scott Kulp, Lara Cushing et al. - Identification of Effective Integrated Indicators for Sustainable Affordable Housing Provision Sura Z. Araji and Bahjat R. Shahin - PAPER • OPEN ACCESS E-mail: vgl@iti.sdu.dk E-mail: vgl@iti.sdu.dk Abstract. The building industry accounts for about 40% of all climate impacts, stemming from construction and renovation processes, use of buildings and demolition, disposal and recycling of buildings and building materials. The Danish Parliament passed a Danish Climate Act in 2020 to reduce greenhouse gas emissions by 70% by 2030, and an action plan in 2021 to create a Circular Economy (CE) in DK that can support the achievement of climate neutrality by 2050. About 20% of the Danish housing stock is affordable housing with approx. 560.000 affordable housing homes, inhabited by nearly 1 million out of 5.8 million people. In 2020 the Danish Government decided on the most significant overall housing agreement to set aside EUR 5,5 billion for ‘Green renovations’ and the building of new affordable housing. Building and renovating affordable housing in Denmark can thus become significant drivers for the Danish building industry's transition to CE. Therefore, developing integrated tools and methodologies for life cycle thinking and CE assessment for the built environment is necessary. We have identified four significant gaps in this endeavour in a previously released literature review: For CE to succeed, it is necessary to take a circular view of the life cycle of buildings, which includes the service life phase, the reuse phase and the recycle phase; To achieve CE, it is necessary to continue research regarding the possibilities of integrating Life Cycle Assessment (LCA), Life Cycle Cost (LCC) and Social Life Cycle Assessment (S-LCA) into Life Cycle Sustainability Assessment (LCSA); S-LCA needs further maturation and development; It is imperative to focus upon operationalising LCSA for practitioners in all phases of a building’s life cycle. This paper aims to outline a strategy for analysing and discussing these four gaps and their interrelation in- depth and suggest an action research proposal to understand better how to bridge the gaps from a research perspective. )LOOLQJWKHJDSV&LUFXODUWUDQVLWLRQRIDIIRUGDEOH KRXVLQJLQ'HQPDUN Sea level rise and coastal flooding threaten affordable housing Maya K Buchanan, Scott Kulp, Lara Cushing et al. - To cite this article: V G Larsen et al 2022 IOP Conf. Ser.: Earth Environ. Sci. 1078 012078 Identification of Effective Integrated Indicators for Sustainable Affordable Housing Provision Sura Z. Araji and Bahjat R. Shahin - View the article online for updates and enhancements. This content was downloaded from IP address 80.166.140.106 on 20/10/2022 at 06:30 This content was downloaded from IP address 80.166.140.106 on 20/10/2022 at 06:30 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 g doi:10.1088/1755-1315/1078/1/012078 doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 doi 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 V G Larsen1, N Tollin1, V Antoniucci2, M Birkved1, P A Sattrup3, T Holmboe4 and G Marella2 V G Larsen1, N Tollin1, V Antoniucci2, M Birkved1, P A Sattrup3, T Holmboe4 and G Marella2 1University of Southern Denmark, Odense, DK 2DICEA, University of Padua, IT 3Danish Association of Architectural Firms, Copenhagen, DK 4Dissing + Weitling A/S, Copenhagen, DK 1University of Southern Denmark, Odense, DK 2DICEA, University of Padua, IT 3Danish Association of Architectural Firms, Copenhagen, DK 4Dissing + Weitling A/S, Copenhagen, DK 1University of Southern Denmark, Odense, DK 2DICEA, University of Padua, IT 3Danish Association of Architectural Firms, Copenhagen, DK 4Dissing + Weitling A/S, Copenhagen, DK SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 The building industry accounts for about 40% of the climate impact in Denmark, stemming from construction and renovation processes, use of buildings and demolition, disposal and recycling of buildings, and building materials [3]. The affordable housing sector is the largest single sector in the Danish building industry. 20% of the Danish housing stock is affordable housing, with approximately five hundred sixty thousand affordable housing units inhabited by almost 1 million people out of a Danish population of 5,8 million people [4]. Throughout the years, increasing funds for construction and renovation of affordable housing has been used as a lever to increase growth during periods of recession to create more jobs in the construction and service sector. Thus, a parliamentary agreement was reached in 2020, allocating 4 billion. EUR from the National Building Fund (LBF) for renovating the existing housing stock of affordable housing in 2020-2026 to support a social and green transformation. Renovation of 72,000 units was initiated in 2021 for EUR 2.4 billion, and EUR 1.5 billion will be set aside for future renovations until 2026. The renovation scheme intends to increase employment in the construction industry and address an economic downturn created by the COVID19 pandemic. In addition, renovations mean up to a 40 per cent reduction in energy consumption and improved indoor climate for tenants in their homes [5, 6]. The renovation scheme is expected to increase employment due to the economic downturn created by the COVID19 pandemic. In addition, renovations mean 30-40 pct less energy consumption and the tenants' indoor health care benefits. Building and renovating affordable housing in Denmark can thus become significant drivers for the Danish building industry's transition to CE. CE in construction is about saving and reusing building materials and keeping materials and construction products in cities and buildings' economic circuits with the highest possible value for the longest time. In addition, CE is a way to ensure sustainable management of the earth's resources and develop new economic opportunities for companies through new business models [7-9]. Therefore, it is necessary for the Danish authorities and stakeholders in the construction value chain to assess the efficiency and effectiveness of CE and how the best possible value for the invested funds is achieved through CE. 1. Introduction The Danish Parliament passed the Danish Climate Act [1] in 2020 to reduce Denmark's greenhouse gas emissions by 70% by 2030. Additionally, in 2021, the Danish Government released an action plan to create a Circular Economy (CE) [2] in Denmark to support the achievement of climate neutrality by 2050 and the decoupling between economic growth and consumption of natural resources. SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.108 The research project [10] aims to combine life cycle thinking methodologies with supporting the evidence-based decision-making and the design process of architectural firms and social housing organisations through value chain innovation. The research project runs through four phases, as shown in Table 1. Each phase responds to research questions, exploring the organisational, sectoral, and political premises to establish recommendations for evaluating CE transition for the affordable housing sector. The purpose of the research project is to support The Danish Association of Architectural Firms (DA)[13] and The National Building Fund in Denmark (LBF)[14] in creating markets for new CE services and solutions for affordable housing, scalable to other housing types in Denmark as well as internationally impacting policy-making. DA is a trade association with approx. seven hundred twenty member firms (by June 2022), and LBF is an independent institution developed by the Danish affordable housing organisations (i.e. ,>>:.4,?4:9> :1 09/@>0=> =09?0=> and regulated by the state through legislation. LBF aims to support and develop the social housing sector, which is self-financing through various subsidy and loan schemes. 3. The research question for this paper. We conducted a literature review, "What are the challenges in assessing Circular Economy for the built environment?"[12] (Table 1, no. 1) to understand the complexity of life cycle assessment as a tool for evaluation and cost-benefit analysis of the efficiency and effectiveness of CE. ?B,>,9 :-50.?4A0:1?3074?0=,?@=0=0A40B$?:8,;?30>?,?@>:149?02=,?4:9:1410D.70 >>0>>809?410D.70:>?492,9/%:.4,7410D.70>>0>>809?%49?:410 D.70%@>?,49,-474?D>>0>>809?%?:@9/0=>?,9/.@==09?7484?,?4:9>,9/69:B70/202,;>=07,?0/ ?:0C4>?4927410.D.70?::7>+9:?30=:-50.?4A0B,>?:/4>.@>>3:B,9/?:B3,?0C?09?7410.D.70 ?3496492.,9-049?02=,?0/49?:?30-@47?09A4=:9809??:>@;;:=??30?=,9>1:=8,?4:9:1?30.:9>?=@.?4:9 >0.?:=1=:87490,=?:.4=.@7,=0.:9:8DB4?3?30;:?09?4,7?:>@;;:=?>@>?,49,-70/0A07:;809?:9,7,=20 >.,70 We appointed four knowledge gaps by recording trends in the scientific literature assessed in the LR, which are lined up in Table 2: Table 2. Knowledge gaps in "What are the challenges in assessing Circular Economy for the built environment?”[12]: Table 2. Knowledge gaps in "What are the challenges in assessing Circular Economy for the built environment?”[12]: Circularity It is necessary to support a systemic shift by developing a comprehensive, genuinely circular view of life cycle phases. A circular argument should include the usage (of buildings) phase, the reuse/recycle (of structures and materials) phase, and the other life cycle phases within the construction and real estate sector. Integration Further research in the integration of LCA, LCC and S-LCA is necessary to develop LCSA for building processes and proper assessment tools for the built environment. Furthermore, efforts should be made to harmonise the three methods and develop coherent data and KPIs, quantitative and qualitative. S-LCA S-LCA is still not very well defined and described in the scientific literature. Therefore, S-LCA needs even further maturation and development. Usability Throughout the reviewed articles, it is stressed that life cycle methodologies are not adequately operational and usable yet, for the actors in the design and building processes in the built environment. Circularity It is necessary to support a systemic shift by developing a comprehensive, genuinely circular view of life cycle phases. A circular argument should include the usage (of buildings) phase, the reuse/recycle (of structures and materials) phase, and the other life cycle phases within the construction and real estate sector. Integration Further research in the integration of LCA, LCC and S-LCA is necessary to develop LCSA for building processes and proper assessment tools for the built environment. Furthermore, efforts should be made to harmonise the three methods and develop coherent data and KPIs, quantitative and qualitative. S-LCA S-LCA is still not very well defined and described in the scientific literature. Therefore, S-LCA needs even further maturation and development. 2. Research background The paper at hand is a part of an overall research project called "Circular transition of affordable housing: Generating environmental, economic and social co-benefits by design"[10]. This research project aims to promote the transition of Danish affordable housing to a CE by developing a framework for assessment, evaluation, and cost-benefit analysis of the efficiency and effectiveness of CE while integrating environmental, economic and social co-benefits [10, 11]. Furthermore, the research project concerned hypothesises that architectural companies play a central role in the CE transition of the affordable housing sector through re-thinking systems by design. Table 1. Research questions in the research project concerned Research questions Status 1. What are the challenges in assessing circular economy for the built environment? [12] Literature review 2. :B.,9?30>0.3,770920>-0,9,7D>0/,9/@9/0=>?::/49/0;?3>:?3,??30 =0>0,=.3.,9>@;;:=??30,11:=/,-703:@>492>0.?:=>?=,9>4?4:9?:?3=:@23 .3,92492-@47/492;=,.?4.0>1=:87490,=?:.4=.@7,=;3,>08:/07> &30;,;0=,?3,9/ 3. How can architectural design processes foster a transition to circular economy for the affordable housing sector? Paper in progress 4. How can the social co-benefits of a transition to circular economy for affordable housing be measured and assessed, and how can end-users and stakeholders' participation through co-design and informed decision-making foster a transition to circular economy for the affordable housing sector? Consecutive paper Research questions Research questions 1. What are the challenges in assessing circular economy for the built environment? [12] 2 Why and how we aim to handle the knowledge gaps through quantitative and qualitative stakeholder surveys, interviews, observations and workshops is described in parts 3.1.-3.4. of the paper at hand. Transition theory will form the research appropriate for understanding change processes in systems and their application within CE. %429414.,9? 9,?4:9,7 policies for sustainability, climate, building and renovation of affordable housing and the existing linear construction and renovation processes guide the direction. These >?@/40>1:=8?30-,>4>1:=,9:A0=7,D:1;:74.40>>;0.414.,?4:9>,9/,.?4A4?40>49,.4=.@7,= ;=:.0>> 8:/07 for masterplan processes to renovate affordable housing in Denmark. Findings are summarised in section 4 to direct the continuous research hereafter to answer the overall project hypothesis about “Circular transition of affordable housing: Generating environmental, economic and social co-benefits by design," [10] elaborated upon in part 5. 3. The research question for this paper. Usability Throughout the reviewed articles, it is stressed that life cycle methodologies are not adequately operational and usable yet, for the actors in the design and building processes in the built environment. The article at hand describes how we examine and understand whether the four knowledge gaps ascertained in the scientific literature also occur when clients / end-users and architectural offices plan and execute affordable housing renovations in concrete planning, design, execution, and usage phases. For this purpose, the following research question (RQ) is raised to guide the research; 3 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 g doi:10.1088/1755-1315/1078/1/012078 doi:10.1088/1755-1315/1078/1/012078 3.1. Circularity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he Danish National Strategy for Sustainable development [18], The Danish Climate Act [1], The Danish Circular Economy action plan [19], and &30),>?0"=/0= + )0,>>@80?3,??3069:B70/202,;,-:@?.4=.@7,=4?DB04/09?4140/49?30>.409?414..:9?0C?,7>::..@=> B309 ;:74?4.> >3,77 -0.:80 :;0=,?4:9,7 49 ?30 .:9>?=@.?4:9 49/@>?=D ?3=:@23 ?30 ;7,99492 /0>429 0C0.@?4:9 ,9/ @>,20 ;3,>0> :1 ,11:=/,-70 3:@>492 =09:A,?4:9 ;=:50.?> :B0A0= -0.,@>0 ?30 .:9>?=@.?4:9>0.?:=4>=07@.?,9??:/0A07:;49?02=,?0/80?3:/>4??09/>?:>?4.6?:-@>490>>,>@>@,7?: 4 SBE-BERLIN-2022 IOP Publishing SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 g doi:10.1088/1755-1315/1078/1/012078 doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 ,A:4/1@=?30=49.=0,>492.:8;70C4?D?:?30,7=0,/D3423.:8;70C4?D:1?30.:9>?=@.?4:9;=:.0>>0>[21]. Therefore, in a consecutive paper (Table 1, no 4), we0C;7:=0?30=0,/490>>1:=.3,920,9/?30;=:>,9/ .:9>:1?30A,7@0.3,49G>0C4>?4927490,=;3,>08:/07>A0=>@>?30.4=.@7,=;3,>08:/07>?3=:@23 >?=@.?@=0//4,7:2@0>B4?33:@>492.:8;,940>,9/,=.34?0.?>49?30,11:=/,-703:@>492>0.?:= ,A:4/1@=?30=49.=0,>492.:8;70C4?D?:?30,7=0,/D3423.:8;70C4?D:1?30.:9>?=@.?4:9;=:.0>>0>[21]. Therefore, in a consecutive paper (Table 1, no 4), we0C;7:=0?30=0,/490>>1:=.3,920,9/?30;=:>,9/ .:9>:1?30A,7@0.3,49G>0C4>?4927490,=;3,>08:/07>A0=>@>?30.4=.@7,=;3,>08:/07>?3=:@23 >?=@.?@=0//4,7:2@0>B4?33:@>492.:8;,940>,9/,=.34?0.?>49?30,11:=/,-703:@>492>0.?:= 3.2. Integration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everal articles see integrating the three methods, LCA, LCC and S-LCA, into a holistic framework, LCSA, as fitting. They claim that documentation of the absolute sustainability of urban development and buildings [29, 30] can be supported in a circular phase model. 3.3. S-LCA %:.4,7 >@>?,49,-474?D ,9/ A,7@0 .=0,?4:9 ,=0 ,-:@? 3:B ?30 -@47? 09A4=:9809? .=0,?0> A,7@0 1:= ?30 /0A07:;0= ?30 @>0=> ,9/ ?30 >:.40?D =02,=/492 49.7@>4:9 .:9>@8;?4:9 ,/A,9?,20> /4>,/A,9?,20> -09014?>,9/.:>?>,>>:.4,?0/B4?3?30-@47?09A4=:9809?%:.4,7>@>?,49,-474?D,9/A,7@0.=0,?4:9,=0,7>: ,-:@??30<@,74?40>?3,?.:9>?=@.?4:9;=:50.?>3,A0=02,=/4921:=0C,8;70.@7?@=,730=4?,20.:30>4:9 ;=:/@.?4A4?D70,=949230,7?3@=-,97410,9/5:-.=0,?4:9&3048;,.?.,?02:=40>.,9-0,>/4A0=>0,> -03,A4:@=>,10?D9@8-0=:1/0,?3>3@8,9=423?>,=.34?0.?@=,7<@,74?D/4A0=>4?D34>?:=4.,7.:9?49@4?D 2:A0=9,9.0>:.4:0.:9:84.48;,.?>,9/7,-:@=8,=60?;:74.40>* + %:.4,7>@>?,49,-474?D,9/A,7@0.=0,?4:9-0492?34>/4A0=>0,=0.3,77092492?:80,>@=0,9//0>.=4-0 @90<@4A:.,77D9,//4?4:9?30/4110=09?.:9>?=@.?4:9,9/=09:A,?4:949/@>?=D;7,D0=>A40B>:.4,7A,7@0 .=0,?4:9/4110=09?7D!0A0=?3070>>>:.4,74>>@0>.,9-074960/?:?300.:9:8D,9/09/@>0=>>@>?,49,-70 B477011:=?,9/-03,A4:@=&3@>>:.4,7A,7@0.=0,?4:94>,>>@80/?:>429414.,9?7D48;,.??30.4=.@7,= 0.:9:8D>>@..0>> % 8,D -0 , B,D :1 /:.@809?492 ,9/ 0A,7@,?492 >:.4,7 >@>?,49,-474?D ,9/ A,7@0 .=0,?4:9 :B0A0= ?30=0 ,=0 9: @9,8-42@:@> 0A,7@,?4:9 >D>?08> 1:= % := 0A,7@,?4:9 :1 >:.4,7 4>>@0> @=?30=8:=0?30$>3:B>?3,??30%2090=,77D>@110=>1=:8,7,.6:1,??09?4:9,9//4114.@7?40> 7,D492 :@? .=4?0=4, ,9/ 49/4.,?:=> :1 >:.4,7 >@>?,49,-474?D 0>4/0> % 49/4.,?:=> 3,A0 /4110=09? ;0=>;0.?4A0>1=:849/4.,?:=>&34>,>;0.?2090=,77D80,9>@9.0=?,49?D49.7,48>1:=?3048;,.? .,?02:=40>,//=0>>0/49?30% %0A0=,7 =0A40B0/ ,=?4.70> 4/09?41D ?30 0A,7@,?4:9 :1 >:.4,7 >@>?,49,-474?D ,9/ A,7@0 .=0,?4:9 ,> 90.0>>,=D1:=,->:7@?0>@>?,49,-70/0A07:;809??4>?30=01:=00>>09?4,7?:-0.:808:=00A4/09?49 .,?02:=4>,?4:9 /01494?4:9> ,9/ .3,=,.?0=4>?4.> :1 >:.4,7 >@>?,49,-474?D ,9/ A,7@0 .=0,?4:9 >: ?3,? ?30 /0A07:;809?:1?301407/.,9?,60;7,.0:9,9491:=80/-,>4> )0 ,>>@80 ?3,? ,=.34?0.?> 3,A0 , @94<@0 =:70 49 09>@=492 >:.4,7 A,7@0 .=0,?4:9 49 ?30 -@47? 09A4=:9809??3=:@23?304=0110.?:9.=0,?492?30=0>@7?>30=0:1* +&30=01:=0B01:.@>>;0.414.,77D:9 ?30,=.34?0.?>=:70,9/?300C;0.?,?4:9>1=:8?303:@>492,>>:.4,?4:9>?:0A,7@,?0>:.4,7>@>?,49,-474?D ,9/A,7@0.=0,?4:949?30=09:A,?4:9:1,11:=/,-703:@>492 &30?:;4.:1.,;,.4?D-@47/492?:0A,7@,?0>:.4,7>@>?,49,-474?D,9/A,7@0.=0,?4:949?30=09:A,?4:9 :1,11:=/,-703:@>4924>0C;7:=0/?3=:@23<@,74?,?4A080?3:/>>@.3,><@0>?4:99,4=0>@=A0D>,9/ <@,74?,?4A049?0=A40B>,9/>?=@.?@=0//4,7:2@0>B4?3,=.34?0.?>,9/3:@>492,>>:.4,?4:9>49.@==09?7D :92:492=0>0,=.3&,-70!: :9;7,99492,9//0>429;=:.0>>0>B4?3,1:.@>:94/09?41D492.=4?0=4, ,9/49/4.,?:=>:1>:.4,7>@>?,49,-474?DA,7@0.=0,?4:9,9/?30B47749290>>?:;,D1:=?34> >,1:@9/,?4:91:=@9/0=>?,9/492,9/,9,7D>492?30>0?@;=02,=/492?30,11:=/,-703:@>492>0.?:= B049.7@/0=070A,9?;:74.D1=,80B:=6>1:=?30,11:=/,-703:@>492>0.?:=,>2@4/0>1:=,.?4:9The Social Housing act [6]Action plan – “Denmark without a parallel society – No ghettos in 2030” [36], and we analyse the affordable housing sector framework: the Federation of Non-profit Housing providers (BL), the National Building Fund (LBF), the affordable housing associations, tenants and the municipalities. 3.4. Usability Despite the relative maturity of the LCA method, there are still many understandings of what an LCA is. Moreover, a shared understanding of what needs to be measured is not yet. However, national LCA and LCC tools (LCAByg and LCCByg) are being developed in Denmark [37-40]. y Despite the relative maturity of the LCA method, there are still many understandings of what an LCA is. Moreover, a shared understanding of what needs to be measured is not yet. However, national LCA and LCC tools (LCAByg and LCCByg) are being developed in Denmark [37-40]. The scientific literature points out that life cycle assessment methods are different and challenging to apply to actors in the construction value chain, which is linked to issues addressed in the description of the three previously mentioned knowledge gaps: Circularity (current practice is linear and not circular and there is no look at value creation); Integration (complex due to the different nature of the three evaluation forms, indicators, time and data) and S-LCA (as an evaluation method S-LCA is immature in comparison with LCA and LCC, and exact definitions of social value creation have not yet matured to an operational level). SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 SBE-BERLIN-2022 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/012078 doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 3.1. Circularity The purpose of integration is to avoid a burden shift from the environmental dimension to the social and economic dimension of sustainability and to promote the ability to evaluate both costs and value creation. In that perspective, it is not enough to assess environmental performance.It is necessary to develop further the ability to evaluate life cycle costs, the total cost of ownership and socio-economic gains and pains and social aspects such as will, effort, consumer patterns and behaviour for CE to succeed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ational Strategy for Sustainable Construction [31] and The Danish voluntary building class [32] to understand the political setup for construction industry regulation. 5 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/012078 doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10 This diversity makes it difficult for actors in the construction value chain to use life cycle methods comparably and consistently. In addition, construction projects consist of many sub-elements that must be assembled into structures and become part of society at micro, meso and macro levels. Therefore, the complexity of evaluating to a comparable, truthful, appropriate and sober level is high due to the many chains and levels of building components. At the same time, innovation in building materials and components is taking place at a high pace, and new knowledge from last year may be old news for next year. Therefore, LCA methods, generally considered technically common property today, may become obsolete when something other than what the methods evaluate becomes essential tomorrow. Moreover, it makes it challenging to manage life-cycle methods that sustainability assumptions change very quickly as new knowledge influences and challenges the beliefs for life cycle assessments [21]. Therefore, we assume that it is necessary to develop building processes to become more agile and to include uncertainties associated with life cycle methods. @=?30=8:=0B0,>>@80?3,??3069:B70/20 2,;>.:9.0=9492?30@>,-474?D:17410.D.70,>>0>>809?80?3:/>4/09?4140/49?30>.409?414..:9?0C?,7>: 080=2049?303:@>492:=2,94>,?4:9>,9/,=.34?0.?>;=,.?4.0?3=:@23?30;7,99492/0>4290C0.@?4:9,9/ @>,20;3,>0>49,11:=/,-703:@>492=09:A,?4:9;=:50.?> &30=01:=0B00C,8490?3070A07:1.:8;0?09.40>900/>,9/0C;0.?,?4:9>.:9.0=9492?30@>,-474?D ,9/<@,74?D:10C4>?492,>>0>>809??::7>?3=:@23 >@=A0D>49?0=A40B>,9/>?=@.?@=0//4,7:2@0>B4?3 /0.4>4:98,60=> 49 ,11:=/,-70 3:@>492 :=2,94>,?4:9> ,9/ ,=.34?0.?> 49 ?30 -@47/492 A,7@0 .3,49 :9 ;7,99492;=:.0>>0>49?30,11:=/,-703:@>492>0.?:=49,.@==09?7D:92:49249A0>?42,?4:9&,-70!: %0A0=,7;:74.40>2@4/0?30;7,99492:1.:9>?=@.?4:9,9/=09:A,?4:9;=:.0>>0>49098,=6&30=01:=0 B049.7@/0?30=070A,9?69:B70/20:1?30-@47/492>0.?:=1=,80B:=6,>,.?4:949>?=@.?4:9> The Danish Building Act [41], Danish Building regulations [42], and Building sector framework and value chain as references in our analysis. 3.3. S-LCA Furthermore, quantitative and qualitative indicators are essential to assess life cycle methods, which are treated differently in all three assessment methods. 6 4. Discussion: Filling the gaps We expect to understand the four knowledge gaps through the activities mentioned in sections 3.1-3.4. We, therefore, summarise an overall assumption that more phases are to be assessed in circular phase models than standardised linear phase models regarding renovation processes in the affordable housing sector construction projects. At the same time, all three dimensions of sustainability should be assessed, preferably as integrated, in circular phase models to comprehend circular building processes and achieve absolute circularity in the renovation processes. We suggest the following vital actions per phase to achieve circularity: We suggest the following vital actions per phase to achieve circularity: • Screening: Assessment of recyclable resources and the environment for an upcoming renovation project should be defined and identified in the initial stages of a renovation process, as this is important for as well considerations of the environment, as upon the economy and the (social) willingness of the end-users to reuse and recycle buildings and building materials; • Building brief: The intention and requirement specification, the level of ambition for circularity, as well as the economy should be assessed during the programming phase; • Design / Innovation: During the design phase, design strategies for user behaviour, circularity and execution, including demolition, should be defined and assessed; • Execution: During the construction phase, coordinated efforts for demolition should be defined and assessed, as well as prepared for delivery and operation; • Usage phase: During the usage phase, assessment and evaluation should be carried out in collaboration with end-users regarding optimal operation and maintenance; • EOL / Disposal: What is considered end-of-life in common considerations and typically related to the demolition and disposal of building materials should instead be seen and assessed as potential 7 SBE-BERLIN-2022 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing SBE BERLIN 2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 doi:10.1088/1755-1315/1078/1/012078 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 doi: resources for future construction and renovation projects. thereby looping to the initial phases in the circular phase model It is worth paying particular attention to defining the screening/building brief, the design/innovation and the usage phases appropriately because of the possible value creation in more areas than solely environmental sustainability. 4. Discussion: Filling the gaps Therefore, we focus on how and when integrated life cycle assessment can support the circular economy in these phases, as shown in Figure 1. :.@>;:49?>1:=;:?09?4,7A,7@0.=0,?4:9?3=:@23,.4=.@7,=0.:9:8D/,;?0/,9/8:/4140/ 1=:8E)3,?,=0?30.3,770920>49,>>0>>4924=.@7,=.:9:8D1:=?30-@47?09A4=:9809?F+ :.@>;:49?>1:=;:?09?4,7A,7@0.=0,?4:9?3=:@23,.4=.@7,=0.:9:8D/,;?0/,9/8:/4140/ 1=:8E)3,?,=0?30.3,770920>49,>>0>>4924=.@7,=.:9:8D1:=?30-@47?09A4=:9809?F+ 5. Perspective – a proposal for a circular life cycle model for the renovation of affordable housing. g &30.4=.@7,=;3,>08:/07?:-02090=,?0/?3=:@23?30=0>0,=.3/0>.=4-0/49>0.?4:9> ,9/B4772@4/0 ?30/0A07:;809?:1,1=,80B:=61:=/0.4>4:9>@;;:=?1:=,11:=/,-703:@>492,>>:.4,?4:9>,9/,=.34?0.?> ,>,9,9>B0=?:?30,48:1?30:A0=,77=0>0,=.3;=:50.??:>@;;:=??30"Circular transition of affordable housing: Generating environmental, economic and social co-benefits by design". 8 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 The described methodology will lay the ground for exploring our hypothesis that architectural companies play a crucial role in the CE transformation of the affordable housing sector by designing socially sustainable housing and implicitly documenting the achieved social value creation. Such a phase model will impact existing decision models for affordable housing renovation. Therefore, the assumption on the importance of the screening/building brief, design/innovation and usage phases is to be confirmed in a case study about planning processes, looking into the current planning practice (by housing associations and architectural offices) of preparing the processes for the renovation of affordable housing, going beyond the scope of the article at hand. &:/,D149,9.4,7,9/?0.394.,7>@;;:=?1:=?30=09:A,?4:9:1,11:=/,-703:@>49249098,=6=0<@4=0> ,;=:.0>>B30=0,9,;;74.,9?,9>B0=>?:?3=00>.3080>%.3080 ,>?0=;7,9%.30800>429 ,9/%.3080:9>?=@.?4:949?3=00,;;74.,?4:9> .4=.@7,=;3,>08:/0749.7@/492%.3080>,9/,9/;:>>4-70,//4?4:9,7>.3080> /,;?0/ ,9/ 8:/4140/ 1=:8 E)3,? ,=0 ?30 .3,770920> 49 ,>>0>>492 4=.@7,= .:9:8D 1:= ?30 -@47? 09A4=:9809?F+ .4=.@7,=;3,>08:/0749.7@/492%.3080>,9/,9/;:>>4-70,//4?4:9,7>.3080> /,;?0/ ,9/ 8:/4140/ 1=:8 E)3,? ,=0 ?30 .3,770920> 49 ,>>0>>492 4=.@7,= .:9:8D 1:= ?30 -@47? 09A4=:9809?F+ &30.:9.=0?0=09:A,?4:9:1,11:=/,-703:@>4924>:=2,94>0/,=:@9/?34>,;;=:A,7;=:.0>>=0>?492 @;:9?304/0,:1:;?484>492.:9>?=@.?4:9.:>?>&30,;;74.,9?4>?30-@47/0=40,3:@>492,>>:.4,?4:9 B3:;=0;,=0>?30,;;74.,?4:9>* +1:=[5]. LBF provides the housing sector with financial support, &30.:9.=0?0=09:A,?4:9:1,11:=/,-703:@>4924>:=2,94>0/,=:@9/?34>,;;=:A,7;=:.0>>=0>?492 @;:9?304/0,:1:;?484>492.:9>?=@.?4:9.:>?>&30,;;74.,9?4>?30-@47/0=40,3:@>492,>>:.4,?4:9 B3:;=0;,=0>?30,;;74.,?4:9>* +1:=[5]. LBF provides the housing sector with financial support, 9 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 SBE-BERLIN-2022 IOP Publishing SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 doi:10.1088/1755-1315/1078/1/012078 expert knowledge and various statistics and IT tools based on a defined and commonly known procedure consisting of: expert knowledge and various statistics and IT tools based on a defined and commonly known procedure consisting of: 4>?301=,80B:=61:=?303:@>492,>>:.4,?4:9>=090B,7,9/1@?@=0;=::1:1, 3:@>492/0;,=?809??1:=8>?30-,>4>1:=?303:@>492,>>:.4,?4:9>.:77,-:=,?4:9B4?3?30,=.34?0.? ?30.:9>@7?,9??308@94.4;,74?D,9/=:8?30900/1:=,=09:A,?4:9-0492.:9>4/0=0/@9?47 %.30804>,;;=:A0/?30:A0=,77;7,99492?D;4.,77D2:0>?3=:@231:@=;3,>0>4#=0;,=,?4:944 /0,,9/;=:50.?/0A07:;809?444#=:50.?.7,=414.,?4:94A>>0>>809?,9/,;;=:A,7%.3080 ,;;74.,?4:9 4> >09? B309 ?30 =0>4/09?> ,9/ ?30 ,>>:.4,?4:9 -:,=/ ,;;=:A0 ?30 149,7 8,>?0= ;7,9 .:9?,49492?30;=:50.?;=074849,=D-@/20?1:=?308,>?0=;7,9149,9.492,9/=09?.:9>0<@09.0>,? ,B,=/800?492 2 Scheme B (Design) application is sent when an explicit project material has been prepared and a price has been obtained for the contractor’s work. The Scheme B application must always be approved by the association board (the client), the municipality and LBF. Once all parties have approved scheme B, the construction work can begin. Scheme C (Construction) application is sent when the construction work has been completed and the terminated construction accounts for the overall plan have been prepared. References 1. Klima-Energi-og-Forsyningsministeriet, Lov om Klima. 2020. 2. Regeringen, Strategi for cirkulær økonomi - Mere værdi og bedre miljø gennem design, forbrug og genanvendelse. 2018. 3. Global-Alliance-for-Buildings-and-Construction, 2021 Global status report for buildings and construction. 2021. 4. Andersen, H.S. and T. Fridberg, Den almene boligsektors rolle i samfundet. Hvad ved vi fra hidtidig forskning og undersøgelser? SBi, 2006. 5. Landsbyggefonden/The_National_Building_Fund, https://lbf.dk/om-lbf/english/. Accessed_12.02.2022. 6. Indenrigs-og-Boligminsteriet, Lov om almene boliger. 2021. 7. Ellen-MacArthur-Foundation, Towards the Circular Economy - Economic and business rationale for an accelerated transition. 2013. 1. 8. Ellen-MacArthur-Foundation, Towards the Circular Economy - Opportunities for the consumer good sector. 2013. 2. 9 Ell M A th F d ti T d th Ci l E A l ti th l SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 Scheme C must be approved by the association board (the client), the municipality and LBF. Scheme C (Construction) application is sent when the construction work has been completed and the terminated construction accounts for the overall plan have been prepared. Scheme C must be approved by the association board (the client), the municipality and LBF. We look into this decision process and explore how the Schemes A, B and C can be inscribed into the circular phase model, as we suggest in Figure 2. As a result, we define how and where to assess CE and whether more or less assessment is necessary (tentatively proposed as Pre-scheme and Scheme D in Figure 2) in a consecutive paper (Table 1, no. 4). Finally, we test the circular phase model in a concrete renovation project. The aims are to identify the drivers and barriers to overcome the four knowledge gaps and assess value creation on all parameters, not solely on environment and economy, but also on social co-benefits through architectural design. g )4?3?34>B0.=4?4.,77D/4>.@>>?30.:9.0;?:1,9/,48?:@9/0=>?,9/3:B,9/B30949?02=,?0/ 7410.D.70,>>0>>809?.:@7/:..@=49,.4=.@7,=.:9>?=@.?4:9;=:.0>>?:>@;;:=?0A4/09.0-,>0//0.4>4:9 8,6492,9/.:9?=4-@?0?:?30?=,9>4?4:9?:,.4=.@7,=0.:9:8D1:=?30,11:=/,-703:@>492>0.?:=07:B ?34>,484>,9:?30=,48?:4/09?41D?30?D;0?480,9/1:=8,?:1491:=8,?4:9?3,?.,9>@;;:=?3:@>492 ,>>:.4,?4:9>G ,9/ ,=.34?0.?>G /0.4>4:98,6492 ,? ?30 A,=4:@> /0>429 >?,20> 49 , >:.4,7 .:>?-09014? ,9,7D>4>=02,=/4920114.409.D,9/0110.?4A090>>*+:1 References References 1. Klima-Energi-og-Forsyningsministeriet, Lov om Klima. 2020. 2. Regeringen, Strategi for cirkulær økonomi - Mere værdi og bedre miljø gennem design, forbrug og genanvendelse. 2018. 3. Global-Alliance-for-Buildings-and-Construction, 2021 Global status report for buildings and construction. 2021. 4. Andersen, H.S. and T. Fridberg, Den almene boligsektors rolle i samfundet. Hvad ved vi fra hidtidig forskning og undersøgelser? SBi, 2006. 5. Landsbyggefonden/The_National_Building_Fund, https://lbf.dk/om-lbf/english/. Accessed_12.02.2022. 6. Indenrigs-og-Boligminsteriet, Lov om almene boliger. 2021. 7. Ellen-MacArthur-Foundation, Towards the Circular Economy - Economic and business rationale for an accelerated transition. 2013. 1. 8. Ellen-MacArthur-Foundation, Towards the Circular Economy - Opportunities for the consumer good sector. 2013. 2. 9. Ellen-MacArthur-Foundation, Towards the Circular Economy - Accelerating the scale-up accross global supply chains 2014 3 9. Ellen-MacArthur-Foundation, Towards the Circular Economy - Accelerating the scale-up accross global supply chains. 2014. 3. Ellen-MacArthur-Foundation, Towards the Circular Economy - Accelerating the scale-up accross global supply chains. 2014. 3. 10 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 SBE-BERLIN-2022 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 g doi:10.1088/1755-1315/1078/1/012078 onf. Series: Earth and Environmental Science 1078 (2022) 012078 doi:10.1088/1755-1315/1078/1/0120 12. Larsen, V.G., et al., What are the challenges in assessing Circular Economy for the built environment? A literature review on integrating LCA, LCC and S-LCA in Life Cycle Sustainability Assessment, LCSA. Journal of Building Engineering, 2022. 13. Danish_Association_of_Architectural_Firms., (https://www.danskeark.com). Accessed_21-06-2022. 14. The_National_Building_Fund, https://lbf.dk/om-lbf/english-read-more-about-us. Accessed_21.06.2022. 13. Danish_Association_of_Architectural_Firms., (https://www.danskeark.com). Accessed_21-06-2 14 Th N i l B ildi F d h //lbf dk/ lbf/ li h d b A d 21 13. Danish_Association_of_Architectural_Firms., (https://www.danskeark.com). Accessed_21-06-2022. 13. Danish_Association_of_Architectural_Firms., (https://www.danskeark.com). Accessed_21-06-2022. 14. The_National_Building_Fund, https://lbf.dk/om-lbf/english-read-more-about-us. Accessed_21.06.2022. 15. Ghisellini, P., M. Ripa, and S. Ulgiati, Exploring environmental and economic costs and benefits of a circular economy approach to the construction and demolition sector. A literature review. Journal of Cleaner Production, 2017. 178: p. 618-643. 15. Ghisellini, P., M. Ripa, and S. Ulgiati, Exploring environmental and economic costs and benefits of a circular economy approach to the construction and demolition sector. A literature review. Journal of Cleaner Production, 2017. 178: p. 618-643. 16. Eberhardt, L., H. Birgisdottir, and M. Birkved, Dynamic Benchmarking of Building Strategies for a Circular Economy, in Sustainable Built Environment D-a-Ch Conference 2019, A. Passer, et al., Editors. 2019. 17. Ministry_for_Climate, E.a.B., General conditions for consultancy services for building and construction works (ABR 18). References 2018. 18. Økonomi-og-Indenrigsministeriet, Et bæredygtigt Danmark - Udvikling i balance. 2018. 19. Miljøministeriet, Handlingsplan for cirkulær økonomi. 2020. j g p f 20. Miljøministeriet, Affaldsbekendtgørelsen. 2020. 20. Miljøministeriet, Affaldsbekendtgørelsen. 2020. Miljøministeriet, Affaldsbekendtgørelsen. 2020 21. Giorgi, S., et al., Drivers and barriers towards circular economy in the building sector: Stakeho interviews and analysis of five European countries policies and practices. 2022. 21. Giorgi, S., et al., Drivers and barriers towards circular economy in the building sector: Stakeholder interviews and analysis of five European countries policies and practices. 2022. 22 A i i T i H l f C l S b l A ld f A 22. Amini Toosi, H., et al., Life Cycle Sustainability Assessment in Building Energy Retrofitting; A Sustainable Cities and Society, 2020. 60. Sustainable Cities and Society, 2020. 60. 23. Roberts, M., S. Allen, and D. Coley, Life cycle assessment in the building design process - A systematic literature review. Building and Environment, 2020. 185. terature review. Building and Environment, 2020. 18 24. Collin, C., G.G.H. Olesen, and A.Q. Secher, A case-based study on the use of life cycle assessment and life cycle costing in the building industry, in Sustainable Built Environment D-a-Ch Conference 2019, A. Passer, et al., Editors. 2019. 25. Dejaco, M.C., et al., Combining LCA and LCC in the early-design stage: a preliminary study for residential buildings technologies. Sustainable Development, 2020. 26. Galimshina et al., Probabilistic LCA and LCC to identify robust and reliable renovation strategies. IOP Conf. Series: Earth and Environmental Science, 2019. 27. Fauzi, RT et al., Exploring the Current Challenges and Opportunities of Life Cycle Sustainability Assessment. Sustainability, 2018. 11(3). 28. Kloepffer, W., Life cycle sustainability assessment of products. The International Journal of Life Cycle Assessment, 2008. 13(2): p. 89-95. 29. Hauschild, M., S. Kara, and I. Røpke, Absolute sustainability: Challenges to life cycle engineer 2020. 30. Hjalsted, A.W., et al., Sharing the safe operating space. HJOurnal of Industrial Ecology, 2021. 2021;25:6–19. 31. Ministry_of_Interior_and_Housing, National Strategy for Sustainable Construction. 2021. Trafik-Bygge-og-Boligstyrelsen, Vejledning om de 33. UNEP-SETAC, Guidelines for Social Life Cycle Assessment of Products. 2009. 34. Benoît-Norris, C., et al., Introducing the UNEP/SETAC methodological sheets for subcategorie social LCA. The International Journal of Life Cycle Assessment, 2011. 16(7): p. 682-690. 35 D i h A i ti f A hit t l Fi A hit t D t V l C ti 2019 social LCA. , p g g g y g j 44. EUROPEAN-COMMISSION, Guide to Cost-Benefit Analysis of Investment Projects. 2014. 43. Almennet, Helhedsplanlægning og myndighedssamarbejde. 2013. 45. EUROPEAN-COMMISSION, Economic Appraisal. Vademecum 2021-2027. General Principles and Sector Applications. 2020. SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 References The International Journal of Life Cycle Assessment, 2011. 16(7): p. 682-690. 35. Danish-Association-of-Architectural-Firms, Architect - Document your Value Creation. 2019. social LCA. The International Journal of Life Cycle Assessment, 2011. 16(7): p. 682-690. 35. Danish-Association-of-Architectural-Firms, Architect - Document your Value Creation. 2019. 35. Danish-Association-of-Architectural-Firms, Architect - Document your Value Creation. 2019. Danish-Association-of-Architectural-Firms, Architect - Document your Value Creation. 2019. Regeringen, Et Danmark uden parallelsamfund. 2018 37. Saridaki, M., M. Psarra, and K. Haugbolle, Implementing life-cycle costing: data integration between design models and cost calculations. Journal of information technology in construction, 2019. 38 Zimmermann R K et al Klimapåvirkning fra 60 bygninger 2020 SBi 2020:04 Zimmermann, R.K., et al., Klimapåvirkning fra 60 bygninger. 2020. SBi 2020:04. 39. Transport-Bygnings-og-Boligministeriet, Introduktion til LCC på bygninger. 2016. 40. Transport-Bygnings-og-Boligministeriet, Introduktion til LCA på bygninger. 2016 p yg g g g p yg g 41. Transport-og-Boligministeriet, Byggeloven. 2016. 41. Transport-og-Boligministeriet, Byggeloven. 2016. Transport-og-Boligministeriet, Byggeloven. 2016. 42. Transport-og-Boligministeriet, Bygningsreglement 2018 (BR18). 2019. 11 SBE-BERLIN-2022 IOP Conf. Series: Earth and Environmental Science 1078 (2022) 012078 IOP Publishing doi:10.1088/1755-1315/1078/1/012078 43. Almennet, Helhedsplanlægning og myndighedssamarbejde. 2013. 44. EUROPEAN-COMMISSION, Guide to Cost-Benefit Analysis of Investment Projects. 2014. 45. EUROPEAN-COMMISSION, Economic Appraisal. Vademecum 2021-2027. General Principles and Sector Applications. 2020. 12
https://openalex.org/W2914820883	https://birmingham.elsevierpure.com/files/74388664/ALICE_collaboration_pp_p_and_correlations_studied_via_femtoscopy_in_pp_reactions_at_s_7_TeV_Physical_Review_C_2019.pdf	Latin	null	<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi>p</mml:mi><mml:mo>−</mml:mo><mml:mi>p</mml:mi></mml:mrow><mml:mo>,</mml:mo><mml:mo> </mml:mo><mml:mrow><mml:mi>p</mml:mi><mml:mo>−</mml:mo><mml:mi mathvariant="normal">Λ</mml:mi></mml:mrow></mml:math> , and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi mathvariant="normal">Λ</mml:mi><mml:mo>−</mml:mo><mml:mi mathvariant="normal">Λ</mml:mi></mml:mrow></mml:math> correlations studied via …	Physical review. C	2,019	cc-by	19,859	p-p, p-Λ, and Λ-Λ correlations studied via femtoscopy in pp reactions at √s = 7\ TeV ALICE Collaboration; Acharya, S.; Andrews, H. A.; Evans, D.; Graham, K. L.; Jevons, O.; Jones, P. G.; Jusko, A.; Krivda, M.; Kvapil, J.; Lietava, R.; Villalobos Baillie, O.; Willsher, E.; Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): ALICE Collaboration, Acharya, S, Andrews, HA, Evans, D, Graham, KL, Jevons, O, Jones, PG, Jusko, A, Krivda, M, Kvapil, J, Lietava, R, Villalobos Baillie, O, Willsher, E & Zardoshti, N 2019, 'p-p, p-Λ, and Λ-Λ correlations studied via femtoscopy in pp reactions at √s = 7\ TeV', Physical Review C, vol. 99, no. 2, 024001. https://doi.org/10.1103/PhysRevC.99.024001 Citation for published version (Harvard): ALICE Collaboration, Acharya, S, Andrews, HA, Evans, D, Graham, KL, Jevons, O, Jones, PG, Jusko, A, Krivda, M, Kvapil, J, Lietava, R, Villalobos Baillie, O, Willsher, E & Zardoshti, N 2019, 'p-p, p-Λ, and Λ-Λ correlations studied via femtoscopy in pp reactions at √s = 7\ TeV', Physical Review C, vol. 99, no. 2, 024001. https://doi.org/10.1103/PhysRevC.99.024001 Link to publication on Research at Birmingham portal Publisher Rights Statement: ALICE collaboration (2019) p-p, p-Λ, and Λ-Λ correlations studied via femtoscopy in pp reactions at √s = 7\ TeV, Physical Review C, volume 99, article no 024001, DOI: 10.1103/PhysRevC.99.024001 Publisher Rights Statement: ll b i ( ) Statement: on (2019) p-p, p-Λ, and Λ-Λ correlations studied via femtoscopy in pp reactions at √s = 7\ TeV, Physical Review C, volum 001, DOI: 10.1103/PhysRevC.99.024001 Publisher Rights Statement: ALICE collaboration (2019) p-p, p-Λ, and Λ-Λ correlations studied via femtoscopy in pp reactions at √s = 7\ TeV, Phys 99, article no 024001, DOI: 10.1103/PhysRevC.99.024001 General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. eely distribute the URL that is used to identify this publication. •Users may freely distribute the URL that is used to identify this publication. •Users may freely distribute the URL that is used to identify this publication. •Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. y y y p •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. U f h d i li i h h f ‘f i d li ’ d h C i h D i d P A 1988 (?) y •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. here a licence is displayed above, please note the terms and conditions of the licence govern your use of this document Where a licence is displayed above, please note the terms When citing, please reference the published version. I. INTRODUCTION and effective ranges for the strong nuclear potential by means of effective models such as the extended-soft-core (ESC08) baryon-baryon model [24] or by means of chiral effective ﬁeld theory (χEFT) approaches at leading order (LO) [25] and next-to-leading order (NLO) [26]. The results obtained from the above-mentioned models are rather different, but all conﬁrm the attractiveness of the -nucleon (-N) interaction for low hyperon momenta. In contrast to the LO results, the NLO solution claims the presence of a negative phase shift in the p- spin singlet channel for momenta larger than p > 600 MeV/c. This translates into a repulsive core for the strong interaction evident at small relative distances. The same repulsive interaction is obtained in the p-wave channel within the ESC08 model [24]. Traditionally femtoscopy is used in heavy-ion collisions at ultrarelativistic energies to investigate the spatial-temporal evolution of the particle emitting source created during the collision [1,2]. Assuming that the interaction for the employed particles is known, a detailed study of the geometrical exten- sion of the emission region becomes possible [3–10]. If one considers smaller colliding systems such as proton- proton (pp) at TeV energies and assumes that the particle emitting source does not show a strong time dependence, one can reverse the paradigm and exploit femtoscopy to study the ﬁnal-state interaction (FSI). This is especially interest- ing in the case where the interaction strength is not well known as for hyperon-nucleon (Y-N) and hyperon-hyperon (Y-Y) pairs [11–19]. Hyperon-nucleon and hyperon-hyperon interactions are still rather poorly experimentally constrained and a detailed knowledge of these interactions is necessary to understand quantitatively the strangeness sector in the low- energy regime of quantum-chromodynamics (QCD) [20]. The existence of hypernuclei [27] conﬁrms that the N- is attractive within nuclear matter for densities below nu- clear saturation ρ0 = 0.16 fm−3. An average value of U(ρ = ρ0, k = 0) ≈−30 MeV [27], with k the hyperon momentum in the laboratory reference system, is extracted from hypernu- clear data on the basis of a dispersion relation for hyperons in a baryonic medium at ρ0. Hyperon-nucleon (p- and p-) scattering experiments have been carried out in the 1960s [21–23] and the measured cross sections have been used to extract scattering lengths The situation for the hyperon is currently rather unclear. (Received 29 June 2018; revised manuscript received 19 November 2018; published 13 February 2019) We report on the ﬁrst femtoscopic measurement of baryon pairs, such as p-p, p-, and -, measured by ALICE at the Large Hadron Collider (LHC) in proton-proton collisions at √s = 7 TeV. This study demonstrates the feasibility of such measurements in pp collisions at ultrarelativistic energies. The femtoscopy method is employed to constrain the hyperon-nucleon and hyperon-hyperon interactions, which are still rather poorly understood. A new method to evaluate the inﬂuence of residual correlations induced by the decays of resonances and experimental impurities is hereby presented. The p-p, p-, and - correlation functions were ﬁtted simultaneously with the help of a new tool developed speciﬁcally for the femtoscopy analysis in small colliding systems: Correlation Analysis Tool using the Schrödinger equation (CATS). Within the assumption that in pp collisions the three particle pairs originate from a common source, its radius is found to be equal to r0 = 1.125 ± 0.018 (stat) +0.058 −0.035 (syst) fm. The sensitivity of the measured p- correlation is tested against different scattering parameters, which are deﬁned by the interaction among the two particles, but the statistics is not sufﬁcient yet to discriminate among different models. The measurement of the - correlation function constrains the phase space spanned by the effective range and scattering length of the strong interaction. Discrepancies between the measured scattering parameters and the resulting correlation functions at LHC and RHIC energies are discussed in the context of various models. DOI: 10.1103/PhysRevC.99.024001 Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 PHYSICAL REVIEW C 99, 024001 (2019) ∗Full author list given at the end of the article. p-p, p-, and - correlations studied via femtoscopy in pp reactions at √s = 7 TeV S. Acharya et al.∗ (ALICE Collaboration) II. DATA ANALYSIS In this paper we present results from studies of the p-p, p-, and - correlations in pp collisions at √s = 7 TeV employing the data collected by ALICE in 2010 during the LHC Run 1. Approximately 3.4 × 108 minimum bias events have been used for the analysis, before event and track se- lection. A detailed description of the ALICE detector and its performance in the LHC Run 1 (2009-2013) is given in Refs. [54,55]. The inner tracking system (ITS) [54] consists of six cylindrical layers of high-resolution silicon detectors placed radially between 3.9 and 43 cm around the beam pipe. The two innermost layers are silicon pixel detectors (SPD) and cover the pseudorapidity range \|η\| < 2. The time projection chamber (TPC) [56] provides full azimuthal cov- erage and allows charged particle reconstruction and identiﬁ- cation (PID) via the measurement of the speciﬁc ionization energy loss dE/dx in the pseudorapidity range \|η\| < 0.9. The time-of-ﬂight (TOF) [57] detector consists of multigap resistive plate chambers covering the full azimuthal angle in \|η\| < 0.9. The PID is obtained by measuring the particle’s velocity β. The above-mentioned detectors are immersed in a B = 0.5 T solenoidal magnetic ﬁeld directed along the beam axis. The V0 are small-angle plastic scintillator detectors used for triggering and placed on either side of the collision vertex along the beam line at +3.3 m and −0.9 m from the nominal interaction point, covering the pseudorapidity ranges 2.8 < η < 5.1 (V0-A) and −3.7 < η < −1.7 (V0-C). The femtoscopy technique was employed by the STAR Collaboration to study - correlations in Au-Au collisions at √sNN = 200 GeV [16]. First a shallow repulsive interac- tion was reported for the - system, but in an alternative analysis, where the residual correlations were treated more ac- curately [38], a shallow attractive interaction was conﬁrmed. These analyses demonstrate the limitations of such measure- ments in heavy-ion collisions, where the source parameters are time dependent and the emission time might not be the same for all hadron species. The need for more experimental data to study the hyperon- nucleon, hyperon-hyperon, and even the hyperon-nucleon- nucleon interaction has become more crucial in recent years due to its connection to the modeling of astrophysical objects such as neutron stars [39–42]. I. INTRODUCTION There are some experimental indications for the formation of hypernuclei [28,29] but different theoretical approaches predict both attractive and repulsive interactions depending on the isospin state and partial wave [24,26,30]. The scarce ex- perimental data for this hypernucleus prevents any validation of the models. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. A -hypernucleus candidate was detected [31] and ongo- ing measurements suggest that the N- interaction is weakly attractive [32]. A recent work by the Lattice HAL-QCD 024001-1 024001-1 ©2019 CERN, for the ALICE Collaboration 2469-9985/2019/99(2)/024001(21) PHYSICAL REVIEW C 99, 024001 (2019) S. ACHARYA et al. Collaboration [33] shows how this attractive interaction could be visible in the p- femtoscopy analysis, in particular by comparing correlation functions for different static source sizes. This further motivates the extension of the femtoscopic studies from heavy ions to pp collisions since in the latter case the source size decreases by about a factor of three at the LHC energies leading to an increase in the strength of the correlation signal [34]. parameters available in the literature for p- and - pairs. The predictions for the correlation function used to ﬁt the data are obtained with the newly developed CATS framework [53]. A common source with a constant size is assumed and the value of the radius is extracted. The paper is organized in the following way. In Sec. II the experiment setup and the analysis technique are brieﬂy intro- duced. In Sec. III the femtoscopy technique and the theoretical models employed are discussed. In Sec. IV the sources of systematic uncertainties are summarized and ﬁnally in Sec. V the results for the p-p, p-, and - correlation function are presented. If one considers hyperon-hyperon interactions, the most prominent example is the - case. The H-dibaryon - bound state was predicted [35] and later a double hyper- nucleus was observed [36]. From this single measurement a shallow - binding energy of few MeV was extracted, but the H-dibaryon state was never observed. Also recent lattice calculations [37] obtain a rather shallow attraction for the - state. II. DATA ANALYSIS In the inner core of these objects the appearance of hyperons is a possible scenario since their creation at ﬁnite density becomes energetically favored in comparison with a purely neutron matter com- position [41]. However, the appearance of these additional degrees of freedom leads to a softening of the nuclear matter equation of state (EOS) [43] making the EOS incompatible with the observation of neutron stars as heavy as two solar masses [44,45]. This goes under the name of the hyperon puzzle. Many attempts were made to solve this puzzle, e.g., by introducing three-body forces for NN leading to an additional repulsion that can counterbalance the large gravita- tional pressure and ﬁnally allow for larger neutron star masses [46–49]. A repulsive core for the two-body forces would also stiffen the EOS containing hyperons. In order to constrain the parameter space of such models a detailed knowledge of the hyperon-nucleon, including and states, and of the hyperon-nucleon-nucleon interaction is mandatory. A. Event selection Selection criterion Value Proton selection criteria Pseudorapidity \|η\| < 0.8 Transverse momentum 0.5 < pT < 4.05 GeV/c TPC clusters nTPC > 80 Crossed TPC pad rows ncrossed > 70 (out of 159) Findable TPC clusters ncrossed/nﬁndable > 0.83 Tracks with shared TPC clusters rejected Distance of closest approach xy \|DCAxy\| < 0.1 cm Distance of closest approach z \|DCAz\| < 0.2 cm Particle identiﬁcation \|nσ,TPC\| < 3 for p < 0.75 GeV/c nσ,combined < 3 for p > 0.75 GeV/c Lambda selection criteria Daughter track selection criteria Pseudorapidity \|η\| < 0.8 TPC clusters nTPC > 70 Distance of closest approach DCA > 0.05 cm Particle identiﬁcation \|nσ,TPC\| < 5 V0 selection criteria Transverse momentum pT > 0.3 GeV/c decay vertex \|ivertex\| < 100 cm, i = x, y, z Transverse radius of the decay vertex rxy 0.2 < rxy < 100 cm DCA of the daughter tracks at the decay vertex DCA(\|p, π\|) < 1.5 cm Pointing angle α cos α > 0.99 K0 rejection 0.48 < Mπ+π−< 0.515 GeV/c2 selection \|Mpπ −M,PDG\| < 4 MeV/c2 \|η\| < 0.8 0.5 < pT < 4.05 GeV/c nTPC > 80 ncrossed > 70 (out of 159) ncrossed/nﬁndable > 0.83 rejected \|DCAxy\| < 0.1 cm \|DCAz\| < 0.2 cm \|nσ,TPC\| < 3 for p < 0.75 GeV/c nσ,combined < 3 for p > 0.75 GeV/c For particle identiﬁcation both the TPC and the TOF detectors are employed. For low momenta (p < 0.75 GeV/c) only the PID selection from the TPC is applied, while for larger momenta the information of both detectors is combined since the TPC does not provide a sufﬁcient separation power in this momentum region. The combination of TPC and TOF signals is done by employing a circular selection criterion nσ,combined ≡ (nσ,TPC)2 + (nσ,TOF)2, where nσ is the number of standard deviations of the measured from the expected signal at a given momentum. The expected signal is computed in the case of the TPC from a parametrized Bethe-Bloch curve, and in the case of the TOF by the expected β of a particle with a mass hypothesis m. A. Event selection In order to further enhance the purity of the proton sample, the nσ is computed assuming different particle hypotheses (kaons, electrons, and pions) and if the corresponding hypothesis is found to be more favorable, i.e., the nσ value found to be smaller, the proton hypothesis and thus the track is rejected. With these selection criteria a pT-averaged proton purity of 99% is achieved. The purity remains above 99% for pT < 2 GeV/c and then decreases to 80% at the momentum cutoff of 4.05 GeV/c. clusters at the same tracklet multiplicity. After application of these selection criteria, about 2.5 × 108 events are available for the analysis. B. Proton candidate selection To ensure a high-purity sample of protons, strict selection criteria are imposed on the tracks. Only particle tracks recon- structed with the TPC without additional matching with hits in the ITS are considered in the analysis in order to avoid biases introduced by the nonuniform acceptance in the ITS. However, the track ﬁtting is constrained by the independently reconstructed primary vertex. Hence, the obtained momentum resolution is comparable to that of globally reconstructed tracks, as demonstrated in Ref. [55]. The selection criteria for the proton candidates are summa- rized in Table I. The selection on the number of reconstructed TPC clusters serves to ensure the quality of the track, to assure a good pT resolution at large momenta and to remove fake tracks from the sample. To enhance the number of protons produced at the primary vertex, a selection is imposed on the distance of closest approach (DCA) in both beam (z) and transverse (xy) directions. In order to minimize the fraction of protons originating from the interaction of primary particles with the detector material, a low transverse momentum cutoff is applied [58]. At high pT a cutoff is introduced to ensure the purity of the proton sample, as the purity drops below 80% for larger pT due to the decreasing separation power of the combined TPC and TOF particle identiﬁcation. A. Event selection The minimum bias interaction trigger requires at least two out of the following three conditions: two pixel chips hit in the outer layer of the silicon pixel detectors, a signal in V0-A, a signal in V0-C [55]. Reconstructed events are required to have at least two associated tracks and the distance along the beam axis between the reconstructed primary vertex and the nominal interaction point should be smaller than 10 cm. Events with multiple reconstructed SPD vertices are consid- ered as pileup. In addition, background events are rejected using the correlation between the number of SPD clusters and the tracklet multiplicity. The tracklets are constrained to the primary vertex, and hence a typical background event is characterized by a large amount of SPD clusters but only few tracklets, while a pileup event contains a larger number of This work presents an alternative to scattering experiments, using the femtoscopy technique to study the Y-N and Y-Y interactions in pp collisions at √s = 7 TeV. We show that pp collisions at the LHC are extremely well suited to investigate baryon-baryon ﬁnal-state interactions and that the measure- ment of the correlation function is not contaminated with the minijet background visible in meson-meson correlations [50,51]. The extracted p-p, p-, and - correlations have been compared to the predicted function obtained by solv- ing the Schrödinger equation exactly by employing the Ar- gonne v18 potential [52] for p-p pairs and different scattering 024001-2 PHYSICAL REVIEW C 99, 024001 (2019) p-p, p-, AND - … TABLE I. Proton (top) and candidate (bottom) selection criteria. TABLE I. Proton (top) and candidate (bottom) selection criteria. III. CORRELATION FUNCTION The observable of interest in femtoscopy is the two-particle correlation function, which is deﬁned as the probability to ﬁnd simultaneously two particles with momenta p1 and p2 divided by the product of the corresponding single-particle probabilities The momentum is calculated as the sum of the daughter momenta. A minimum transverse momentum requirement on the candidate is applied to reduce the contribution of fake candidates. Finally, a selection is applied on the opening angle α between the momentum and the vector pointing from the primary vertex to the secondary V0 decay vertex. The rather broad PID selection of the daughter tracks introduces a residual pion contamination of the proton daughter sample that in combination with the charge-conjugate pion of the V0 leads to the misidentiﬁcation of K0 S as candidates. These K0 S candidates are removed by a selection on the π+π−invariant mass. C(p1, p2) ≡ P(p1, p2) P(p1) · P(p2). (1) (1) These probabilities are directly related to the inclusive Lorentz invariant spectra P(p1, p2) = E1E2 d6N d3p1d3p2 and P(p1,2) = E1,2 d3N d3p1,2 . In absence of a correlation signal the value of C(p1, p2) equals unity. These probabilities are directly related to the inclusive Lorentz invariant spectra P(p1, p2) = E1E2 d6N d3p1d3p2 and P(p1,2) = E1,2 d3N d3p1,2 . In absence of a correlation signal the value of C(p1, p2) equals unity. Approximating the emission process and the momenta of the particles, the size of the particle emitting source can be studied. Following [2], Eq. (1) can then be rewritten as The reconstructed invariant mass, its resolution and purity are determined by ﬁtting eight spectra of the same size in pT ∈[0.3, 4.3] GeV/c with the sum of two Gaussian func- tions describing the signal and a second-order polynomial to emulate the combinatorial background. The obtained values for the mean and variance of the two Gaussian functions are combined with an arithmetic average. The determined mass is in agreement with the PDG value for the and particles [59]. A total statistics of 5.9 × 106 and 5.5 × 106 and a signal to background ratio of 20 and 25 at a pT-averaged purity of 96% and 97% is obtained for and , respectively. It should be noted that the purity is constant within the investigated pT range. C. candidate selection The weak decay →pπ−(BR = 63.9%, cτ = 7.3 cm [59]) is exploited for the reconstruction of the candi- date, and accordingly the charge-conjugate decay for the identiﬁcation. The reconstruction method forms so-called V0 decay candidates from two charged particle tracks using a procedure described in Ref. [60]. The selection criteria for 024001-3 PHYSICAL REVIEW C 99, 024001 (2019) S. ACHARYA et al. the candidates are summarized in Table I. The V0 daughter tracks are globally reconstructed tracks and, in order to maxi- mize the efﬁciency, selected by a broad particle identiﬁcation cut employing the TPC information only. Additionally, the daughter tracks are selected by requiring a minimum impact parameter of the tracks with respect to the primary vertex. After the selection all positively charged daughter tracks are combined with a negatively charged partner to form a pair. The resulting vertex ivertex, i = x, y, z is then deﬁned as the point of closest approach between the two daughter tracks. This distance of closest approach of the two daughter tracks with respect to the decay vertex DCA(\|p, π\|) is used as an additional quality criterion of the candidate. is applied. To avoid any contribution from autocorrelations, all candidates are checked for shared daughter tracks. If this condition is found to be true, the candidate with the smaller cosine pointing angle is removed from the sample. If a primary proton is also used as a daughter track of a candidate, the latter is rejected. Figure 1 shows the pT- integrated invariant mass of the and candidates. III. CORRELATION FUNCTION Finally, a selection on the pπ−(pπ+) invariant mass C(k∗) = d3r∗S(r∗)\|ψ(r∗, k∗)\|2, (2) (2) where k∗is the relative momentum of the pair deﬁned as k∗= 1 2 · \|p∗ 1 −p∗ 2\|, with p∗ 1 and p∗ 2 the momenta of the two particles in the pair rest frame (PRF, denoted by the ∗), S(r∗) contains the distribution of the relative distance of particle pairs in the pair rest frame, the so-called source function, and ψ(r∗, k∗) denotes the relative wave function of the particle pair. The latter contains the particle interaction term and determines the shape of the correlation function. In this work, the p-p correlation function, which is theoretically well understood, is employed to obtain the required information about the source function and this information will be used to study the p- and - interaction. FIG. 1. Invariant mass distribution of pπ −(pπ +) to obtain the () signal. The dashed lines set the selection width used in the analysis. In order to relate the correlation function to experimentally accessible quantities, Eq. (1) is reformulated [2] as C(k∗) = N A(k∗) B(k∗). (3) (3) The distribution of particle pairs from the same event is denoted with A(k∗) and B(k∗) is a reference sample of un- correlated pairs. The latter is obtained using event mixing techniques, in which the particle pairs of interest are combined from single particles from different events. To avoid accep- tance effects of the detector system, the mixing procedure is conducted only between particle pairs stemming from events with similar z position of the primary vertex and similar multiplicity [2]. The normalization parameter for mixed and same event yields N is chosen such that the mean value of the correlation function equals unity for k∗∈[0.2, 0.4] GeV/c. FIG. 1. Invariant mass distribution of pπ −(pπ +) to obtain the () signal. The dashed lines set the selection width used in the analysis. As correlation functions of all studied baryon-baryon pairs, i.e., p-p, p-, and -, exhibit identical behavior compared 024001-4 PHYSICAL REVIEW C 99, 024001 (2019) p-p, p-, AND - … TABLE II. Weight parameters of the individual components of the p-p, p-, and - correlation function. III. CORRELATION FUNCTION p-p p- - Pair λ parameter [%] Pair λ parameter [%] Pair λ parameter [%] pp 74.18 p 47.13 29.94 pp 15.52 p− 9.92 0 19.96 pp 0.81 p0 9.92 00 3.33 p+ p 6.65 p0 15.71 0 12.61 p+ p+ 0.15 p 4.93 00 1.33 pp+ 0.70 p− 1.04 − 12.61 ˜pp 1.72 p0 1.04 −− 1.33 ˜pp 0.18 p0 1.64 00 4.20 ˜pp+ 0.08 p+ 2.11 0− 4.20 ˜p˜p 0.01 p+− 0.44 0− 2.65 p+0 0.44 ˜ 4.38 p+0 0.70 ˜0 1.46 ˜p 0.55 ˜0 0.92 ˜p− 0.18 ˜− 0.92 ˜p0 0.12 ˜ ˜ 0.16 ˜p0 0.12 p ˜ 3.45 p ˜ 0.36 p+ ˜ 0.15 ˜p ˜ 0.04 TABLE II. Weight parameters of the individual components of the p-p, p-, and - correlation function. TABLE II. Weight parameters of the individual components of the p-p, p-, and - correlation function. to those of their respective antibaryon-antibaryon pairs, the corresponding samples are combined to enhance the statistical signiﬁcance. Therefore, in the following p-p denotes the combination of p-p ⊕p-p, and accordingly for p- and -. where ˜X refers to misidentiﬁed particles of specie X. p, and p+ correspond to protons stemming from the weak decay of the corresponding hyperons. The →π →pππ decays are explicitly considered in the feed-down contribution of the p- correlation and hence are omitted in Eq. (5) to avoid double counting. As shown in Appendix, the fraction of primary protons and their feed-down fractions are required to calculate the λ parameters of the different contributions to Eq. (5). The information about the origin of the protons, i.e., whether the particles are of primary origin, originating from feed down or from the interactions with the detector material, is obtained by ﬁtting Monte Carlo (MC) templates to the experimental distributions of the distance of closest approach of the track to the primary vertex. The MC templates and the purity are extracted from PYTHIA [61] simulations using the Perugia 2011 tune [62], which were ﬁltered through the ALICE detector and the reconstruction algorithm [54]. The pT averages are then calculated by weighting the quantities of interest by the respective particle yields dN/d pT. III. CORRELATION FUNCTION The resulting fraction of primary protons averaged over pT is 87% (where in this fraction we also include the protons stemming from strong decays of broad resonances), with the other 13% of the total yield associated to weak decays of resonances and the contribution from the detector material is found to be negligible. A. Decomposition of the correlation function Since the and N∗resonances have typical widths above 120 MeV the decay length is in the order of 1 fm so that the decay particles do still experience the ﬁnal-state interaction with the neighboring particles as the primaries do. We estimate that about 65% of all primary protons and stem from the strong decay of resonances [68] and we have simulated how the source could be modiﬁed by such effects. A difference of 5% in the results of the Gaussian ﬁt is found when comparing p-p to p- pairs. These effects are in the same order of the present uncertainty on the radius and are herewith neglected. A quantitative study including all resonances is planned in the analysis of the LHC Run 2 sample. + p ˜ + p ˜ + p+ ˜ + ˜p ˜. (6) (6) The purity is obtained from ﬁts to the invariant mass spectra in eight bins of pT and deﬁned as S/(S + B), where S denotes the actual signal and B the background. The feed- down contribution is determined from MC template ﬁts of the experimental distributions of the cosine pointing angle, in which a total of four templates are considered corresponding to direct, feed-down, material, and impurity contributions. The production probability dN/d pT is employed in order to obtain pT weighted average values. Around 73% of the s are primaries (where in this fraction we also include the stemming from strong decays of broad resonances) and 23% originate from weakly decaying reso- nances, which is in line with the values quoted in Ref. [63]. The remaining yield is associated to combinatorial back- ground and s produced in the detector material. The main contribution to the feed-down fraction is expected to originate from the states with no preference for the neutral or the charged, respectively. This hypothesis is supported by PYTHIA simulations where the secondary particles arise from the weak decay of the 0 (48%) and ± (49%) resonances. The remaining contribution in the simulation arises from the 0, which, however, is treated separately. Since the latter decays electromagnetically almost exclusively into γ [59], it has a very short life time and cannot be experimentally differen- tiated from the sample of primary s. Measurements of the ratio R0/ = σ0/σ have obtained values around 1/3 [64– 67], however, with large uncertainties for hadronic collisions at high energies. A. Decomposition of the correlation function The experimental determination of the correlation func- tion is distorted by two distinct mechanisms. The sample of genuine particle pairs include misidentiﬁed particles and feed-down particles from strong and weak decays. In this work a new method to separate all the individual components contributing to a measured correlation signal is proposed. The correlation functions arising from resonances or impurities of the sample are weighted with the so-called λ parameters and in this way are taken into account in the total correlation function of interest C(k∗) = 1 + λgenuine · [Cgenuine(k∗) −1] + i j λi j[Ci j(k∗) −1], (4) (4) where the i, j denote all possible impurity and feed-down contributions. These λ parameters can be obtained employing exclusively single-particle properties such as the purity and feed-down probability. The underlying mathematical formal- ism is outlined in the Appendix. The feed down from weakly decaying resonances is eval- uated by using cross sections from PYTHIA and for the proton sample consists of the (70%) and + (30%) contributions. The individual contributions to the total correlation function are presented in Table II. For the case of p-p correlation the following contributions must be taken into account: {pp} = pp + pp + pp + p+ p + p+ p+ + pp+ + ˜pp + ˜pp + ˜pp+ + ˜p˜p, (5) The decomposition of the p- correlation function is conducted in a similar manner as for the p-p pair, however, (5) 024001-5 PHYSICAL REVIEW C 99, 024001 (2019) S. ACHARYA et al. considering the purities and feed-down fractions of both par- ticles tion is well established. The good agreement between the mT- integrated data and the femtoscopic ﬁt demonstrates that the assumption of a Gaussian source is pertinent. A comparison of the mT distribution for p-p and p- pairs shows a good agreement between the two. The limited experimental sample, however, does not allow conducting a differential analysis yet. {p} = p + p−+ p0 + p0 + p + p− + p0 + p0 + p+ + p+−+ p+0 + p+0 + ˜p + ˜p−+ ˜p0 + ˜p0 + p ˜ + p ˜ + p+ ˜ + ˜p ˜. (6) Additionally, we have considered the effect of the strong decays such as →N + π and N∗→ + K on the pro- duction of protons and . C. Nonfemtoscopic background For sufﬁciently large relative momenta (k∗> 200 MeV/c) and increasing separation distance, the FSI among the parti- cles is suppressed and hence the correlation function should approach unity. As shown in Fig. 2, however, the measured correlation function for p-p and p- exhibits an increase for k∗larger than about 200 MeV/c for the two systems. Such nonfemtoscopic effects, probably due to energy-momentum conservation, are in general more pronounced in small col- liding systems where the average particle multiplicity is low [2]. In the case of meson-meson correlations at ultrarelativistic energies, the appearance of long-range structures in the corre- lation functions for moderately small k∗(k∗< 200 MeV/c) is typically interpreted as originating from minijetlike structures [50,69]. For the - correlation function the following pair contri- butions are taken into account: {} = + 0 + 00 + 0 + 00 + −+ −−+ 00 + 0− + 0−+ ˜ + ˜0 + ˜− + ˜0 + ˜ ˜. (7) (7) The resulting λ parameters are shown in Table II. Notable is that the actual pair of interest contributes only to about one- third of the signal, while pair fractions involving in particular 0 and give a signiﬁcant contribution. The statistical uncer- tainties of these parameters are negligible and their inﬂuence on the systematic uncertainties will be evaluated in Sec. IV. The resulting λ parameters are shown in Table II. Notable is that the actual pair of interest contributes only to about one- third of the signal, while pair fractions involving in particular 0 and give a signiﬁcant contribution. The statistical uncer- tainties of these parameters are negligible and their inﬂuence on the systematic uncertainties will be evaluated in Sec. IV. PYTHIA also shows the same nonfemtoscopic correlation for larger k∗but fails to reproduce quantitatively the behavior shown in Fig. 2, as already observed for the angular correla- tion of baryon-baryon and antibaryon-antibaryon pairs [58]. Possible effects that were considered in this analysis and that could be inﬂuencing the source are either the decay of strong resonances, or a mT scaling. The latter is related to collective effects and may result in a non-Gaussian behavior of the source. The p-p correlation function is here considered as a benchmark since the theoretical description of the interac- Energy-momentum conservation leads to a contribution to the signal, which can be reproduced with a formalism described in Ref. A. Decomposition of the correlation function For lack of better estimates the value of 1/3 is used in the following. The resulting λ parameters for the p- pair are shown in Table II. B. Detector effects The shape of the experimentally determined correlation function is affected by the ﬁnite momentum resolution. This is taken into account when the experimental data are compared to model calculations in the ﬁtting procedure by transforming the modeled correlation function, see Eq. (15), to the recon- structed momentum basis. When tracks of particle pairs involved in the correlation function are almost collinear, i.e., have a low k∗, detector effects can affect the measurement. No hint for track merging or splitting is found and therefore no explicit selection criteria are introduced. 1. Genuine correlation function For the p-p correlation function the Coulomb and the strong interaction as well as the antisymmetrization of the wave functions are considered [71]. The strong interaction part of the potential is modeled employing the Argonne v18 [52] potential considering the s and p waves. The source is assumed to be isotropic with a Gaussian proﬁle of radius r0. The resulting Schrödinger equation is then solved with the CATS [53]. While the CATS framework can provide an exact solution for any source and local interaction potential, the Lednicky- Lyuboshitz approach uses the known analytical solution out- side the range of the strong interaction potential and takes into account its modiﬁcation in the inner region in an approximate way only. That is why this approach may not be valid for small systems. In the case of p- and - we employ the Lednický and Lyuboshitz analytical model [72] to describe these correlation functions. This model is based on the assumption of an isotropic source with Gaussian proﬁle S(r0) = 1 4πr2 0 3/2 exp −r2 4r2 0 , (8) (8) C. Nonfemtoscopic background [70] and is accordingly also considered in 024001-6 PHYSICAL REVIEW C 99, 024001 (2019) p-p, p-, AND - … FIG. 2. The raw correlation function compared to PYTHIA6 Perugia 2011 simulations for (a) p-p, (b) p-, and (c) - pairs. FIG. 2. The raw correlation function compared to PYTHIA6 Perugia 2011 simulations for (a) p-p, (b) p-, and (c) - pairs. function for uncharged particles [72] reads function for uncharged particles [72] reads this work. Therefore, a linear function C(k∗)nonfemto = ak∗+ b where a, b are ﬁt parameters, is included to the global ﬁt as C(k∗) = C(k∗)femto × C(k∗)nonfemto to improve the description of the signal by the femtoscopic model. The ﬁt parameters of the baseline function are obtained in k∗∈[0.3, 0.5] GeV/c for p-p and p- pairs. For the case of the - correlation function, the uncertainties of the data do not allow to ad- ditionally add a baseline, which is therefore omitted in the femtoscopic ﬁt. this work. Therefore, a linear function C(k∗)nonfemto = ak∗+ b where a, b are ﬁt parameters, is included to the global ﬁt as C(k∗) = C(k∗)femto × C(k∗)nonfemto to improve the description of the signal by the femtoscopic model. The ﬁt parameters of the baseline function are obtained in k∗∈[0.3, 0.5] GeV/c for p-p and p- pairs. For the case of the - correlation function, the uncertainties of the data do not allow to ad- ditionally add a baseline, which is therefore omitted in the femtoscopic ﬁt. C(k∗)Lednicky C(k∗)Lednicky = 1 + S ρS 1 2 f (k∗)S r0 2 1 − dS 0 2√πr0 + 2Re f (k∗)S √πr0 F1(Qinvr0) −Im f (k∗)S r0 F2(Qinvr0) , (10) where Re f (k∗)S [Im f (k∗)S] denotes the real (imaginary) part of the complex scattering amplitude, respectively. The F1(Qinvr0) and F2(Qinvr0) are analytical functions resulting from the approximation of isotropic emission with a Gaussian source and the factor ρS contains the pair fraction emitted into a certain spin state S. For the p- pair unpolarized emission is assumed. The - pair is composed of identical particles and hence additionally quantum statistics needs to be considered, which leads to the introduction of an additional term to the Lednický model, as employed, e.g., in Ref. [16]. 2. Residual correlations Table II demonstrates that a signiﬁcant admixture of resid- uals is present in the experimental sample of particle pairs. A ﬁrst theoretical investigation of these so-called residual correlations was conducted in Ref. [73]. This analysis relies on the procedure established in Ref. [19], where the initial correlation function of the residual is calculated and then transformed to the new momentum basis after the decay. where r0 is the size of the source. Additionally, the complex scattering amplitude is evaluated by means of the effective range approximation f (k∗)S = 1 f S 0 + 1 2dS 0 k∗2 −ik∗ −1 , (9) (9) For the p-p channel only the feed down from the p- correlation function is considered, which is obtained by ﬁtting the p- experimental correlation function and then trans- forming it to the p-p momentum basis. All contributions are weighted by the corresponding λ parameters and the modeled correlation function for this pair Cmodel,p-p(k∗) can be written with the scattering length f S 0 , the effective range dS 0 and S denoting the total spin of the particle pair. In the following, the usual sign convention of femtoscopy is employed where an attractive interaction leads to a positive scattering length. With these assumptions the analytical description of the correlation 024001-7 PHYSICAL REVIEW C 99, 024001 (2019) S. ACHARYA et al. TABLE III. Selection parameter variation and the resulting relative systematic uncertainty on the p-p, p-, and - correlation function. Variable Default Variation p-p[%] p-[%] -[%] Min. pT proton (GeV/c ) 0.5 0.4, 0.6 1 0.2 – \|η\| proton 0.8 0.7, 0.9 0.4 0.2 – nσ proton 3 2, 5 1.8 0.2 – Proton tracks TPC only Global 2.4 0 – nCluster proton 80 90 0.3 0.1 – Min. pT V0 (GeV/c ) 0.3 0.24, 0.36 – 0 0 cos(α) V0 0.99 0.998 – 0 1.8 nσ V0 daughter 5 4 – 0.1 0.3 nCluster V0 daughter 70 80 – 0.1 0.7 \|η\| V0 0.8 0.7, 0.9 – 0.6 0.8 DCA(\|p, π\|) (cm) 1.5 1.2 – 0.5 0 DCA (cm) 0.05 0.06 – 0.7 0.6 TABLE III. Selection parameter variation and the resulting relative systematic uncertainty on the p-p, p-, and - correlation function. TABLE III. Selection parameter variation and the resulting relative systematic uncertainty on the p-p, p-, and - correlation function. 3. Total correlation function model as Cmodel,p-p(k∗) =1+λpp · [Cpp(k∗) −1]+λpp Cpp(k∗) −1 . (11) The correlation function modeled according to the con- siderations discussed above is then multiplied by a linear function to correct for the baseline as discussed in Sec. III C and weighted with a normalization parameter N All other residual correlations are assumed to be ﬂat. 0 All other residual correlations are assumed to be ﬂat. 0 For the p-, residual correlations from the p-0, p-, and - pairs are taken into account. As the - correlation function is rather ﬂat no further transformation is applied. The p-0 correlation function is obtained using predictions from Ref. [74]. Ctot(k∗) = N · (a + b · k∗) · Cmodel(k∗), (15) (15) where Cmodel(k∗) incorporates all considered theoretical cor- relation functions, weighted with the corresponding λ param- eters as discussed in Secs. III A and III D. As the decay products of the reaction →π are charged and therefore accessible by ALICE, we measure the p- correlation function. The experimental data are parametrized with a phenomenological function The inclusion of a baseline is further motivated by the presence of a linear but nonﬂat correlation observed in the data outside the femtoscopic region (see Fig. 2 for k∗∈[0.3, 0.5] GeV/c). When attempting to use a higher-order polynomial to model the background, the resulting curves are still compatible with a linear function, while their interpolation into the lower k∗region leads to an overall poorer ﬁt quality. Cp−−(k∗) = 1 + exp(−k∗a) k∗a , (12) (12) where the parameter a is employed to scale the function to the data and has no physical meaning. Its value is found to be a = 3.88 fm. IV. SYSTEMATIC UNCERTAINTIES The modeled correlation functionCmodel,p-(k∗) for the pair is obtained by B. Femtoscopic ﬁt To evaluate the systematic uncertainty of the femtoscopic ﬁt, and hence on the measurement of the radius r0, the ﬁt is performed applying the following variations. Instead of the common ﬁt, the radius is determined separately from the p-p and p- correlation functions. - is excluded because it imposes only a shallow constraint on the radius, in particular since the scattering parameters are unconstrained for the ﬁt. Furthermore, the input to the λ parameters are varied by 25%, while keeping the purity and the fraction of primaries and sec- ondaries constant since this would correspond to a variation of the particle selection and thus would require a different experimental sample as discussed above. Additionally, all ﬁt ranges of both the femtoscopic and the baseline ﬁts are varied individually by up to 50% and 10%, respectively. The lower bound of the femtoscopic ﬁt is always left at its default value. For the p- correlation function the dependence on the ﬁt model is studied by replacing the Lednický and Lyuboshitz analytical model with the potential introduced by Bodmer, Usmani, and Carlson [75] for which the Schrödinger equation is explicitly solved using CATS. Additionally, the ﬁt for the p-p and p- correlation function is performed without the linear baseline. The radius is determined for 2000 random combinations of the above-mentioned variations. The result- ing distribution of radii is not symmetric and the systematic uncertainty is therefore extracted as the boundaries of the 68% conﬁdence interval around the median of the distribution and accounts to about 4% of the determined radius. In absence of residual contributions and any FSI, the - correlation function is expected to approach 0.5 as k∗→0. While the data suggest that the - correlation exceeds the value expected considering only quantum statistic effects, the limited amount of data of the herewith presented sample does not allow us to draw strong conclusions on the attractive nature of the - interaction [27,38]. The experimental data are ﬁtted using CATS and hence the exact solution of the Schrödinger equation for the p-p correlation and the Lednický model for the p- and - correlation. The three ﬁts are done simultaneously and in this way the source radius is extracted and different scattering parameters for the p- and - interactions can be tested. A. Correlation function Cmodel,p-(k∗) = 1 + λp[Cp(k∗) −1] + λp0 Cp0 (k∗) −1 + λp− Cp−(k∗) −1 . (13) The systematic uncertainties of the correlation functions are extracted by varying the proton and candidate selection criteria according to Table III. Due to the low number of particle pairs, in particular at low k∗, the resulting variations of the correlation functions are in general much smaller than the statistical uncertainties. In order to still estimate the system- atic uncertainties the data are rebinned by a factor of 10. The systematic uncertainty on the correlation function is obtained by computing the ratio of the default correlation function to the one obtained by the respective cut variation. Whenever this results in two systematic uncertainties, i.e., by a variation up and downwards, the average is taken into account. Then all systematic uncertainties from the cut variations are summed up quadratically. This is then extrapolated to the ﬁner binning of the correlation function by ﬁtting a polynomial of second order. The obtained systematic uncertainties are found to be largest in the lowest k∗bin. The individual contributions in (13) As the present knowledge on the hyperon-hyperon interac- tion is scarce, in particular regarding the interaction of the with other hyperons, all residual correlations feeding into the - correlation function are considered to be consistent with unity, Cmodel,-(k∗) = 1 + λ[C(k∗) −1]. (14) (14) It should be noted, that the residual correlation functions, after weighting with the corresponding λ parameter, transfor- mation to the momentum base of the correlation of interest and taking into account the ﬁnite momentum resolution, only barely contribute to the total ﬁt function. 024001-8 PHYSICAL REVIEW C 99, 024001 (2019) p-p, p-, AND - … FIG. 3. The (a) p-p, (b) p-, and (c) - correlation function with a simultaneous ﬁt with the NLO expansion (red line) for the scattering parameter of p- [26]. The dashed line denotes the linear baseline. After the ﬁt is performed the LO [25] parameter set (green curve) is plugged in for the p- system and the scattering length obtained from [16] for the - system (cyan curve). FIG. 3. The (a) p-p, (b) p-, and (c) - correlation function with a simultaneous ﬁt with the NLO expansion (red line) for the scattering parameter of p- [26]. The dashed line denotes the linear baseline. V. RESULTS that bin are summarized in Table III and the resulting total sys- tematic uncertainty accounts to about 4% for p-p, 1% for p-, and 2.5% for -. Variations of the proton DCA selection are not taken into account for the computation of the systematic uncertainty since it dilutes (enhances) the correlation signal by introducing more (less) secondaries in the sample. This effect is recaptured by a change in the λ parameter. The obtained p-p, p-, and - correlation functions are shown in Fig. 3. For each of the correlation functions we do not observe any minijet background in the low k∗region, as observed in the case of neutral [76] and charged [51] kaon pairs and charged pion pairs [50]. This demonstrates that the femtoscopic signal in baryon-baryon correlations is dominant in ultrarelativistic pp collisions. The signal am- plitude for the p-p and p- correlations are much larger than the one observed in analogous studies from heavy-ion collisions [1,12,13,15], due to the small particle emitting source formed in pp collisions, allowing a higher sensitivity to the FSI. A. Correlation function After the ﬁt is performed the LO [25] parameter set (green curve) is plugged in for the p- system and the scattering length obtained from [16] for the - system (cyan curve). PHYSICAL REVIEW C 99, 024001 (2019) S. ACHARYA et al. TABLE IV. Scattering parameters for the p- system from various theoretical calculations [25,26,77–83] and the corresponding degree of consistency with the experimentally determined correlation function expressed in numbers of standard deviations nσ. The χEFT scattering parameters are obtained at a cutoff scale = 600 MeV. The usual sign convention of femtoscopy is employed where an attractive interaction leads to a positive scattering length. TABLE IV. Scattering parameters for the p- system from various theoretical calculations [25,26,77–83] and the corresponding degree of consistency with the experimentally determined correlation function expressed in numbers of standard deviations nσ. The χEFT scattering parameters are obtained at a cutoff scale = 600 MeV. The usual sign convention of femtoscopy is employed where an attractive interaction leads to a positive scattering length. Model f S=0 0 (fm) f S=1 0 (fm) dS=0 0 (fm) dS=1 0 (fm) nσ ND [77] 1.77 2.06 3.78 3.18 1.1 NF [78] 2.18 1.93 3.19 3.358 1.1 NSC89 [79] 2.73 1.48 2.87 3.04 0.9 a 0.71 2.18 5.86 2.76 1.0 b 0.9 2.13 4.92 2.84 1.0 c 1.2 2.08 4.11 2.92 1.0 NSC97 [80] d 1.71 1.95 3.46 3.08 1.0 e 2.1 1.86 3.19 3.19 1.1 f 2.51 1.75 3.03 3.32 1.0 ESC08 [81] 2.7 1.65 2.97 3.63 0.9 LO [25] 1.91 1.23 1.4 2.13 1.8 χEFT NLO [26] 2.91 1.54 2.78 2.72 1.5 A [82] 1.56 1.59 1.43 3.16 1.0 Jülich J04 [83] 2.56 1.66 2.75 2.93 1.4 J04c [83] 2.66 1.57 2.67 3.08 1.1 repulsive interactions. The p- strong interaction is modeled employing scattering parameters obtained using the next-to- leading order expansion of a chiral effective ﬁeld theory at a cutoff scale of = 600 MeV [26]. The simultaneous ﬁt of the p-p, p-, and - correlation functions yields a common radius of r0 = 1.125 ± 0.018 (stat) +0.058 −0 035 (syst) fm. ones reﬂects on the p- correlation function. The green curve corresponds to the results obtained employing LO scattering parameters and the theoretical correlation function is clearly sensitive for k∗→0 to the input parameter. In order to probe which scattering parameters are com- patible with the measured - correlation function, the ef- fective range and the scattering length of the potential are varied within d0 ∈[0, 18] fm and 1/ f0 ∈[−2, 5] 1/fm, while keeping the renormalization constant N as the only free ﬁt parameter. PHYSICAL REVIEW C 99, 024001 (2019) It should be noted that the resulting variations of N are on the percent level. The resulting correlation func- tions obtained by employing the Lednický and Lyuboshitz analytical model [72] and considering also the secondaries and impurities contributions are compared to the data. The degree of consistency is expressed in the number of standard deviations nσ, as displayed in Fig. 4 together with an overview of the present knowledge about the - interaction. For a detailed overview of the currently available models see, e.g., Ref. [38], from which we have obtained the collection of scattering parameters. Additionally to the Nijmegen meson exchange models mentioned above, the data are compared to various other theoretical calculations. An exemplary boson- exchange potential is Ehime [84,85], whose strength is ﬁtted to the outdated double hypernuclear bound energy, B = 4 MeV [86] and accordingly known to be too attractive. As an exemplary quark model including baryon-baryon interactions with meson exchange effects, the fss2 model [87,88] is used. Moreover, the potentials by Filikhin and Gal (FG) [89] and by Hiyama, Kamimura, Motoba, Yamada, and Yamamoto (HK- MYY) [90], which are capable of describing the NAGARA event [36] are employed. 0.035 The blue line in the left panel in Fig. 3 shows the result of the femtoscopic ﬁt to the p-p correlation function using the Argonne v18 potential that describes the experimental data in a satisfactory way. The red curve in the central panel shows the result of the NLO calculation for p-. In the case of - (right panel), the yellow curve represents the femtoscopic ﬁt with free scattering parameters. The width of the femtoscopic ﬁts corresponds to the systematic uncertainty of the correlation function discussed in Sec. IV. After the ﬁt with the NLO scattering parameters has con- verged, the p- correlation function for the same source size is compared to the data using various theoretically ob- tained scattering parameters [25,26,77–83] as summarized in Table IV. The degree of consistency is expressed in the num- ber of standard deviations nσ. The employed models include several versions of meson exchange models proposed such as the Nijmegen model D (ND) [77], model F (NF) [78], soft core (NSC89 and NSC97) [79,80], and extended soft core (ESC08) [81]. B. Femtoscopic ﬁt While in the case of the p-p and p- correlation function the existence of a baseline is clearly visible in the data, the low amount of pairs in the - channel do not allow for such a conclusion. Therefore, the baseline is not included in the model for the - correlation function. The simultaneous ﬁt is carried out by using a combined χ2 and with the radius as a free parameter common to all correlation functions. The ﬁt range is k∗∈[0, 0.16] GeV/c for p-p and k∗∈[0, 0.22] GeV/c for p- and -. Here- after we adopt the convention of positive scattering lengths for attractive interactions and negative scattering lengths for 024001-9 PHYSICAL REVIEW C 99, 024001 (2019) PHYSICAL REVIEW C 99, 024001 (2019) This demonstrates that these scattering parameters intervals combined with the Lednický model are not suited to describe the correlations functions measured in small systems. As for the region of negative scattering length f0 this is connected in scattering theory either to a repulsive in- teraction or to the existence of a bound state close to the threshold and a change in the sign of the scattering length. Since the - interaction is known to be slightly attractive above the threshold [36], the measurement of a negative scattering lengths would strongly support the existence of the H-dibaryon. Notably the correlation function modeled employing the scattering parameters obtained by the STAR Collaboration in Au-Au collisions at √sNN = 200 GeV [16] and all the secondaries and impurities contributions deviates by 6.8 standard deviations from the data. This is also shown by the cyan curve displayed in the right panel of Fig. 3, which is obtained using the source radius and the λ parameters from this analysis and the scattering parameters from Ref. [16]. On the other hand these parameters and all those corresponding to the gray-shaded area in Fig. 4 lead to a negative genuine - correlation function if the Lednický model is employed. The total correlation function that is compared to the experimental data is not negative because the impurities and secondaries contributions lead to a total correlation function that is always positive. This means that the combination of large effective ranges and negative scattering lengths translate into unphys- This study is the ﬁrst measurement with baryon pairs in pp collisions at √s = 7 TeV, while other femtoscopic analyses were conducted with neutral [76] and charged [51] kaon pairs and charged pion pairs [50] with the ALICE experiment. The radius obtained from baryon pairs is found to be slightly larger than that measured from meson-meson pairs at comparable transverse mass as shown in Fig. 5 PHYSICAL REVIEW C 99, 024001 (2019) Additionally, models considering contributions from one- and two-pseudoscalar-meson exchange diagrams and from four-baryon contact terms in χEFT at leading [25] and next-to-leading order [26] are employed, together with the ﬁrst version of the Jülich Y-N meson exchange model [82], which in a later version [83] also features one-boson exchange. All employed models describe the data equally well and hence the available data does not allow yet for a discrimina- tion. As an example, we show in the central panel of Fig. 3 how employing scattering parameters different than the NLO In contrast to the p- case, the agreement with the data increases with every revision of the Nijmegen potential, while the introduction of the extended soft core slightly increases the deviation. In particular solution NSC97f yields the overall 024001-10 PHYSICAL REVIEW C 99, 024001 (2019) p-p, p-, AND - … FIG. 5. Comparison of radii obtained for different charged par- ticle multiplicity intervals in the pp collision system at √s = 7 TeV [50,51,76]. The error bars correspond to statistical and the shaded regions to the systematic uncertainties. The black point is the radius obtained in this analysis with p-p, p-, and - pairs, while the gray bar corresponds to the range of covered mT in this analysis. FIG. 4. Number of standard deviations nσ of the modeled cor- relation function for a given set of scattering parameters (effective range d0 and scattering length f0) with respect to the data, together with various model calculations [77–81,84,85,87–90] and measure- ments [16]. The gray shaded area corresponds to the region where the Lednický model predicts a negative correlation function for pp collisions at √s = 7 TeV. FIG. 4. Number of standard deviations nσ of the modeled cor- Number of standard deviations nσ of the modeled co FIG. 4. Number of standard deviations nσ of the modeled cor- relation function for a given set of scattering parameters (effective range d0 and scattering length f0) with respect to the data, together with various model calculations [77–81,84,85,87–90] and measure- ments [16]. The gray shaded area corresponds to the region where the Lednický model predicts a negative correlation function for pp collisions at √s = 7 TeV. FIG. 5. Comparison of radii obtained for different charged par- ticle multiplicity intervals in the pp collision system at √s = 7 TeV [50,51,76]. The error bars correspond to statistical and the shaded regions to the systematic uncertainties. PHYSICAL REVIEW C 99, 024001 (2019) The black point is the radius obtained in this analysis with p-p, p-, and - pairs, while the gray bar corresponds to the range of covered mT in this analysis. best agreement with the data. The correlation function mod- eled using scattering parameters of the Ehime model, which is known to be too attractive deviates by about two standard deviations from the data. best agreement with the data. The correlation function mod- eled using scattering parameters of the Ehime model, which is known to be too attractive deviates by about two standard deviations from the data. For an attractive interaction (positive f0) the correlation function is pushed from the quantum statistics distribution for two fermions (correlation function equal to 0.5 for k∗= 0) to unity. As a result within the current uncertainties the - correlation function is rather ﬂat and close to 1 and this lack of structure makes it impossible to extract the two scattering parameters with a reasonable uncertainty. This means that even by increasing the data by a factor 10, as expected from the RUN2 data, it will be very complicated to constrain precisely the region f0 > 0. ical correlation functions, for small colliding systems as pp. This effect is not immediate visible in larger colliding system such as Au-Au at √sNN = 200 GeV measured by STAR, where the obtained correlation function does not become negative. This demonstrates that these scattering parameters intervals combined with the Lednický model are not suited to describe the correlations functions measured in small systems. One could test the corresponding local potentials with the help of CATS [53], since the latter does not suffer from the limitations of the Lednický model due to the employment of the asymptotic solution. On the other hand we have directly compared the correlation functions obtained employing CATS and the - local potentials reported in [38] with the corre- lation functions obtained using the corresponding scattering parameters and the Lednický model. For the typical source radii of 1.3 fm the deviations are within 10%. This disfavours the region of negative scattering lengths and large effective ranges for the - correlation. ical correlation functions, for small colliding systems as pp. This effect is not immediate visible in larger colliding system such as Au-Au at √sNN = 200 GeV measured by STAR, where the obtained correlation function does not become negative. VI. SUMMARY and Education, Croatia; Ministry of Education, Youth and Sports of the Czech Republic, Czech Republic; The Danish Council for Independent Research \| Natural Sciences, the Carlsberg Foundation and Danish National Research Founda- tion (DNRF), Denmark; Helsinki Institute of Physics (HIP), Finland; Commissariat à l’Energie Atomique (CEA) and In- stitut National de Physique Nucléaire et de Physique des Particules (IN2P3) and Centre National de la Recherche Sci- entiﬁque (CNRS), France; Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF) and GSI Helmholtzzentrum für Schwerionenforschung GmbH, Ger- many; General Secretariat for Research and Technology, Min- istry of Education, Research and Religions, Greece; National Research, Development and Innovation Ofﬁce, Hungary; De- partment of Atomic Energy Government of India (DAE), Department of Science and Technology, Government of India (DST), University Grants Commission, Government of India (UGC) and Council of Scientiﬁc and Industrial Research (CSIR), India; Indonesian Institute of Science, Indonesia; Centro Fermi - Museo Storico della Fisica e Centro Studi e Ricerche Enrico Fermi and Istituto Nazionale di Fisica Nucleare (INFN), Italy; Institute for Innovative Science and Technology, Nagasaki Institute of Applied Science (IIST), Japan Society for the Promotion of Science (JSPS) KAK- ENHI and Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; Consejo Nacional de Ciencia (CONACYT) y Tecnología, through Fondo de Cooperación Internacional en Ciencia y Tecnología (FON- CICYT) and Dirección General de Asuntos del Personal Academico (DGAPA), Mexico; Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO), Netherlands; The Re- search Council of Norway, Norway; Commission on Science and Technology for Sustainable Development in the South (COMSATS), Pakistan; Pontiﬁcia Universidad Católica del Perú, Peru; Ministry of Science and Higher Education and National Science Centre, Poland; Korea Institute of Science and Technology Information and National Research Founda- tion of Korea (NRF), Republic of Korea; Ministry of Edu- cation and Scientiﬁc Research, Institute of Atomic Physics and Romanian National Agency for Science, Technology and Innovation, Romania; Joint Institute for Nuclear Research (JINR), Ministry of Education and Science of the Russian Federation and National Research Centre Kurchatov Institute, Russia; Ministry of Education, Science, Research and Sport of the Slovak Republic, Slovakia; National Research Founda- tion of South Africa, South Africa; Centro de Aplicaciones Tecnológicas y Desarrollo Nuclear (CEADEN), Cubaenergía, Cuba and Centro de Investigaciones Energéticas, Medioambi- entales y Tecnológicas (CIEMAT), Spain; Swedish Research Council (VR) and Knut & Alice Wallenberg Foundation (KAW), Sweden; European Organization for Nuclear Re- search, Switzerland; National Science and Technology De- baryon-baryon and antibaryon-antibaryon pairs is feasible. VI. SUMMARY and Education, Croatia; Ministry of Education, Youth and Sports of the Czech Republic, Czech Republic; The Danish Council for Independent Research \| Natural Sciences, the Carlsberg Foundation and Danish National Research Founda- tion (DNRF), Denmark; Helsinki Institute of Physics (HIP), Finland; Commissariat à l’Energie Atomique (CEA) and In- stitut National de Physique Nucléaire et de Physique des Particules (IN2P3) and Centre National de la Recherche Sci- entiﬁque (CNRS), France; Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF) and GSI Helmholtzzentrum für Schwerionenforschung GmbH, Ger- many; General Secretariat for Research and Technology, Min- istry of Education, Research and Religions, Greece; National Research, Development and Innovation Ofﬁce, Hungary; De- partment of Atomic Energy Government of India (DAE), Department of Science and Technology, Government of India (DST), University Grants Commission, Government of India (UGC) and Council of Scientiﬁc and Industrial Research (CSIR), India; Indonesian Institute of Science, Indonesia; Centro Fermi - Museo Storico della Fisica e Centro Studi e Ricerche Enrico Fermi and Istituto Nazionale di Fisica Nucleare (INFN), Italy; Institute for Innovative Science and Technology, Nagasaki Institute of Applied Science (IIST), Japan Society for the Promotion of Science (JSPS) KAK- ENHI and Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; Consejo Nacional de Ciencia (CONACYT) y Tecnología, through Fondo de Cooperación Internacional en Ciencia y Tecnología (FON- CICYT) and Dirección General de Asuntos del Personal Academico (DGAPA), Mexico; Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO), Netherlands; The Re- search Council of Norway, Norway; Commission on Science and Technology for Sustainable Development in the South (COMSATS), Pakistan; Pontiﬁcia Universidad Católica del Perú, Peru; Ministry of Science and Higher Education and National Science Centre, Poland; Korea Institute of Science and Technology Information and National Research Founda- tion of Korea (NRF), Republic of Korea; Ministry of Edu- cation and Scientiﬁc Research, Institute of Atomic Physics and Romanian National Agency for Science, Technology and Innovation, Romania; Joint Institute for Nuclear Research (JINR), Ministry of Education and Science of the Russian Federation and National Research Centre Kurchatov Institute, Russia; Ministry of Education, Science, Research and Sport of the Slovak Republic, Slovakia; National Research Founda- tion of South Africa, South Africa; Centro de Aplicaciones Tecnológicas y Desarrollo Nuclear (CEADEN), Cubaenergía, Cuba and Centro de Investigaciones Energéticas, Medioambi- entales y Tecnológicas (CIEMAT), Spain; Swedish Research Council (VR) and Knut & Alice Wallenberg Foundation (KAW), Sweden; European Organization for Nuclear Re- search, Switzerland; National Science and Technology De- velopment Agency (NSDTA), Suranaree University of Tech- nology (SUT) and Ofﬁce of the Higher Education Com- mission under NRU project of Thailand, Thailand; Turkish Atomic Energy Agency (TAEK), Turkey; National Academy of Sciences of Ukraine, Ukraine; Science and Technology VI. SUMMARY This paper presents the ﬁrst femtoscopic measurement of p-p, p-, and - pairs in pp collisions at √s = 7 TeV. No evidence for the presence of minijet background is found and it is demonstrated that this kind of study with 024001-11 PHYSICAL REVIEW C 99, 024001 (2019) S. ACHARYA et al. VI. SUMMARY With a newly developed method to compute the contributions arising from impurities and weakly decaying resonances to the correlation function from single particles quantities only, the genuine correlation functions of interest can be extracted from the signal. These correlation functions contribute with 74% for p-p, 47% for p-, and 30% for - to the total signal. A simultaneous ﬁt of all correlation functions with a femtoscopic model featuring residual correlations stemming from the above-mentioned effects yields a radius of the parti- cles emitting source of r0 = 1.125 ± 0.018 (stat) +0.058 −0.035 (syst) fm. For the ﬁrst time, the Argonne v18 NN potential with the s and p waves was used to successfully describe the p-p correlation and so obtain a solid benchmark for our investigation. For the case of the p- correlation function, the NLO parameter set obtained within the framework of chiral effective ﬁeld theory is consistent with the data, but other models are also found to be in agreement with the data. The present pair data in the - channel allows us to constrain the available scattering parameter space. Large effective ranges d0 in combination with negative scattering parameters lead to unphysical correlations if the Lednický model is employed to compute the correlation function. This also holds true for the average values published by the STAR collaboration in Au-Au collisions at √sNN = 200 GeV, that are found to be incompatible with the measurement in pp collisions within the Lednický model. The larger data sample of the LHC Run 2 and Run 3, where we expect up to a factor ten and 100 more data respectively, will enable us to extend the method also to , , and hyperons and thus further constrain the hyperon-nucleon interaction. APPENDIX: DERIVATION OF THE λ PARAMETERS (A10) (A9) XF = NF i−1 Xi, (A1) (A1) Hence the total correlation function becomes: as the total number of particles that stem from feed down and C(XY ) = i,j N(XiYj) M(XY ) = i,j N(XiYj) M(XY ) M(XiYj) M(XiYj) (A11) = i,j N(XiYj) M(XiYj) Ci,j(XY ) M(XiYj) M(XY ) λi,j(XY ) = i,j λi,j(XY )Ci,j(XY ), (A12) C(XY ) = i,j N(XiYj) M(XY ) = i,j N(XiYj) M(XY ) M(XiYj) M(XiYj) (A11) XM = NM NF +1 Xi, (A2) (A11) (A2) = i,j N(XiYj) M(XiYj) Ci,j(XY ) M(XiYj) M(XY ) λi,j(XY ) = i,j λi,j(XY )Ci,j(XY ), as the total number of particles that were misidentiﬁed (i.e., impurities). X0 is the number of correctly identiﬁed primary particles that are of interest for the femtoscopy analysis. The purity P is the fraction of correctly identiﬁed parti- cles, not necessarily primary, to the total number of particles in the sample [Eq. (A3)]. (A12) where Ci,j(XY ) is the contribution to the total correlation of the i, jth channel of origin of the particles X,Y and λi,j(XY ) is the corresponding weight coefﬁcient. How to obtain the individual functions Ci,j(XY ) is discussed in the main body of the paper. The weights λi,j can be derived from the purities and channel fractions of the particles X and Y. This is pos- sible since λi,j depends only on the mixed event sample for which the underlying assumption is that the particles are not correlated. In that case the two-particle yield M(XY ) can be factorized and according to Eq. (A11) the λ coefﬁcients can be expressed as where Ci,j(XY ) is the contribution to the total correlation of the i, jth channel of origin of the particles X,Y and λi,j(XY ) is the corresponding weight coefﬁcient. How to obtain the individual functions Ci,j(XY ) is discussed in the main body of the paper. The weights λi,j can be derived from the purities and channel fractions of the particles X and Y. This is pos- sible since λi,j depends only on the mixed event sample for which the underlying assumption is that the particles are not correlated. In that case the two-particle yield M(XY ) can be factorized and according to Eq. ACKNOWLEDGMENTS The ALICE Collaboration would like to thank all its en- gineers and technicians for their invaluable contributions to the construction of the experiment and the CERN accelerator teams for the outstanding performance of the LHC complex. The ALICE Collaboration gratefully acknowledges the re- sources and support provided by all Grid centers and the Worldwide LHC Computing Grid (WLCG) collaboration. The ALICE Collaboration acknowledges the following funding agencies for their support in building and running the AL- ICE detector: A. I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation (ANSL), State Com- mittee of Science and World Federation of Scientists (WFS), Armenia; Austrian Academy of Sciences and Nationalstiftung für Forschung, Technologie und Entwicklung, Austria; Min- istry of Communications and High Technologies, National Nuclear Research Center, Azerbaijan; Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq), Universi- dade Federal do Rio Grande do Sul (UFRGS), Financiadora de Estudos e Projetos (Finep) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil; Min- istry of Science & Technology of China (MSTC), National Natural Science Foundation of China (NSFC) and Ministry of Education of China (MOEC), China; Ministry of Science 024001-12 PHYSICAL REVIEW C 99, 024001 (2019) p-p, p-, AND - … Next we will relate P(Xi) and f (Xi) to the correlation function between particle pairs, which is deﬁned as States Department of Energy, Ofﬁce of Nuclear Physics (DOE NP), United States of America. C(XY ) = N(XY ) M(XY ), (A8) APPENDIX: DERIVATION OF THE λ PARAMETERS (A8) Let X be a speciﬁc particle type and X is the number of particles of that species. For each particle different subsets Xi are deﬁned, each representing a unique origin of the particle, where i = 0 corresponds to the case of a primary particle, the rest are either particles originating from feed down or misidentiﬁcation. In particular indexes 1 ⩽i ⩽NF should be associated with feed-down contributions and NF + 1 ⩽i ⩽ NF + NM should be associated with impurities, where NF is the number of feed-down channels and NM the number of impurity channels. In the present work we assume that all impurity channels contribute with a ﬂat distribution to the total correlation, therefore we do not study differentially the origin of the impurities and combine them in a single channel, i.e., NM = 1. Further we deﬁne where N and M are the yields of an XY particle pair in same and mixed events respectively. Note that this is a raw correlation function, which is not properly normalized. The normalization is discussed in the main body of the paper, but is irrelevant in the current discussion and it will be omitted. Both N and M are yields, which can be decomposed into the sum of their ingredients. Using the previously discussed notion of different channels of origin where N and M are the yields of an XY particle pair in same and mixed events respectively. Note that this is a raw correlation function, which is not properly normalized. The normalization is discussed in the main body of the paper, but is irrelevant in the current discussion and it will be omitted. Both N and M are yields, which can be decomposed into the sum of their ingredients. Using the previously discussed notion of different channels of origin N(XY ) = N ⎛ ⎝ i,j XiYj ⎞ ⎠= i,j N(XiYj), (A9) M(XY ) = M ⎛ ⎝ i,j XiYj ⎞ ⎠= i,j M(XiYj). APPENDIX: DERIVATION OF THE λ PARAMETERS (A11) the λ coefﬁcients can be expressed as P(X ) = (X0 + XF )/X. (A3) (A3) The impurity is ¯ P(X ) = XM/X. (A4) (A4) For the later discussion it is beneﬁcial to combine the two deﬁnitions and refer to the purity as P(Xi) = P(X ) = (X0 + XF )/X for i ⩽NF, ¯ P(X ) = XM/X else. (A5) (A5) λi,j(XY ) = M(XiYj) M(XY ) = M(Xi) M(X ) M(Yi) M(Y ) = P(Xi)P(Yi). (A13) Another quantity of interest will be the channel fraction fi, which is deﬁned as the fraction of particles originating from the ith channel relative to the total number of either correctly identiﬁed or misidentiﬁed particles: The last step follows directly from Eq. (A7) applied to the mixed event samples of X and Y. Equation (A7) relates P to the known quantities P and f , hence the λ coefﬁcients can be rewritten as f (Xi) = Xi/(X0 + XF ) for i ⩽NF, Xi/XM else. (A6) (A6) As discussed in the main body of the paper both the purity and the channel fractions can be obtained either from MC simulations or MC template ﬁts. The product of the two reads λi,j(XY ) = P(Xi) f (Xi)P(Yj) f (Yj). (A14) (A14) We would like to point out that due to the deﬁnition of P(Xi) the sum of all λ parameters is automatically normalized to unity. P(Xi) = P(Xi) f (Xi) = Xi X . (A7) (A7) 024001-13 PHYSICAL REVIEW C 99, 024001 (2019) S. ACHARYA et al. [1] S. Pratt, Pion interferometry of quark-gluon plasma, Phys. Rev. D 33, 1314 (1986). [1] S. Pratt, Pion interferometry of quark-gluon plasma, Phys. Rev. D 33, 1314 (1986). [20] W. Weise, Low-energy QCD and hadronic structure, Nucl. Phys. A 827, 66C (2009). [2] M. A. Lisa, S. Pratt, R. Soltz, and U. Wiedemann, Femtoscopy in relativistic heavy ion collisions, Ann. Rev. Nucl. Part. Sci. 55, 357 (2005). [21] B. Sechi-Zorn, B. Kehoe, J. Twitty, and R. A. Burnstein, Low-energy -Proton elastic scattering, Phys. 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Lietava,108 B. Lim,18 S. Lindal, V. Lindenstruth, S. W. Lindsay, C. Lippmann, M. A. Lisa, V. Litichevskyi, A. Liu, H. M. Ljunggren, W. J. Llope,141 D. F. Lodato,63 V. Loginov,91 C. Loizides,94,79 P. Loncar,35 X. Lopez,132 E. López Torres,8 A. Lowe,143 P. Luettig,69 J. R. Luhder,142 M. Lunardon,29 G. Luparello,59 M. Lupi,34 A. Maevskaya,62 M. Mager,34 S. M. Mahmood,21 A. Mastroserio,52,136 A. M. Mathis,116,103 P. F. T. Matuoka,120 A. Matyja,117,128 C. Mayer,117 M. Mazzilli,33 M. A. Mazzoni,57 F. Meddi,23 Y. Melikyan,91 A. Menchaca-Rocha,72 E. Meninno,30 J. Mercado Pérez,102 M. Meres,14 S. Mhlanga,124 Y. Miake,131 L. Micheletti,26 M. M. Mieskolainen,43 D. L. Mihaylov,103 K. Mikhaylov,64,75 A. Mischke,63 A. N. Mishra,70 D. Mi´skowiec,104 J. Mitra,139 C. M. Mitu,68 N. Mohammadi,34 A. P. Mohanty,63 B. Mohanty,85 M. Mohisin Khan,17,d 142 44 29 113 34 34 51 y y y y B. APPENDIX: DERIVATION OF THE λ PARAMETERS Zichichi,27,10 M. B. Zimmermann,34 G. Zinovjev,2 J. Zmeskal,112 and S. Zou6 M. A. Völkl,101 K. Voloshin,64 S. A. Voloshin,141 G. Volpe,33 B. von Haller,34 I. Vorobyev,116,103 D. Voscek,115 D. Vranic,104,34 J. Vrláková,38 B. Wagner,22 H. Wang,63 M. Wang,6 Y. Watanabe,131 M. Weber,112 S. G. Weber,104 A. Wegrzynek,34 J. Wilkinson,53 G. A. Willems,142,34 M. C. S. Williams,53 E. Willsher,108 B. Windelband,102 W. E. Witt,128 K. Yamakawa,45 S. Yano,45 Z. Yin,6 H. Yokoyama,131,78 I.-K. Yoo,18 J. H. Yoon,60 V. Yurchenko,2 V. Zaccolo,58 A. Zaman,15 C. Zampolli,34 H. J. C. Zanoli,120 N. Zardoshti,108 A. Zarochentsev,111 P. Závada,67 N. Zaviyalov,106 H. Zbroszczyk,140 M. Zhalov,96 X. Zhang,6 Y. Zhang,6 Z. Zhang,6,132 C. Zhao,21 V. Zherebchevskii,111 N. Zhigareva,64 D. Zhou,6 Y. Zhou,88 Z. Zhou,22 H. Zhu,6 J. Zhu,6 Y. Zhu,6 A. Zichichi,27,10 M. B. Zimmermann,34 G. Zinovjev,2 J. Zmeskal,112 and S. Zou6 M. Shimomura,82 S. Shirinkin,64 Q. Shou,6,110 K. Shtejer,26 Y. Sibiriak,87 S. Siddhanta,54 K. M. Sielewicz,34 T. Siemiarczuk,84 D. Silvermyr,80 G. Simatovic,89 G. Simonetti,34,103 R. Singaraju,139 R. Singh,85 R. Singh,99 V. Singhal,139 T. Sinha,107 B. Sitar,14 M. Sitta,32 T. B. Skaali,21 M. Slupecki,126 N. Smirnov,144 R. J. M. Snellings,63 T. W. Snellman,126 J. Sochan,115 C. Soncco,109 J. Song,18 F. Soramel,29 S. Sorensen,128 F. Sozzi,104 I. Sputowska,117 J. Stachel,102 I. Stan,68 P. Stankus,94 E. Stenlund,80 D. Stocco,113 M. M. Storetvedt,36 P. Strmen,14 A. A. P. Suaide,120 T. Sugitate,45 C. Suire,61 M. Suleymanov,15 M. Suljic,34,25 R. Sultanov,64 M. Šumbera,93 S. Sumowidagdo,50 K. Suzuki,112 S. Swain,66 A. Szabo,14 I. Szarka,14 U. Tabassam,15 J. Takahashi,121 G. J. Tambave,22 N. Tanaka,131 M. Tarhini,113 M. Tariq,17 M. G. Tarzila,47 A. Tauro,34 G. Tejeda Muñoz,44 A. Telesca,34 C. Terrevoli,29 B. Teyssier,133 D. Thakur,49 S. Thakur,139 D. Thomas,118 F. Thoresen,88 R. Tieulent,133 A. Tikhonov,62 A. R. Timmins,125 A. Toia,69 N. Topilskaya,62 M. Toppi,51 F. Torales-Acosta,20 S. R. Torres,119 S. Tripathy,49 S. Trogolo,26 G. Trombetta,33 L. Tropp,38 V. Trubnikov,2 W. H. Trzaska,126 T. P. Trzcinski,140 B. A. Trzeciak,63 T. Tsuji,130 A. Tumkin,106 R. Turrisi,56 T. S. Tveter,21 K. Ullaland,22 E. N. Umaka,125 A. Uras,133 G. L. Usai,24 A. Utrobicic,97 M. Vala,115 J. W. Van Hoorne,34 M. van Leeuwen,63 P. Vande Vyvre,34 D. Varga,143 A. Vargas,44 M. Vargyas,126 R. Varma,48 M. Vasileiou,83 A. Vasiliev,87 A. Vauthier,78 O. Vázquez Doce,103,116 V. Vechernin,111 A. M. Veen,63 E. Vercellin,26 S. Vergara Limón,44 L. Vermunt,63 R. Vernet,7 R. Vértesi,143 L. Vickovic,35 J. Viinikainen,126 Z. Vilakazi,129 O. Villalobos Baillie,108 A. Villatoro Tello,44 A. Vinogradov,87 T. Virgili,30 V. Vislavicius,88,80 A. Vodopyanov,75 M. A. Völkl,101 K. Voloshin,64 S. A. Voloshin,141 G. Volpe,33 B. von Haller,34 I. Vorobyev,116,103 D. Voscek,115 D. Vranic,104,34 J. Vrláková,38 B. Wagner,22 H. Wang,63 M. Wang,6 Y. Watanabe,131 M. Weber,112 S. G. Weber,104 A. Wegrzynek,34 D. F. Weiser,102 S. C. Wenzel,34 J. P. Wessels,142 U. Westerhoff,142 A. M. Whitehead,124 J. Wiechula,69 J. Wikne,21 G. Wilk,84 J. Wilkinson,53 G. A. Willems,142,34 M. C. S. Williams,53 E. Willsher,108 B. Windelband,102 W. E. Witt,128 R. Xu,6 S. Yalcin,77 K. Yamakawa,45 S. Yano,45 Z. Yin,6 H. Yokoyama,131,78 I.-K. Yoo,18 J. H. Yoon,60 V. Yurchenko,2 V. Zaccolo,58 A. Zaman,15 C. Zampolli,34 H. J. C. Zanoli,120 N. Zardoshti,108 A. Zarochentsev,111 P. Závada,67 N. Zaviyalov,106 H. Zbroszczyk,140 M. Zhalov,96 X. Zhang,6 Y. Zhang,6 Z. Zhang,6,132 C. Zhao,21 V. Zherebchevskii,111 N. Zhigareva,64 D. Zhou,6 Y. Zhou,88 Z. Zhou,22 H. Zhu,6 J. Zhu,6 Y. Zhu,6 A. Zichichi,27,10 M. B. Zimmermann,34 G. Zinovjev,2 J. Zmeskal,112 and S. Zou6 APPENDIX: DERIVATION OF THE λ PARAMETERS Naik,48 R. Nair,84 B. K. Nandi,48 R. Nania,53,10 E. Nappi,52 A. Narayan,48 M. U. Naru,15 A. F. Nassirpour,80 H. Natal da Luz,120 C. Nattrass,128 S. R. Navarro,44 K. Nayak,85 R. Nayak,48 T. K. Nayak,139 S. Nazarenko,106 R. A. Negrao De Oliveira,69,34 L. Nellen,70 S. V. Nesbo,36 G. Neskovic,39 F. Ng,125 M. Nicassio,104 J. Niedziela,140,34 B. S. Nielsen,88 S. Nikolaev,87 S. Nikulin,87 V. Nikulin,96 F. Noferini,10,53 P. Nomokonov,75 G. Nooren,63 J. C. C. Noris,44 J. Norman,78 A. Nyanin,87 J. Nystrand,22 H. Oh,145 A. Ohlson,102 J. Oleniacz,140 A. C. Oliveira Da Silva,120 M. H. Oliver,144 J. Onderwaater,104 C. Oppedisano,58 R. Orava,43 M. Oravec,115 A. Ortiz Velasquez,70 A. Oskarsson,80 J. Otwinowski,117 K. Oyama,81 Y. Pachmayer,102 V. Pacik,88 D. Pagano,138 G. Pai´c,70 P. Palni,6 J. Pan,141 A. K. Pandey,48 S. Panebianco,135 V. Papikyan,1 P. Pareek,49 J. Park,60 J. E. Parkkila,126 S. Parmar,98 A. Passfeld,142 S. P. Pathak,125 R. N. Patra,139 B. Paul,58 6 63 6 71 135 87 69 69 H. Pei,6 T. Peitzmann,63 X. Peng,6 L. G. Pereira,71 H. Pereira Da Costa,135 D. Peresunko,87 E. Perez Lezama,69 V. Peskov,69 g Y. Pestov,4 V. Petráˇcek,37 M. Petrovici,47 C. Petta,28 R. P. Pezzi,71 S. Piano,59 M. Pikna,14 P. Pillot,113 L. O. D. L. Pimentel,88 O. Pinazza,53,34 L. Pinsky,125 S. Pisano,51 D. B. Piyarathna,125 M. Płosko´n,79 M. Planinic,97 F. Pliquett,69 J. Pluta,140 H. Poppenborg,142 S. Porteboeuf-Houssais,132 V. Pozdniakov,75 S. K. Prasad,3 R. Preghenella,53 F. Prino,58 C. A. Pruneau,141 I. Pshenichnov,62 M. Puccio,26 V. Punin,106 J. Putschke,141 S. Raha,3 S. Rajput,99 J. Rak,126 A. Rakotozaﬁndrabe,135 L. Ramello,32 F. Rami,134 R. Raniwala,100 S. Raniwala,100 S. S. Räsänen,43 B. T. Rascanu,69 R. Rath,49 V. Ratza,42 31 94 128 84 22 69 34 6 69 62 J. Saini, S. Sakai, M. A. Saleh, S. Sambyal, V. Samsonov, A. Sandoval, A. Sarkar, D. Sarkar, N. Sarkar, P. Sarma,41 M. H. P. Sas,63 E. Scapparone,53 F. Scarlassara,29 B. Schaefer,94 H. S. Scheid,69 C. Schiaua,47 R. Schicker,102 C. Schmidt,104 H. R. Schmidt,101 M. O. Schmidt,102 M. Schmidt,101 N. V. Schmidt,69,94 J. Schukraft,34 Y. Schutz,34,134 K. Schwarz,104 K. Schweda,104 G. Scioli,27 E. Scomparin,58 M. Šefˇcík,38 J. E. Seger,16 Y. Sekiguchi,130 D. Sekihata,45 p g g I. Selyuzhenkov,91,104 S. Senyukov,134 E. Serradilla,72 P. Sett,48 A. Sevcenco,68 A. Shabanov,62 A. Shabetai,113 R. Shahoyan,34 W. Shaikh,107 A. Shangaraev,90 A. Sharma,98 A. Sharma,99 M. Sharma,99 N. Sharma,98 A. I. Sheikh,139 K. Shigaki,45 024001-17 024001-17 PHYSICAL REVIEW C 99, 024001 (2019) S. ACHARYA et al. M. Shimomura,82 S. APPENDIX: DERIVATION OF THE λ PARAMETERS Shirinkin,64 Q. Shou,6,110 K. Shtejer,26 Y. Sibiriak,87 S. Siddhanta,54 K. M. Sielewicz,34 T. Siemiarczuk,84 D. Silvermyr,80 G. Simatovic,89 G. Simonetti,34,103 R. Singaraju,139 R. Singh,85 R. Singh,99 V. Singhal,139 T. Sinha,107 B. Sitar,14 M. Sitta,32 T. B. Skaali,21 M. Slupecki,126 N. Smirnov,144 R. J. M. Snellings,63 T. W. Snellman,126 J. Sochan,115 C. Soncco,109 J. Song,18 F. Soramel,29 S. Sorensen,128 F. Sozzi,104 I. Sputowska,117 J. Stachel,102 I. Stan,68 P. Stankus,94 E. Stenlund,80 D. Stocco,113 M. M. Storetvedt,36 P. Strmen,14 A. A. P. Suaide,120 T. Sugitate,45 C. Suire,61 M. Suleymanov,15 M. Suljic,34,25 R. Sultanov,64 M. Šumbera,93 S. Sumowidagdo,50 K. Suzuki,112 S. Swain,66 A. Szabo,14 I. Szarka,14 U. Tabassam,15 J. Takahashi,121 G. J. Tambave,22 N. Tanaka,131 M. Tarhini,113 M. Tariq,17 M. G. Tarzila,47 A. Tauro,34 G. Tejeda Muñoz,44 A. Telesca,34 C. Terrevoli,29 B. Teyssier,133 D. Thakur,49 S. Thakur,139 D. Thomas,118 F. Thoresen,88 R. Tieulent,133 A. Tikhonov,62 A. R. Timmins,125 A. Toia,69 N. Topilskaya,62 M. Toppi,51 F. Torales-Acosta,20 S. R. Torres,119 S. Tripathy,49 S. Trogolo,26 G. Trombetta,33 L. Tropp,38 V. Trubnikov,2 W. H. Trzaska,126 T. P. Trzcinski,140 B. A. Trzeciak,63 T. Tsuji,130 A. Tumkin,106 R. Turrisi,56 T. S. Tveter,21 K. Ullaland,22 E. N. Umaka,125 A. Uras,133 G. L. Usai,24 A. Utrobicic,97 M. Vala,115 J. W. Van Hoorne,34 M. van Leeuwen,63 P. Vande Vyvre,34 D. Varga,143 A. Vargas,44 M. Vargyas,126 R. Varma,48 M. Vasileiou,83 A. Vasiliev,87 A. Vauthier,78 O. Vázquez Doce,103,116 V. Vechernin,111 A. M. Veen,63 E. Vercellin,26 S. Vergara Limón,44 L. Vermunt,63 R. Vernet,7 R. Vértesi,143 L. Vickovic,35 J. Viinikainen,126 Z. Vilakazi,129 O. Villalobos Baillie,108 A. Villatoro Tello,44 A. Vinogradov,87 T. Virgili,30 V. Vislavicius,88,80 A. Vodopyanov,75 M. A. Völkl,101 K. Voloshin,64 S. A. Voloshin,141 G. Volpe,33 B. von Haller,34 I. Vorobyev,116,103 D. Voscek,115 D. Vranic,104,34 J. Vrláková,38 B. Wagner,22 H. Wang,63 M. Wang,6 Y. Watanabe,131 M. Weber,112 S. G. Weber,104 A. Wegrzynek,34 D. F. Weiser,102 S. C. Wenzel,34 J. P. Wessels,142 U. Westerhoff,142 A. M. Whitehead,124 J. Wiechula,69 J. Wikne,21 G. Wilk,84 J. Wilkinson,53 G. A. Willems,142,34 M. C. S. Williams,53 E. Willsher,108 B. Windelband,102 W. E. Witt,128 R. Xu,6 S. Yalcin,77 K. Yamakawa,45 S. Yano,45 Z. Yin,6 H. Yokoyama,131,78 I.-K. Yoo,18 J. H. Yoon,60 V. Yurchenko,2 V. Zaccolo,58 A. Zaman,15 C. Zampolli,34 H. J. C. Zanoli,120 N. Zardoshti,108 A. Zarochentsev,111 P. Závada,67 N. Zaviyalov,106 H. Zbroszczyk,140 M. Zhalov,96 X. Zhang,6 Y. Zhang,6 Z. Zhang,6,132 C. Zhao,21 V. Zherebchevskii,111 N. Zhigareva,64 D. Zhou,6 Y. Zhou,88 Z. Zhou,22 H. Zhu,6 J. Zhu,6 Y. Zhu,6 A. (ALICE Collaboration) 1A. I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2Bogolyubov Institute for Theoretical Physics, National Academy of Sciences of Ukraine, Kiev, Ukraine 1A. I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2Bogolyubov Institute for Theoretical Physics, National Academy of Sciences of Ukraine, Kiev, Ukraine nstitute, Department of Physics and Centre for Astroparticle Physics and Space Scien 4Budker Institute for Nuclear Physics, Novosibirsk, Russia 4Budker Institute for Nuclear Physics, Novosibirsk, Russia 5California Polytechnic State University, San Luis Obispo, California, United States 6Central China Normal University, Wuhan, China 7Centre de Calcul de l’IN2P3, Villeurbanne, Lyon, France 8Centro de Aplicaciones Tecnológicas y Desarrollo Nuclear (CEADEN), Havana, Cuba 9Centro de Investigación y de Estudios Avanzados (CINVESTAV), Mexico City and Mérida, Mexico 10Centro Fermi - Museo Storico della Fisica e Centro Studi e Ricerche “Enrico Fermi”, Rome, Italy 11Chicago State University, Chicago, Illinois, United States 12China Institute of Atomic Energy, Beijing, China 13Chonbuk National University, Jeonju, Republic of Korea 14Comenius University Bratislava, Faculty of Mathematics, Physics and Informatics, Bratislava, Slovakia 15COMSATS Institute of Information Technology (CIIT), Islamabad, Pakistan 16Creighton University, Omaha, Nebraska, United States 17Department of Physics, Aligarh Muslim University, Aligarh, India 18Department of Physics, Pusan National University, Pusan, Republic of Korea 19Department of Physics, Sejong University, Seoul, Republic of Korea 20Department of Physics, University of California, Berkeley, California, United States 21Department of Physics, University of Oslo, Oslo, Norway 22Department of Physics and Technology, University of Bergen, Bergen, Norway 23Dipartimento di Fisica dell’Università ’La Sapienza’ and Sezione INFN, Rome, Italy 24Dipartimento di Fisica dell’Università and Sezione INFN, Cagliari, Italy 25Dipartimento di Fisica dell’Università and Sezione INFN, Trieste, Italy 26Dipartimento di Fisica dell’Università and Sezione INFN, Turin, Italy 27Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Bologna, Italy 28Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Catania, Italy 29Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Padova, Italy 30Dipartimento di Fisica ‘E. R. Caianiello’ dell’Università and Gruppo Collegato INFN, Salerno, Italy 31Dipartimento DISAT del Politecnico and Sezione INFN, Turin, Italy 5California Polytechnic State University, San Luis Obispo, California, United States 6Central China Normal University Wuhan China 024001-18 PHYSICAL REVIEW C 99, 024001 (2019) p-p, p-, AND - … 32Dipartimento di Scienze e Innovazione Tecnologica dell’Università del Piemonte Orientale and INFN Sezione di Torino, Alessandria, Italy 33Dipartimento Interateneo di Fisica ‘M. (ALICE Collaboration) Merlin’ and Sezione INFN, Bari, Italy 34European Organization for Nuclear Research (CERN), Geneva, Switzerland 35Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, University of Split, Split, Croatia 36Faculty of Engineering and Science, Western Norway University of Applied Sciences, Bergen, Norway 37Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic 38Faculty of Science, P. J. (ALICE Collaboration) Šafárik University, Košice, Slovakia 39Frankfurt Institute for Advanced Studies, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany 40Gangneung-Wonju National University, Gangneung, Republic of Korea 41Gauhati University, Department of Physics, Guwahati, India 42Helmholtz-Institut für Strahlen- und Kernphysik, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany 43Helsinki Institute of Physics (HIP), Helsinki, Finland 44High Energy Physics Group, Universidad Autónoma de Puebla, Puebla, Mexico 45Hiroshima University, Hiroshima, Japan 46Hochschule Worms, Zentrum für Technologietransfer und Telekommunikation (ZTT), Worms, Germany 47Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 48Indian Institute of Technology Bombay (IIT), Mumbai, India 49Indian Institute of Technology Indore, Indore, India 50Indonesian Institute of Sciences, Jakarta, Indonesia 51INFN, Laboratori Nazionali di Frascati, Frascati, Italy 52INFN, Sezione di Bari, Bari, Italy 53INFN, Sezione di Bologna, Bologna, Italy 54INFN, Sezione di Cagliari, Cagliari, Italy 55INFN, Sezione di Catania, Catania, Italy 56INFN, Sezione di Padova, Padova, Italy 57INFN, Sezione di Roma, Rome, Italy 58INFN, Sezione di Torino, Turin, Italy 59INFN, Sezione di Trieste, Trieste, Italy 60Inha University, Incheon, Republic of Korea 61Institut de Physique Nucléaire d’Orsay (IPNO), Institut National de Physique Nucléaire et de Physique des Particules (IN2P3/CNRS), Université de Paris-Sud, Université Paris-Saclay, Orsay, France 62Institute for Nuclear Research, Academy of Sciences, Moscow, Russia 63Institute for Subatomic Physics, Utrecht University/Nikhef, Utrecht, Netherlands 64Institute for Theoretical and Experimental Physics, Moscow, Russia 65Institute of Experimental Physics, Slovak Academy of Sciences, Košice, Slovakia 66Institute of Physics, Homi Bhabha National Institute, Bhubaneswar, India 67Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 68Institute of Space Science (ISS), Bucharest, Romania 69Institut für Kernphysik, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany 70Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Mexico City, Mexico 71Instituto de Física, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil 72Instituto de Física, Universidad Nacional Autónoma de México, Mexico City, Mexico 73iThemba LABS, National Research Foundation, Somerset West, South Africa 74Johann-Wolfgang-Goethe Universität Frankfurt Institut für Informatik, Fachbereich Informatik und Mathematik, Frankfurt, Germany 75Joint Institute for Nuclear Research (JINR), Dubna, Russia 76Korea Institute of Science and Technology Information, Daejeon, Republic of Korea 77KTO Karatay University, Konya, Turkey 78Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS-IN2P3, Grenoble, France 79Lawrence Berkeley National Laboratory, Berkeley, California, United States 80Lund University Department of Physics, Division of Particle Physics, Lund, Sweden 81Nagasaki Institute of Applied Science, Nagasaki, Japan 82Nara Women’s University (NWU), Nara, Japan 83National and Kapodistrian University of Athens, School of Science, Department of Physics, Athens, Greece 84National Centre for Nuclear Research, Warsaw, Poland 85National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India 86National Nuclear Research Center, Baku, Azerbaijan 54INFN, Sezione di Cagliari, Cagliari, Italy 55INFN, Sezione di Catania, Catania, Italy 56INFN, Sezione di Padova, Padova, Italy 57INFN, Sezione di Roma, Rome, Italy 58INFN, Sezione di Torino, Turin, Italy 85National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India 86National Nuclear Research Center, Baku, Azerbaijan 024001-19 PHYSICAL REVIEW C 99, 024001 (2019) S. (ALICE Collaboration) ACHARYA et al. 87National Research Centre Kurchatov Institute, Moscow, Russia 88Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 89Nikhef, National institute for subatomic physics, Amsterdam, Netherlands 90NRC Kurchatov Institute IHEP, Protvino, Russia 91NRNU Moscow Engineering Physics Institute, Moscow, Russia 92Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, United Kingdom 93Nuclear Physics Institute of the Czech Academy of Sciences, ˇRež u Prahy, Czech Republic 94Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States 95Ohio State University, Columbus, Ohio, United States 96Petersburg Nuclear Physics Institute, Gatchina, Russia 97Physics department, Faculty of science, University of Zagreb, Zagreb, Croatia 98Physics Department, Panjab University, Chandigarh, India 99Physics Department, University of Jammu, Jammu, India 100Physics Department, University of Rajasthan, Jaipur, India 101Physikalisches Institut, Eberhard-Karls-Universität Tübingen, Tübingen, Germany 102Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 103Physik Department, Technische Universität München, Munich, Germany 104Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany 105Rudjer Boškovi´c Institute, Zagreb, Croatia 106Russian Federal Nuclear Center (VNIIEF), Sarov, Russia 107Saha Institute of Nuclear Physics, Homi Bhabha National Institute, Kolkata, India 108School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 109Sección Física, Departamento de Ciencias, Pontiﬁcia Universidad Católica del Perú, Lima, Peru 110Shanghai Institute of Applied Physics, Shanghai, China 111St. Petersburg State University, St. (ALICE Collaboration) Petersburg, Russia 112Stefan Meyer Institut für Subatomare Physik (SMI), Vienna, Austria 113SUBATECH, IMT Atlantique, Université de Nantes, CNRS-IN2P3, Nantes, France 114Suranaree University of Technology, Nakhon Ratchasima, Thailand 115Technical University of Košice, Košice, Slovakia 116Technische Universität München, Excellence Cluster ‘Universe’, Munich, Germany 117The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Cracow, Poland 118The University of Texas at Austin, Austin, Texas, United States 119Universidad Autónoma de Sinaloa, Culiacán, Mexico 120Universidade de São Paulo (USP), São Paulo, Brazil 121Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil 122Universidade Federal do ABC, Santo Andre, Brazil 123University College of Southeast Norway, Tonsberg, Norway 124University of Cape Town, Cape Town, South Africa 125University of Houston, Houston, Texas, United States 126University of Jyväskylä, Jyväskylä, Finland 127University of Liverpool, Liverpool, United Kingdom 128University of Tennessee, Knoxville, Tennessee, United States 129University of the Witwatersrand, Johannesburg, South Africa 130University of Tokyo, Tokyo, Japan 131University of Tsukuba, Tsukuba, Japan 132Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 133Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, Lyon, France 134Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000 Strasbourg, France, Strasbourg, France 135Université Paris-Saclay Centre d’etudes de Saclay (CEA), IRFU, Department de Physique Nucléaire (DPhN), Saclay, France 136Università degli Studi di Foggia, Foggia, Italy 137Università degli Studi di Pavia, Pavia, Italy 138Università di Brescia, Brescia, Italy 139Variable Energy Cyclotron Centre, Homi Bhabha National Institute, Kolkata, India 140Warsaw University of Technology, Warsaw, Poland 141Wayne State University, Detroit, Michigan, United States 142Westfälische Wilhelms-Universität Münster, Institut für Kernphysik, Münster, Germany 87National Research Centre Kurchatov Institute, Moscow, Russia 88Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 89Nikhef, National institute for subatomic physics, Amsterdam, Netherlands 90NRC Kurchatov Institute IHEP, Protvino, Russia 91NRNU Moscow Engineering Physics Institute, Moscow, Russia 92Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, United Kingdom 93Nuclear Physics Institute of the Czech Academy of Sciences, ˇRež u Prahy, Czech Republic 94Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States 95Ohio State University, Columbus, Ohio, United States 96Petersburg Nuclear Physics Institute, Gatchina, Russia 97Physics department, Faculty of science, University of Zagreb, Zagreb, Croatia 98Physics Department, Panjab University, Chandigarh, India 99Physics Department, University of Jammu, Jammu, India 100Physics Department, University of Rajasthan, Jaipur, India 101Physikalisches Institut, Eberhard-Karls-Universität Tübingen, Tübingen, Germany 102Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 103Physik Department, Technische Universität München, Munich, Germany 104Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany 105Rudjer Boškovi´c Institute, Zagreb, Croatia 106Russian Federal Nuclear Center (VNIIEF), Sarov, Russia 107Saha Institute of Nuclear Physics, Homi Bhabha National Institute, Kolkata, India 108School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 109Sección Física, Departamento de Ciencias, Pontiﬁcia Universidad Católica del Perú, Lima, Peru 110Shanghai Institute of Applied Physics, Shanghai, China 111St. 143Wigner Research Centre for Physics, Hungarian Academy of Sciences, Budapest, Hungary 144Yale University, New Haven, Connecticut, United States 145Yonsei University, Seoul, Republic of Korea aDeceased. bDipartimento DET del Politecnico di Torino, Turin, Italy. cM. V. Lomonosov Moscow State University, D. V. Skobeltsyn Institute of Nuclear, Physics, Moscow, Russia. dDepartment of Applied Physics, Aligarh Muslim University, Aligarh, India. eInstitute of Theoretical Physics, University of Wroclaw, Poland. (ALICE Collaboration) Petersburg State University, St. Petersburg, Russia 112Stefan Meyer Institut für Subatomare Physik (SMI), Vienna, Austria 113SUBATECH, IMT Atlantique, Université de Nantes, CNRS-IN2P3, Nantes, France 114Suranaree University of Technology, Nakhon Ratchasima, Thailand 115Technical University of Košice, Košice, Slovakia 116Technische Universität München, Excellence Cluster ‘Universe’, Munich, Germany 117The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Cracow, Poland 118The University of Texas at Austin, Austin, Texas, United States 119Universidad Autónoma de Sinaloa, Culiacán, Mexico 120Universidade de São Paulo (USP), São Paulo, Brazil 121Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil 122Universidade Federal do ABC, Santo Andre, Brazil 123University College of Southeast Norway, Tonsberg, Norway 124University of Cape Town, Cape Town, South Africa 125University of Houston, Houston, Texas, United States 126University of Jyväskylä, Jyväskylä, Finland 127University of Liverpool, Liverpool, United Kingdom 128University of Tennessee, Knoxville, Tennessee, United States 129University of the Witwatersrand, Johannesburg, South Africa 130University of Tokyo, Tokyo, Japan 131University of Tsukuba, Tsukuba, Japan 132Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 133Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, Lyon, France 134Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000 Strasbourg, France, Strasbourg, France 135Université Paris-Saclay Centre d’etudes de Saclay (CEA), IRFU, Department de Physique Nucléaire (DPhN), Saclay, France 136Università degli Studi di Foggia, Foggia, Italy 137Università degli Studi di Pavia, Pavia, Italy 138Università di Brescia, Brescia, Italy 139V i bl E C l t C t H i Bh bh N ti l I tit t K lk t I di 024001-20 PHYSICAL REVIEW C 99, 024001 (2019) PHYSICAL REVIEW C 99, 024001 (2019) p-p, p-, AND - … p-p, p-, AND - … 024001-21
https://openalex.org/W2022466795	https://www.scielo.br/j/rbz/a/8J7mhLn86yFpnST9fWNK3kH/?lang=pt&format=pdf	Portuguese	null	Efeito da técnica de curtimento e do método utilizado para remoção da pele da tilápia-do-nilo sobre as características de resistência do couro	Revista Brasileira de Zootecnia	2,006	cc-by	5,386	Maria Luiza Rodrigues de Souza1, Jorge de Matos Casaca2, Laura Satiko Okada Nakaghi3, Nilson do Prado Franco2, Leocília Oliveira da Silva4, Doroty Mesquita Dourado5, Elisabete Maria Macedo Viegas6 1 Depto de Zootecnia - Universidade Estadual de Maringá. Av. Colombo 5790, CEP: 87020-900, Maringá - PR, Brasil. 2 Pós-graduação em Aqüicultura - CAUNESP - Jaboticabal - SP. 1 Depto de Zootecnia - Universidade Estadual de Maringá. Av. Colombo 5790, CEP: 87020-900, Maringá - PR, Brasil. 2 Pós-graduação em Aqüicultura - CAUNESP - Jaboticabal - SP. 3 Depto de Morfologia e Fisiologia Animal da FCAV/UNESP - Jaboticabal - SP. 4 Biológa. Biológa. 5 Centro de Ciências Biológicas, Agrárias e da Saúde da Universidade para o Desenvolvimento do Estado e da Região do Pantanal (UNIDERP). 6 Depto de Zootecnia/FZEA-USP e CAUNESP/Jaboticabal. Av. Duque de Caxias Norte, 225, CEP: 13635-900, Pirassununga - SP. Apoio: Secretaria Especial de Aqüicultura e Pesca da Presidência da República. RESUMO - Um estudo foi realizado com o objetivo de avaliar os efeitos do método de remoção da pele do filé (com alicate e com máquina) e da técnica de curtimento (com ou sem sais de cromo, bioleather) sobre a resistência da pele de tilápia-do- nilo (Oreochromis niloticus). Foram retiradas as peles de 40 tilápias (450 g), 20 com uso de alicates e 20 com auxílio de máquina. Após o curtimento e a retirada dos corpos-de-prova, as peles foram encaminhadas a um laboratório aclimatizado (23ºC e 50% de umidade relativa do ar) para realização dos testes de resistência em dinamômetro EMIC, com velocidade de afastamento entre cargas de 100 ± 20 mm/mm. O método de retirada da pele do filé não afetou os resultados dos testes de tração e rasgamento progressivo. A pele retirada com a máquina apresentou elongação superior (88,48%) à removida com alicate (71,09%). Peles curtidas com sais de cromo (25,54 N/mm; 12,18 N/mm2) foram mais resistentes que as curtidas sem sais de cromo (bioleather) (13,11 N/mm; 9,89 N/mm2). Peles curtidas por estas técnicas (com cromo e bioleather) podem ser utilizadas na confecção de vestuários, pois as médias dos testes de tração, alongamento e rasgamento progressivo foram superiores a 9,80 N/mm2, 60% e 14,72 N/mm, respectivamente, valores mínimos recomendados para couros curtidos ao cromo. Palavras-chave: pele ecológica, qualidade da pele, retirada da pele do filé, testes físicos-mecânicos Effect of tanning technique and methods used for skin removal on leather resistance characteristics of Nile Tilapia ABSTRACT - This research evaluated the effect of different methods (fillet skin removal and tanning methods [with or without chromium salts and bioleather]) on leather resistance characteristics of Nile Tilapia (Oreochromis niloticus). Skin from 40 tilapias (450 g) were removed: 20 by pliers and 20 by machine. After skin tanning, the samples were removed for resistance tests, which were done, in a climatized environment (23ºC) and 50% air relative humidity, with EMIC dynamometer, at removal speed of 100 ± 20 mm/mm. No effect of fillet skin removal method on progressive tearing and strength was observed. The method of skin removal by machine resulted in greater (88.48%) elongation than that removed by pliers (71.09%). Skin tanned with chromium salts (25.54 N/mm; 12.18N/mm2) had higher resistance than that tanned without chromium (bioleather) (13.11 N/mm; 9.89 N/mm2). Chromium- and bioleather tanned skins may be used in clothes manufacture since average values of strength, elongation and progresive tearing are higher than 9.80N/mm², 60% and 14.72 N/mm, respectively, which are values recommended for leathers tanned with chromium salts. Key Words: bioleather skin, fillet skin removal, physical and mechanical tests, skin quality, skin resistance CCorrespondências devem ser enviadas para: mlrsouza@uem.br Revista Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia ISSN impresso: 1516-3598 ISSN on-line: 1806-9290 www.sbz.org.br Revista Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia ISSN impresso: 1516-3598 ISSN on-line: 1806-9290 www.sbz.org.br R. Bras. Zootec., v.35, n.4, p.1273-1280, 2006 Material e Métodos veis seria a adequação na linha de filetagem, utilizando-se equipamentos regulados, para alta produção em curto prazo, como ocorre quando a retirada da pele dos filés é realizada com máquina apropriada. Os ensaios foram realizados no Laboratório de Curti- mento da Universidade Estadual de Maringá (Maringá –PR) e os testes físico-químicos, na BASF, Empresa Multinacional produtora e fornecedora de insumos químicos para a indús- tria de curtimento. A pele é um subproduto que pode ter elevado valor econômico com o processo de curtimento (Souza, 2004). Porém, é importante que esse processo seja realizado de forma a não agredir o meio ambiente. Atualmente, tem-se valorizado a produção do “couro ecológico”, buscando alternativas para processamento da pele com produtos mais naturais, direcionando a atenção dos produtores para um futuro em que se possa dispensar o uso de sais de cromo. Portanto, de acordo com o CATC – BASF (1999), deve-se dar atenção especial ao “wet-white” a partir do uso de dialdeído glutárico, ponto de partida mais generalizado e aprovado para os “couros ecológicos” ou o “couro bioleather”. Quarenta tilápias-do-nilo de aproximadamente 450 g foram abatidas por choque térmico (água e gelo na propor- ção 1:1) para retirada da pele e do filé. Em 20 tilápias, primeiramente retirou-se a pele (com auxílio de alicate) e, em seguida, o filé (M1). Nas demais, retirou-se o filé e, em seguida, a pele, utilizando-se máquina apropriada (M2). As peles foram identificadas individualmente com miçangas (fixadas com linha mercê nº 40) e distribuídas aleatoriamente em dois fulões (equipamento de formato cilíndrico, apresen- tando nas paredes internas travessas ou tornos destinados a favorecer a movimentação da carga), com velocidade de rotação 16 rpm/min. Em um fulão, foram colocadas 20 peles retiradas com uso de alicate e 20 retiradas com máquina, para curtimento pela técnica com sais de cromo. No outro fulão, distribuíram-se as outras 20 peles retiradas com alicate e as 20 extraídas com máquina, para curtimento pela técnica sem sais de cromo ou bioleather (à base de sulfona e ácidos sulfônicos aromáticos, composta por taninos fenólicos, aldeído glutárico e cloreto básico de alumínio). Nas Figuras 1A e B são descritas as duas técnicas de curtimento. O impacto ambiental é um fator muito discutido em qualquer área da produção, tanto na aqüicultura, agricultura e pecuária quanto na atividade industrial, como os curtumes. Material e Métodos Buscam-se, portanto, alternativas tecnológicas para subs- tituição do cromo por outros agentes de curtimento (curtentes vegetais, sais minerais alternativos, compostos orgânicos reativos e substituição parcial do cromo pela obtenção de wet-white), bem como a otimização no processo de curtimento ao cromo, utilizando-se métodos de auto- esgotamento de cromo, de sistemas de re-ciclos e outros (Gutterres, 1997). Após o curtimento das peles, foram retirados dez corpos- de-prova no sentido longitudinal ao comprimento do corpo do peixe, para determinação da resistência à tração, ao alongamento (ABNT – NBR 11041, 1997) e ao rasgamento progressivo (ABNT – NBR 11055, 1997) (Figura 2). Como não existem normas específicas para realização dos testes de resistência dos couros de peixes, foram estabelecidos determinados parâmetros para retirada dos corpos-de-prova, incluindo local, posição e número de amostragens. As atuais manifestações ecológicas estimularam novos segmentos de mercado a aderirem ao “couro ecoló- gico”, muito mais importante para uso em artefatos que não exigem determinadas resistências físicas, como estabilida- de em temperaturas de 100oC ou mais (Souza, 2004). Na confecção de estofamento, não existe esse problema e couros “livres de cromo”, elaborados a partir de “wet- white”, são consumidos em larga escala pela indústria automobilística, especialmente os Dialdeído Glutárico (CATC – BASF, 1999). Os corpos-de-prova foram retirados do couro (ABNT – NBR 11035, 1990) com uso de um balancim. Em seguida, foram encaminhados a um laboratório climatizado (23ºC e 50% de umidade relativa do ar) por 24 horas, conforme ABNT (NBR 10455, 1988), e determinadas as espessuras de cada amostra (ABNT – NBR 11062, 1997). Para os testes de resistência, foi utilizado dinamômetro EMIC com velocidade de afastamento entre cargas de 100 ± 20 mm/mm. Graffunder & Kolling (2002) citam resinas recurtentes como um avanço técnico e ecológico para proporcionar tecnologia limpa no curtimento de couros. Os compostos recurtentes apresentam elevada solidez à luz e ao calor e promovem enchimento ao couro sem prejuízo à maciez, além de melhora significativa na resistência ao rasgamento progressivo. Neste sentido, este estudo foi realizado com o objetivo de avaliar o efeito do método de retirada da pele do filé e da técnica de curtimento aplicada (com ou sem sais de cromo -bioleather) sobre a resistência e a qualidade do couro da tilápia-do-nilo (Oreochromis niloticus). Introdução mento das peles pode ser uma forma de agregar valor a um resíduo que, de outra forma, seria descartado. A unidade curtida hoje atinge R$1,00 a R$2,50 conforme o tamanho e a forma de acabamento. Com a intensificação da piscicultura e com a tendência de algumas empresas brasileiras exportarem o filé de peixe sem pele, as peles de peixes são descartadas ou direcionadas, junto com os demais resíduos, à produção de farinha ou silagem de peixe. Todavia, o processo de curti- Com essa ampliação na comercialização, alternativas para maior produção na linha de processamento do peixe tornam-se extremamente importantes. Uma das formas viá- 1274 Souza et al. Material e Métodos A preparação de amostras de couro para análise química foi realizada de acordo com as condições exigidas pelas normas da ABNT (NBR 11034, 2001), para determinação do óxido de cromo Cr2O3 (ABNT - NBR 11054, 1999), das substâncias extraíveis com diclorometano (CH2Cl2) (ABNT © 2006 Sociedade Brasileira de Zootecnia curtimento e do método utilizado para remoção da pele da tilápia-do-nilo sobre as características de... 1275 A. Sais de cromo % Produtos Tempo (min) Obs: pH Remolho 200 Água 25ºC 0,4 Tensoativo1 20 Esgotar/lavar 15’ Caleiro 200 Água 25ºC 2,5 Sulfeto de sódio 2,5 Cal hidratada 0,3 Tensoativo1 120 Esgotar Desencalagem 100 Água 25ºC 2 Sulfato de amônio 20 Esgotar 9 Purga 100 Água 25ºC 0,05 Enzima pancreática2 60 1,5 Sulfato de amônio 20 Esgotar e lavar 100 Água 25ºC 0,05 Enzima pancreática2 60 0,2 Desencalante3 – 2 Sulfato de amônio 20 0,3 Tensoativo1 20 Esgotar e lavar Píquel e curtimento 100 Água 25ºC 14 Sal grosso 15 8ºBé 0,9 Ácido sulfúrico 3x15-120 3,5 8 Sais de cromo 50 2 Bicarbonato de sódio 3x15 3,9 Pernoitar Neutralização 100 Água 25ºC 0,7 Neutralizante8 10 0,5 Bicarbonato de sódio 3x15 0,7 Formiato de sódio 30 5,7 Esgotar/lavar Recurtimento, tingimento e engraxe 50 Água 25ºC 3 Hidrofugante10 1 Hidrofugante11 0,1 Hidrofugante12 40 1,5 Tanino de acácia (mimosa) Conc. 95% 1,5 Taninos de quebracho não-adstringente. Conc. 95% 1,5 Curtente8 30 3 Corante vermelho 60 200 Água 60ºC 2 Ácido fórmico 2x15 Esgotar 3,4 Engraxe 200 Água 60ºC 3 Hidrofugante10 2x15 Esgotar 3,5 1 Hidrofugante11 60 1 Ácido fórmico 20 1,5 Sais de cromo 30 1 Ácido fórmico 20 Esgotar 50 Água 30ºC 1 Sais de cromo 30 2 Sais de cromo 60 Esgotar/lavar - Bioleather (B). B. Figura 1 - Formulação com sais de cromo (A) e sem sais de cromo - Bioleather (B). Material e Métodos Figure 2 - Leather samples removal of Nile Tilapia (Oreochromis niloticus) hide longitudinal direction to the body length. Longitudinal - referente ao comprimento do corpo do peixe Corpo-de-prova para determinação da Corpo-de-prova para determinação da resistência à Corpo-de-prova para determinação da resistência ao rasgamento Sample for tests of tearing strenght Corpo-de-prova para determinação da resistência à tração e ao alongamento Sample for tests of tensile strenght and elogation Longitudinal - referente ao comprimento do corpo do peixe Longitudinal - in relation to the fish body length Longitudinal - referente ao comprimento d corpo do peixe Longitudinal - referente ao comprimento do corpo do peixe Longitudinal - in relation to the fish body length Corpo-de-prova para determinação da Corpo-de-prova para determinação da resistência ao rasgamento Sample for tests of tearing strenght Corpo-de-prova para determinação da Corpo-de-prova para determinação da resistência ao rasgamento Sample for tests of tearing strenght Corpo-de-prova para determinação da Corpo-de-prova para determinação da resistência ao rasgamento Sample for tests of tearing strenght Figura 2 - Retirada dos corpos-de-prova do couro de tilápia-do-nilo (Oreochromis niloticus) no sentido longitudinal ao comprimento do corpo do peixe. Figure 2 - Leather samples removal of Nile Tilapia (Oreochromis niloticus) hide longitudinal direction to the body length. - NBR 11030, 1997), do pH e da cifra diferencial do pH de um extrato aquoso (ABNT- NBR 11057, 1999). sidade de mais mão-de-obra para a execução do descarne (remoção do músculo da pele). Souza et al. (1999) analisaram a influência do método de filetagem e das categorias de peso sobre o rendimento de carcaça, filé e pele de tilápia-do-nilo e constataram que, em peixes de 250 a 450 g, quando a pele foi retirada com alicate (5,32%), a porcentagem de pele foi menor que quando retirada com faca (8,51%). Os autores afirmaram que a porcentagem de descarne foi maior para as peles retiradas com faca. Portanto, na pele retirada com alicate, a porcentagem de pele é sempre menor que naquelas retiradas com faca ou máquina, visto que menor quantidade de músculos permanece na pele. Portanto, quanto menor a proporção de músculo na pele após retirada do filé mais rapidamente será descarnada, necessitando de menos mão- de-obra para essa operação. Material e Métodos Sem sais de cromo - Bioleather % Produtos Tempo (min) Obs: pH Remolho 200 Água 25ºC 0,4 Tensoativo1 20 Esgotar/lavar 15’ Caleiro 200 Água 25ºC 2,5 Sulfeto de sódio 2,5 Cal hidratada 0,3 Tensoativo1 120 Esgotar Desencalagem 100 Água 25ºC 2 Sulfato de amônio 20 Esgotar 9 Purga 100 Água 25ºC 0,05 Enzima pancreática2 60 1,5 Sulfato de amônio 20 Esgotar/lavar 100 Água 25º 0,05 Enzima pancreática2 60 0,2 Desencalante3 – 2 Sulfato de amônio 20 0,3 20 Esgotar/lavar Píquel e curtimento 100 Água 25ºC 14 Sal grosso 15 8ºBé 0,45 Ácido sulfúrico 3x15 1,7 Ácido fórmico 3x15 120 3,6 0,5 Óleo4 4 Curtente5 50 3,7 2,5 Neutralizante6 5 1 Curtente7 50 1 Formiato de sódio 60 4,2 1,5 Curtente8 60 Esgotar Neutralização 150 Água 30ºC 0,7 Neutralizante8 10 0,5 Bicarbonato de sódio2x15+30 Esgotar/lavar 6,9 Recurtimento, tingimento e engraxe 50 Água 30º 3 Hidrofugante10 1 Hidrofugante11 0,1 Hidrofugante12 30 4 Curtente8 20 4 Curtente8 20 1 Tensoativo - compostos orgânicos surfactantes iônicos e não-iônicos. Concentração 82%; 2 Enzima pancreática com 10.000 UFG. 3 Desencalante - mistura ac. Orgânicos e inorgânicos isentos de nitrogênio. 4 Óleo - mistura de óleo sintético e natural (lecitina) predominando óleo sintético. Concen- tração 70%. 5 Curtente - aldeído glutárico + polímero acrílico. Concentração 95%. 6 Neutralizante à base de curtente sintético e ácidos orgânicos. Concentração 95%. 7 Curtente produto de condensação à base de sulfona e ácidos sulfônicos aromáticos. Concentração 40%, pH 4 (1:10). 8 Curtente de substituição, à base de condensação de fenol (fenólico ). Concentração 9 % pH 3,3 (1:10). 9 Neutralizante à base de sulfitos, formiato e ftalato de sódio. 10 Hidrofugante - combinação de graxa sintética hidrofugante e emulgadores especiais. Concentração 45% pH 8,5 (1:10). 11 Hidrofugante - engraxante sintético contendo silicone dotado de um sistema emulsionante especial. Concentração 50% pH 8 (1:10). 12 Hidrofugante - solução polimérica aquosa aniônica isenta de AOX. Concentração 40%. pH 7,5 (1:10). 13 Curtente à base de alumínio – cloreto básico de alumínio. Concentração de Al2O3 17% basicidade 66% pH 3,5 (1:10). B. Sem sais de cromo - Bioleather Figura 1 - Formulação com sais de cromo (A) e sem sais de cromo - Bioleather (B). © 2006 Sociedade Brasileira de Zootecnia 1276 Souza et al. Figura 2 - Retirada dos corpos-de-prova do couro de tilápia-do-nilo (Oreochromis niloticus) no sentido longitudinal ao comprimento do corpo do peixe. Material e Métodos O delineamento experimental adotado foi o inteira- mente casualizado, em esquema fatorial 2 x 2, dois métodos de retirada da pele do filé (com alicate e com a máquina) e duas técnicas de curtimento (com e sem sais de cromo - bioleather), considerando o couro a unidade experimental. Os resultados dos testes físico-mecânicos foram subme- tidos à análise de variância, comparando-se as médias pelo teste Tukey a 5% de probabilidade (Banzatto & Kronka, 1995). Os resultados dos testes físico-químicos foram utiliza- dos apenas para caracterização do produto final (couro). © 2006 Sociedade Brasileira de Zootecnia Resultados e Discussão O método de retirada da pele (alicate ou máquina) influenciou a porcentagem de pele, calculada em relação ao peso do peixe inteiro. Quando retirada com alicate, a por- centagem de pele de tilápia-do-nilo foi significativamente (P<0,01) inferior (6,54 vs 7,64% - com máquina). Não houve efeito significativo (P>0,05) do método de retirada da pele do filé sobre a resistência do couro à tração (alicate = 11,55 N/mm2; máquina = 10,52 N/mm2) e ao rasgamento progressivo (alicate = 20,53 N/mm; máquina = 18,11 N/mm). Todavia, a remoção da pele do filé com uso de alicate influenciou os resultados do teste de alongamento do couro. A diferença quanto ao método de retirada da pele do filé demonstra que maior quantidade de musculatura do filé ficou retida na pele quando se fez a retirada com máquina, o que dificulta o processo de curtimento, por haver neces- As peles retiradas com alicate apresentaram, após cur- timento, menor flexibilidade ao alongamento, que foi signifi- Efeito da técnica de curtimento e do método utilizado para remoção da pele da tilápia-do-nilo sobre as características de... 1277 foram utilizados os óleos hidrofugantes, todavia, o couro bioleather (sem sais de coromo) foi menos resistente aos testes de rasgamento, tração e alongamento (Tabela 1). Certamente, mesmo com essa proteção dos óleos hidrofugantes sobre as fibras colágenas das peles subme- tidas às duas técnicas de curtimento, houve ação de outros fatores, como os tipos de agentes curtentes utilizados na etapa de curtimento ou recurtimento, a combinação de produtos químicos, a concentração e o tempo de cada etapa do processo. cativamente superior para os couros retirados com máquina (alicate = 71,09%; máquina = 88,48%). Talvez a força empre- gada para remoção da pele tenha proporcionado maior estiramento das fibras colágenas, que, após o elevado estiramento, não apresentavam a mesma elasticidade de uma pele que ainda não tenha sido esticada. Os couros curtidos com sais de cromo (12,18 N/mm2, 93,08% e 25,54 N/mm) apresentaram resultados significa- tivamente superiores aos curtidos sem sais de cromo (9,89 N/mm2, 66,52% e 13,11 N/mm2, respectivamente para os testes de tração, alongamento e rasgamento progressivo) (Tabela 1). A carga de força aplicada até ruptura no teste de tração não apresentou diferença significativa entre os couros quando analisado o efeito dos métodos de retirada da pele e dos tipos de curtimento (com e sem sais de cromo) (Tabela 1). © 2006 Sociedade Brasileira de Zootecnia Resultados e Discussão No curtimento com sais de cromo, foram utilizados os óleos hidrofugantes da BASF, que proporcionam especial característica ao couro, aumentando a tensão superficial limite entre as fibras colágenas do couro e a água, pela envoltura das fibras com uma película hidrofugada (CATC – BASF, 1999), possibilitando aumento na resistência do couro. Nas peles curtidas sem sais de cromo, também De acordo com Souza et al. (2002), as peles de carpa espelho curtidas sem sais de cromo apresentaram resistên- cia à tração de 18,75 N/mm2, enquanto, nesse experimento, a resistência medida nas peles de tilápia foi de 9,89 N/mm2. Tabela 1 - Resultados do teste de resistência do couro de tilápia-do-nilo, de acordo com o método de retirada da pele do filé (alicate ou máquina) e com a técnica de curtimento (com ou sem sais de cromo) Table 1 - Mean values of resistance test of Nile tilapia leather in function of the methods used in fillet removal (pliers and machine) and in tanning technique (chromium salts and bioleather) Fonte de variação Tração e alongamento Rasgamento progressivo Source of variation Traction and elongation Progressive tearing Carga de ruptura (N) Tração (N/mm2) Alongamento (%) Carga de força (N) Rasgo (N/mm) Força máxima (N) Rupture load Traction Elongation Force load Tearing Maximum force Método de retirada da pele do filé Methods of skin fillet removal Alicate 70,33 a(1) 11,55a 71,09b 14,92a 20,53a 39,22a Plies Máquina 70,54a 10,52a 88,48a 12,20a 18,11a 30,37b Machine Técnica de curtimento Tanning technique Com cromo 80,33a 12,18a 93,08a 17,60a 25,54a 44,70a Chromium Sem cromo 60,94a 9,89b 66,52b 9,51b 13,11b 24,89b Without chromiun Teste F F test Retirada da pele 0,002ns 2,27ns 32,97** 6,62* 3,37ns 9,23 Removal skin Técnica de curtimento 25,28 11,13 76,80 8,43 88,94 46,24 Tanning technique Interação método de retirada 0,17ns 1,01ns 1,97ns 1,82ns 2,03ns 1,94ns × técnica de curtimento Removal skin × tanning technique interaction CV (%) 17,27 19,71 11,22 24,69 21,42 26,47 1 Em cada coluna, valores seguidos de mesma letra não diferem pelo teste Tukey a 5% de probabilidade. 1 Values followed by the same letter in a column do not differ by Tukey test at a 5% probability. ns - não-significativo (P>0,05); significativo (P<0,01). ns - not significant (P>0.05); **significant (P<0.01). 1278 Souza et al. Quanto ao teste de alongamento, o valor observado foi de 73,38%, superior ao obtido para a tilápia (66,52%), pela mesma técnica de curtimento, no sentido longitudinal do corpo-de-prova. Resultados e Discussão A resistência ao rasgamento progressivo para o couro da tilápia-do-nilo com curtimento bioleather foi de 13,11 N/mm, superior à relatada por Souza et al. (2002) para pele de carpa espelho (12,06 N/mm) curtida sem sais de cromo. A técnica sem sais de cromo utilizada neste experi- mento foi a mesma aplicada por Souza et al. (2002). Todavia, os resultados de resistência obtidos neste estudo, de 9,89 N/mm2 para resistência à tração e 66,52% para resistên- cia ao alongamento, foram diferentes dos relatados por Souza et al. (2002), de 18,75 N/mm2 e 73,38%, respectiva- mente, curtidos pela técnica sem sais de cromo. Esta dife- rença pode ser atribuída à espécie de peixe, que difere quanto à distribuição e orientação das fibras colágenas. dos por Hoinacki, 1989). Todavia, as peles curtidas sem sais de cromo apresentaram valor inferior ao mínimo exigido apenas no teste do rasgamento progressivo (13,11 N/mm). Portanto, quanto à resistência ao rasgamento progressivo são consideradas inadequadas para o uso na confecção de vestuário. Esse valor, no entanto, foi obtido no sentido longitudinal do couro e provavelmente seria diferente no sentido transversal, talvez superior, com média para ser utilizado na confecção de vestuário. De acordo com Souza (2003), em pele de tilápia-do-nilo (de 500 a 800 g) submetida a sete diferentes técnicas de curtimento, os valores encontrados no teste de rasgamento progressivo variaram de 9,44 a 24,12 N/mm, conforme a técnica de cur- timento aplicada. Esse autor avaliou os couros nos sentidos longitudinal e transversal ao comprimento do peixe. Os couros analisados para essa mesma variável apresentaram resis- tência significativamente menor no sentido longitudinal (14,76 N/mm) em comparação ao transversal (17,86 N/mm). Talvez, pela média (sentido longitudinal e transversal) no teste de rasgamento progressivo obtida em peles de tilápia curtidas sem sais de cromo, esse couro também atenda às condições exigidas para confecção de vestuários (compara- dos ao couro bovino curtido com sais de cromo). A carga de força e a força máxima aplicada na determi- nação da resistência ao rasgamento progressivo foi supe- rior (P<0,01) nos couros curtidos com sais de cromo (17,60 e 44,70 N) quando comparados àqueles curtidos sem sais de cromo (couro bioleather) (9,51 e 24,89 N, respectivamente, para a carga e força máxima aplicada no teste aplicada, a qual foi maior para as peles retiradas com alicate – 39,22 N). Resultados e Discussão A pele retirada com máquina, por apresentar maior elasticida- de (88,48%), necessitou de menor intensidade de força para o rasgamento progressivo. Todavia, isso também pode estar diretamente relacionando à espécie do peixe. Em trabalho semelhante, desenvolvido com a carpa espelho, não houve efeito do método de retirada da pele sobre a força máxima para rasgamento progressivo (Souza et al., 2002). A força máxima aplicada para a pele de carpa espelho retirada com máquina foi de 27,89 N, enquanto para aquela retirada com alicate foi de 24,93 N. Como não foram utilizados sais de cromo no curtimento do couro bioleather, não foi determinado o teor de óxido de cromo. A quantidade de óxido de cromo nos couros curti- dos com sais de cromo foi de 3,78% (Tabela 2). O teor de óxido de cromo obtido na pele da tilápia-do- nilo está relacionado à proporção deste curtente fixado às fibras colágenas. Segundo Hoinacki (1989), o valor mínimo de óxido de cromo no couro semi-acabado deve ser de 3,0%. A análise quantitativa de óxido de cromo do couro indicou capacidade para suportar elevadas temperaturas, sem retrair-se, fato comprovado pelo teste de fervura ou retração realizado durante o processo de curtimento. Portanto, a espécie de peixe está também diretamente relacionada aos resultados de resistência, provavelmente em relação à estrutura histológica da pele, pois as espessu- ras dos couros das duas espécies são próximas. Segundo Souza et al. (2002), a espessura da pele da carpa espelho variou de 0,70 a 0,76 mm, enquanto a da tilápia, observada neste experimento, foi de 0,67 a 0,76 mm. A quantidade de substâncias extraíveis em diclorometano foi de 19,42% pela técnica com sais de cromo e de 20,02% pela técnica sem sais de cromo (bioleather). Entretanto, segundo Vademécum (1995), as substâncias extraíveis em diclorometano devem ser de, no máximo, 16 a 18% para que o couro possa ser utilizado em vestuário. Souza et al. (2002) observaram, em peles de carpa espelho curtidas sem sais de cromo, valores de substâncias extraíveis em diclorometano de 13,78 a 15,76%, inferiores aos encontrados neste experimento para os couros de tilápia-do-nilo. © 2006 Sociedade Brasileira de Zootecnia Resultados e Discussão Os couros curtidos sem sais de cromo, ou bioleather, apresentaram resistência à tração e ao alongamento supe- rior à exigida para couro de bovinos curtidos com sais de cromo, destinados à confecção de vestuários, que são, no mínimo de 9,80 N/mm2, 60% e 14,72 N/mm, para resistência à tração, ao alongamento até a ruptura e ao rasgamento progressivo, respectivamente (Niveles de Calidad Aceptables en la Indústria del Cuero de Organización de las Naciones Unidas para el Desarrollo Industrial, 1976, relata- O pH das peles curtidas foi de 2,9 e 3,9, enquanto os valores de cifra diferencial das duas técnicas foram 0,7 e 1,0, respectivamente, para peles curtidas com e sem sais de © 2006 Sociedade Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia de curtimento e do método utilizado para remoção da pele da tilápia-do-nilo sobre as características de... 1279 1279 Efeito da técnica de curtimento e do método utilizado para remoção da pele da tilápia-do-nilo sobre as características de... Tabela 2 - Teor de óxido de cromo, graxa, pH e cifra diferencial das amostras de couro de tilápia-do-nilo curtido com e sem sais de cromo (Bioleather) Table 2 - Mean contents of chromium oxide pH and diferential cifra of Nile tilapia leather samples tanned with and without chromium salt (Bioleather) Análise físico-química Técnica de curtimento Physical and chemical analysis Tanning technique Cromo Bioleather Chromium Bioleather Determinação do óxido de cromo (Cr2O3) 3,78% - Determination of chromium oxide (Cr2O3) Determinação de substâncias extraíveis em diclorometano 19,42% 20,02% Determination of extractive substances in diclorometan Determinação do pH e da cifra diferencial em extrato aquoso pH 2,9 3,9 Determination of pH and diferencial cifra in water extract Cifra 0,70 1,0 omo, graxa, pH e cifra diferencial das amostras de couro de tilápia-do-nilo curtido com e sem sais de cromo mium oxide pH and diferential cifra of Nile tilapia leather samples tanned with and without chromium salt (Bioleather) As peles curtidas com a técnica bioleather apresen- taram resistência à tração e ao alongamento suficiente para utilização na confecção de vestuários em geral. cromo (Tabela 2). De acordo com Hoinacki (1989), o valor mínimo de pH no couro semi-acabado deve ser de 3,5 e a cifra diferencial de, no máximo, 0,7. Para os couros de carpa espelho curtidos pela mesma técnica sem sais de cromo, Souza et al. Agradecimento À Secretaria Especial da Aqüicultura e Pesca da Presi- dência da República, pelo apoio na obtenção dos equipa- mentos do laboratório de curtimento. A José Telmo Agostini Schmidt, da Leather Solutions, e a Antonio Manuel Camim, pelo auxílio na execução do trabalho. À Epagri, à Empresa Cardume e à BASF, pelo inestimável apoio. Considerando o pH do couro curtido ao cromo (2,9), pode-se inferir que esse couro pode enfraquecer com o tempo de estocagem e uso. Portanto, a formulação utilizada deve ser modificada, reduzindo-se a quantidade de ácido aplicado no momento da fixação dos corantes e óleos de engraxe. A porcentagem de ácido na formulação do tingimento ao engraxe foi de 4% em relação ao peso das peles, o que deveria ser de aproximadamente 2,5% ou, no máximo, 3,0% pela técnica com sais de cromo, conforme observado na formulação 1. Literatura Citada ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 10455: climatização de materiais usados na fabricação de calçados e correlatos. Rio de Janeiro: 1988. p.1-2. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 11030: couro - determinação de substâncias extraíveis com diclorometano (CH2Cl2). Rio de Janeiro: 1997. p.1-3. É As análises feitas nos couros de tilápia neste experi- mento foram sempre no sentido longitudinal, embora os valores relatados por alguns autores para o rasgamento progressivo sejam inferiores aos observados no sentido transversal. Talvez para análise mais apurada da resistên- cia das técnicas de curtimento aplicadas, os testes de resistência deveriam ter sido realizados também no sentido transversal, possibilitando o valor médio entre ambos os sentidos (longitudinal e transversal). ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 11034: couro - preparação de amostras de couro para análise química. Rio de Janeiro, 2001. p.1-2. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 11035: corte de corpos-de-prova em couro. Rio de Janeiro: 1990. p.1. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 11041: couros – determinação da resistência à tração e alongamento. Rio de Janeiro: 1997. p.1-5. É ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 11054: couros – determinação de óxido crômico (Cr2O3). Rio de Janeiro: 1999. p.1-5. Resultados e Discussão (2002) encontraram valores de pH de 3,9 e cifra diferencial de 0,7, ou seja, melhores que os obtidos neste experimento em peles de tilápia (pH 3,9 e cifra diferencial 1,0). SOUZA, M.L.R; CASACA, J.M.; FERREIRA, I.C. et al. Análise da resistência do couro da carpa espelho curtido sem utilização de sais de cromo. Tecnicouro, v.23, n.8, p.57-69, 2002. VADEMÉCUM para el técnico em curtición. 3.ed.rev. Ludwigshanfen: Basf, 1995. 477p. © 2006 Sociedade Brasileira de Zootecnia Conclusões ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 11055: couro - determinação da força de rasgamento progressivo. Rio de Janeiro: 1997. p.1-4. A técnica de retirada da pele do peixe (com alicate ou máquina) não interferiu nas características de resistência do couro, exceto quanto à flexibilidade (alongamento), que foi maior para as peles retiradas mecanicamente (máquina). ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 11057: couro - determinação do pH e da cifra diferencial. Rio de Janeiro: 1999. p.1-3. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS - ABNT. NBR 11062: determinação da espessura. Rio de Janeiro, 1997. p.1. A resistência ao rasgamento progressivo, à tração e ao alongamento foi superior nos couros curtidos com sais de cromo. BANZATTO, D.A.; KRONKA, S.N. Experimentação agrícola. 3.ed. Jaboticabal: FUNEP, 1995. 247p. CATC - BASF – Wet como ponto de partida para “couro ecológico”. Revista do couro, n.237, p.32, 1999. © 2006 Sociedade Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia Souza et al. 1280 SOUZA, M.L.R; CASACA, J.M.; FERREIRA, I.C. et al. Análise da resistência do couro da carpa espelho curtido sem utilização de i d T i 23 8 57 69 2002 GRAFFUNDER, V.; KOLLING, C.A. Resinas recurtentes: evolução e ecologia. Revista do Couro, n.154, p.34-38, 2002. GUTTERRES, M. Considerações sobre curtimento ao cromo e meio ambiente. Revista do couro, n.122, p.28-29, 1997. sais de cromo. Tecnicouro, v.23, n.8, p.57-69, 2002. VADEMÉCUM para el técnico em curtición. 3.ed.rev. Ludwigshanfen: Basf, 1995. 477p. HOINACKI, E. Peles e couros: origens, defeitos, e industrialização. 2.ed.rev. Porto Alegre: Henrique d‘Ávila Bertaso, 1989. 319p. SOUZA, M.L.R. Processamento do filé e da pele da tilápia do Nilo (Oreochromis niloticus): aspectos tecnológicos, composição centesimal, rendimento, vida útil do filé defumado e testes de resistência da pele curtida. Jaboticabal: Universidade Estadual Paulista, 2003. 169p. Tese (Doutorado em Aqüicultura) - Universidade Estadual Paulista, 2003. SOUZA, M.L.R. Tecnologia para processamento das peles de peixe. Maringá: Eduem, 2004. 59p. (Coleção Fundamentum, 11). SOUZA, M.L.R; MACEDO-VIEGAS, E.M.; KRONKA, S.N. Influência do método de filetagem e categorias de peso sobre rendimento de carcaça, filé e pele da tilápia do Nilo (Oreochromis niloticus). Revista Brasileira de Zootecnia, v.28, n.1, p.1-6, 1999. Recebido: 17/02/05 Aprovado: 30/01/06 © 2006 Sociedade Brasileira de Zootecnia
https://openalex.org/W2083133298	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0037944&type=printable	English	null	Dufulin Activates HrBP1 to Produce Antiviral Responses in Tobacco	PloS one	2,012	cc-by	11,659	Abstract Background: Dufulin is a new antiviral agent that is highly effective against plant viruses and acts by activating systemic acquired resistance (SAR) in plants. In recent years, it has been used widely to prevent and control tobacco and rice viral diseases in China. However, its targets and mechanism of action are still poorly understood. Methodology/Principal Findings: Here, differential in-gel electrophoresis (DIGE) and classical two-dimensional electrophoresis (2-DE) techniques were combined with mass spectrometry (MS) to identify the target of Dufulin. More than 40 proteins were found to be differentially expressed ($1.5 fold or #1.5 fold) upon Dufulin treatment in Nicotiana tabacum K326. Based on annotations in the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, these proteins were found to be related to disease resistance. Directed acyclic graph (DAG) analysis of the various pathways demonstrated harpin binding protein-1 (HrBP1) as the target of action of Dufulin. Additionally, western blotting, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and real time PCR analyses were also conducted to identify the specific mechanism of action of Dufulin. Our results show that activation of HrBP1 triggers the salicylic acid (SA) signaling pathway and thereby produces antiviral responses in the plant host. A protective assay based on lesion counting further confirmed the antiviral activity of Dufulin. Conclusion: This study identified HrBP1 as a target protein of Dufulin and that Dufulin can activate the SA signaling pathway to induce host plants to generate antiviral responses. eng M, Song B, Hou C, Hu D, et al. (2012) Dufulin Activates HrBP1 to Produce Antiviral Responses in Tobacco. PLoS ONE 7(5): e37 one.0037944 Editor: Anand S. Mehta, Drexel University College of Medicine, United States of America Editor: Anand S. Mehta, Drexel University College of Medicine, United States of America Received January 4, 2012; Accepted April 26, 2012; Published May 25, 2012 Copyright: 2012 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by the following funding sources: The National Basic Research Program of China (Nos. 2010CB126105) URL: http://www. 973.gov.cn, the National Basic Research Preliminary Program of China (Grant No. 2010CB134504) URL: http://www.973.gov.cn and the National Natural Science Foundation of China (Nos. 21132003, 20872021) URL: http://www.nsfc.gov.cn. Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: CH is employed by Shanghai Applied Protein Technology Co. Ltd. There are no patents, products in developm to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. CH is employed by Shanghai Applied Protein Technology Co. Ltd. There are no patents, products in development or marketed products alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: basong@gzu.edu.cn (BS); fcc.syang@gzu.edu.cn (SY) related genes 1(NPR1)/unknown effective protein pathway, the EDS/SA/NPR1/AOX/PR pathway, and the ethylene (ET)/ jasmonic acid (JA)/PDF1.2/Thi2.1 pathway. Some studies have shown that changes in the activation state of HrBP1 are caused by changes in its spatial structure, which can vary depending on the ligand [2–5]. HrBP1 has also been shown to up-regulate Harpin-induced gene (H1N1), a newly discovered pathogenesis- related gene, which induces mitogen-activated protein kinase (MAPK) activity to generate disease resistance [6]. Moreover, production of hypersensitivity reaction (HR) and SAR via the harpin–HrBP1 interaction has been found to be closely associated with the nonrace-specific disease resistance (NDR1) and enhanced disease susceptibility-1 (EDS1) genes [7], and to be inhibited by exogenous auxin [8]. Dufulin Activates HrBP1 to Produce Antiviral Responses in Tobacco Zhuo Chen1, Mengjiao Zeng2, Baoan Song1, Chengrui Hou3, Deyu Hu1, Xiangyang Li1, Zhenchao Wang1, Huitao Fan1, Liang Bi1, Jiaju Liu1, Dandan Yu1, Linhong Jin1, Song Yang1 1 State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang, China, 2 School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, China, 3 Shanghai Applied Protein Technology Co. Ltd., Shanghai, China Introduction related genes 1(NPR1)/unknown effective protein pathway, the EDS/SA/NPR1/AOX/PR pathway, and the ethylene (ET)/ jasmonic acid (JA)/PDF1.2/Thi2.1 pathway. Some studies have shown that changes in the activation state of HrBP1 are caused by changes in its spatial structure, which can vary depending on the ligand [2–5]. HrBP1 has also been shown to up-regulate Harpin-induced gene (H1N1), a newly discovered pathogenesis- related gene, which induces mitogen-activated protein kinase (MAPK) activity to generate disease resistance [6]. Moreover, production of hypersensitivity reaction (HR) and SAR via the harpin–HrBP1 interaction has been found to be closely associated with the nonrace-specific disease resistance (NDR1) and enhanced disease susceptibility-1 (EDS1) genes [7], and to be inhibited by exogenous auxin [8]. Harpin binding protein-1 (HrBP1), which is found in plant cell walls, induces plants to generate systemic acquired resistance (SAR) and has important biological significance for pest control [1]. HrBP1 was first discovered in plant cell walls while studying the biological activity of harpin, a protein secreted by Gram- negative bacteria and other microorganisms and capable of inducing hypersensitive response [1]. HrBP1 was subsequently detected in Arabidopsis thaliana, soybean, barley, tomatoes, rice, potatoes, wheat, corn, and other plants. It is encoded by 284 amino acids and has a molecular weight (MW) of approximately 30 kDa with conserved primary structures in many plants [1]. In its activated state, HrBP1 can selectively up-regulate several signaling pathways and generate anti-bacterial, anti-viral, anti- fungal and anti-insect responses in the plant host. Independent signaling pathways induced by HrBP1 include the enhanced disease susceptibility-5 (EDS5)-salicylic acid (SA)/nonexpressor of pathogenesis- Dufulin, an amino phosphonate compound, is a plant antiviral agent with a novel molecular structure and good antiviral activity against tobacco mosaic virus (TMV), cucumber mosaic virus, and Potato Virus Y, among many other plant viruses (Figure 1). PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 PLoS ONE \| www.plosone.org 1 May 2012 \| Volume 7 \| Issue 5 \| e37944 Enhanced Disease Resistance Enhanced Disease Resistance Figure 1. Molecular structure of Dufulin. doi:10.1371/journal.pone.0037944.g001 pathways associated with its antiviral functions, the expression levels of some key genes such as EDS and pathogenesis-related protein (PR) were assessed using RT-PCR in plants treated with Dufulin. Our results validate HrBP1 as the target of action of Dufulin. 2D-DIGE Analysis Figure 1. Molecular structure of Dufulin. doi:10.1371/journal.pone.0037944.g001 Samples from all treatment groups were labeled with fluorescent dyes (Cy2, Cy3, or Cy5) [10,11] and run on the following conditions to eliminate their inherent biological and systemic variations: (1) the same sample was divided into two parts, labeled with Cy3 and Cy5, and run on two gels; (2) different treatment groups from the same plant were not run on the same gel; (3) two samples within the same gel were placed as far apart as possible; and (4) internal standards were mixed with equal amounts of all samples, labeled with Cy2, and run on each gel [10,11] (Figure 2). In order to eliminate differences between gels and improve the statistical reliability of our data, the value of each protein spot was calculated as the ratio of the measured values of the protein spot to the total value from all protein spots in the internal standard Preliminary studies have shown that it exerts its antiviral effects by activating SAR, but the specific mechanisms of its action and its molecular targets remain unclear [9]. In the present study, differential in-gel electrophoresis (DIGE)/two-dimensional elec- trophoresis (2-DE) combined with mass spectrometry (MS), bioinformatics, western blotting and reverse transcription poly- merase chain reaction (RT-PCR) was used to identify the target of action of Dufulin. Its antiviral activity was found to involve SAR, and HrBP1 was identified as its target protein. Based on the current understanding of HrBP1 and the downstream regulatory Figure 2. 2D-DIGE image analysis of total proteins from the tobacco leaves of Groups B–E. (A) Composite image of Cy2, Cy3 and Cy5 images. (B), (C), and (D) represent the Cy2, Cy3 and Cy5 fluorescent dye images, respectively. doi:10.1371/journal.pone.0037944.g002 Figure 2. 2D-DIGE image analysis of total proteins from the tobacco leaves of Groups B–E. (A) Composite image of Cy2, Cy3 and Cy5 images. (B), (C), and (D) represent the Cy2, Cy3 and Cy5 fluorescent dye images, respectively. doi:10.1371/journal.pone.0037944.g002 PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 PLoS ONE \| www.plosone.org 2 Enhanced Disease Resistance Figure 3. Expression levels and differential expression of some proteins. Each part of the figure shows the standard log abundance of spots on the y-axis for each protein spot of Groups B, C, D, and E (pool groups 2, 3, 4, and 5, respectively) on the x-axis. 2D-DIGE Analysis The dotted line represents the mean abundance for three repeated measurements in Groups B–D, whereas the continuous line represents the mean abundance for three repeated treatments in any single treatment group. doi:10.1371/journal.pone.0037944.g003 Figure 3. Expression levels and differential expression of some proteins. Each part of the figure shows the standard log abundance of spots on the y-axis for each protein spot of Groups B, C, D, and E (pool groups 2, 3, 4, and 5, respectively) on the x-axis. The dotted line represents the mean abundance for three repeated measurements in Groups B–D, whereas the continuous line represents the mean abundance for three repeated treatments in any single treatment group. doi:10.1371/journal.pone.0037944.g003 group. In addition, the same protein labeled with all three fluorescent dyes showed identical protein spots at the same position in the gel, thereby enhancing the matching rate of the protein spots. DeCyder software was used for image analysis to avoid errors caused by the user’s subjective influence in the selection of points. Protein points without appropriate matches were considered to arise from impurities or random errors in the experiment. A total of 14 protein spots showed statistically significant changes in expression across the four treatment groups (B, C, D, and E; one-way ANOVA, P,0.05). These spots were mainly within the pH range of 5–8 (Figure 3). could not search for corresponding information related to tobacco likely due to lack of information on plant proteins in the database. Of the 14 protein spots, 1 protein (spot 457) was annotated as ‘‘putative b-galactosidase’’ from a different species (Solanum lycopersicum); another protein (spot 310) was designated as the CF1-a subunit of ATP synthase from yet another species (Nicotiana sylvestris). The trends in changes in protein expression across the four treatment groups are illustrated in Figure 3. Spots 187, 769, 990, and 1041 were ascribed to genes associated with disease resistance and were upregulated in groups C and D. Four other proteins (spots 210, 422, 701, and 1413) identified were found to control the biological functions of carbon metabolism, photosyn- thesis, as well as photorespiration and cell division. Spots 457 (putative b-galactosidase) and 598 (carboxylase/oxygenase) showed trends towards downregulation. The 14 protein spots were identified as belonging to 13 protein products using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS and linear trap quadru- pole (LTQ) MS (Table 1, Table 2 and Table S1). PLoS ONE \| www.plosone.org Classic 2-DE Analysis Table 1. Differentially expressed proteins identified by DIGE and MS. DIGE spot no. Protein description Species GI no. Function Subcellular location Theoretical MW (Da)/pI Experimental MW (kDa)/pI Peptide Count Protein Score Protein Score C.I % 301 ATP synthase CF1-a subunit Nicotiana sylvestris gi\|78102516 energy metabolism mitochondrial membranes 55388/5.14 62.4/5.15 19 686 100 178 Chain A of ribulose-1,5- bisphosphate carboxylase oxygenase N. tabacum gi\|515239 photosynthesis and photorespiration chloroplast 49526.1/6.19 70.1/6.57 14 250 100 457 putative b-galactosidase Solanum lycopersicum gi\|7939623 carbon metabolism lysosome 93183.9/6.81 46.9/7.28 11 339 100 598 carboxylase/oxygenase N. tabacum gi\|223593 carbon metabolism cytoplasm 14490.2/4.99 40.5/5.05 5 65 78.524 769 HrBP1 N. tabacum gi\|38679323 disease resistance cell membrane 29980/8.8 34.4/4.66 8 242 100 990 germin-like protein N. tabacum gi\|222051768 disease resistance cytoplasm 21408.2/5.84 28.2/5.77 4 257 100 1015 oxygen-evolving complex 33-kDa photosystem II N. tabacum gi\|30013657 photosynthesis and photorespiration chloroplast 35176.9/5.63 27.9/6.17 11 284 100 1041 24 K germin-like protein N. tabacum gi\|31711507 disease resistance cytoplasm 21954.4/7.82 27.1/7.78 4 350 100 187 truncated N-protein N. tabacum gi\|45544515 disease resistance cytoplasm 74429 70.15/5.53 30 4.9e+02 4 210 putative cell cycle protein N. tabacum gi\|82775180 cell division nucleus 20523 68.55/7.28 22 2.8e+03 2 y In order to make up for the shortcoming of the DIGE assay, which involves lower loading quantities than other methods, 2-DE analysis combined with silver staining and coomassie brilliant blue staining was performed in three replicates on group B (tobacco infected by TMV for 1 day) and E (tobacco treated with Dufulin for 5 day in group B). The goal of these experiments was to identify some low-abundance proteins by loading 800 mg of sample in each well [12,13], and to investigate temporal changes in protein profiles that occur during the infection of tobacco leaves by TMV and after their 5-day treatment with Dufulin (group E). Each sample was run in gels in triplicates, and they showed a high degree of reproducibility (Figure 4). Our quantitative image analysis showed 21 protein spots whose intensities changed (P,0.05) by at least 1.2-fold (Table S2 and Figure S1). The molecular weights of the differentially expressed proteins were mainly in areas corresponding to the MW markers 20–40 kDa. Intensities of the protein spots in group E were as high as 79.3% relative to the group B, and their pH ranged from 4–7. Classic 2-DE Analysis g p p g In total, 21 spots representing 20 proteins were successfully identified using MALDI-TOF/TOF MS (Table 3 and Table S3). Four proteins were annotated either as unknown and hypothet- ical proteins, or as proteins without any specific function assigned in the database; other proteins were identified to be associated with photosynthesis, photorespiration, disease resistance, stress response, as well as the metabolism of redox components, lipids, and energy. For example, the spots 5942, 6138, 6087, 6288 6291, 6527, 6556, and 6675 were identified as proteins associated with photorespiration and photosynthesis, while the spots 6071, 6257, and 6388 were identified as being associated with disease resistance. Interestingly, two spots (6388 and 6395) were identified as HrBP1, which was also identified by DIGE/ MS to have a trend of up-regulation (Figure 3). A comparison of our results of the 2-DE and DIGE assays showed that four proteins [GenInfo identifier (GI) nos. 100380, 515239, 30013663, and 38679323] were identified in both assays, indicating that our 2-DE assays complemented the DIGE assay well. However, some proteins related to disease resistance (spots 6071 and 6257) were identified by 2-DE/MS, but not by DIGE/MS, indicating that the results of the two assays don’t correspond fully with each other. 2D-DIGE Analysis The software PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 3 Enhanced Disease Resistance y y GE spot o. Protein description Species GI no. Function Subcellular location Theoretical MW (Da)/pI Experimental MW (kDa)/pI Peptide Count Protein Score Protein Score C.I % 1 ATP synthase CF1-a subunit Nicotiana sylvestris gi\|78102516 energy metabolism mitochondrial membranes 55388/5.14 62.4/5.15 19 686 100 8 Chain A of ribulose-1,5- bisphosphate carboxylase oxygenase N. tabacum gi\|515239 photosynthesis and photorespiration chloroplast 49526.1/6.19 70.1/6.57 14 250 100 7 putative b-galactosidase Solanum lycopersicum gi\|7939623 carbon metabolism lysosome 93183.9/6.81 46.9/7.28 11 339 100 8 carboxylase/oxygenase N. tabacum gi\|223593 carbon metabolism cytoplasm 14490.2/4.99 40.5/5.05 5 65 78.524 9 HrBP1 N. tabacum gi\|38679323 disease resistance cell membrane 29980/8.8 34.4/4.66 8 242 100 0 germin-like protein N. tabacum gi\|222051768 disease resistance cytoplasm 21408.2/5.84 28.2/5.77 4 257 100 15 oxygen-evolving complex 33-kDa photosystem II N. tabacum gi\|30013657 photosynthesis and photorespiration chloroplast 35176.9/5.63 27.9/6.17 11 284 100 41 24 K germin-like protein N. tabacum gi\|31711507 disease resistance cytoplasm 21954.4/7.82 27.1/7.78 4 350 100 7 truncated N-protein N. tabacum gi\|45544515 disease resistance cytoplasm 74429 70.15/5.53 30 4.9e+02 4 0 putative cell cycle protein N. tabacum gi\|82775180 cell division nucleus 20523 68.55/7.28 22 2.8e+03 2 Upregulation of the SA-mediated Signaling Pathway Identified Using Bioinformatic Analysis of Proteomic Dat Identified Using Bioinformatic Analysis of Proteomic Data The wealth of discrete proteomic data available today has made their bioinformatic analysis both necessary and feasible. To this end, proteins that were found to have differential expression in our proteomics assay were annotated based on the Genbank database. Excluding data for 13 redundant sequences and 2 sequences that could not be retrieved (accession codes 76869447 and 51964984), information related to 33 sequences was downloaded from GenBank and the sequences were analyzed using Blast2GO [14–16]. The non-redundant (NR) database was searched using BLAST, with the E-value (expectation) set as 1e210. Our results showed that Gene Ontology (GO) codes corresponding to 7 sequences were not annotated (Table S4). Gene Ontology (GO) categorization based on molecular function (MF), biological process (BP) and cellular component (CC) was done based on GO numbers of differentially expressed proteins using BLAST (see Table S5, Table S6, Table S7, Table S8, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6 and Figure S7). The pie graphs were made according to the distribution of the target sequences (Figure 5A, Figure 5B, and Figure 5C). Binding activity and catalytic activity, among others, accounted May 2012 \| Volume 7 \| Issue 5 \| e37944 4 Enhanced Disease Resistance Table 2. Differentially expressed proteins identified by DIGE and LTQ spectrometry. DIGE spot no. Protein Description Species GI no. Function Subcellular location Peptide count Unique peptide count CoverPercent MW PI XC DeltaCn Peptide Sequence MH+ Diff(MH+) Charge Rank 422 ribulose-bisphosphate carboxylase activase N. tabacum gi\|19992 photosynthesis and photorespiration Chloroplast 4 2 8.19% 25929.41 5.01 R.TDNVPEEAVVK.I 1201.309 0.39451 2 1 2.4651 0.6007 R.TDNVPEEAVVK.I 1201.309 0.48751 2 1 2.4786 0.5422 R.VYDDEVRK.W 1024.109 –0.41359 2 1 2.6064 0.5159 R.VYDDEVRK.W 1024.109 0.18941 2 1 2.2002 0.4997 701 Phosphomannomutase N. tabacum gi\|90762161 carbon metabolism Cytoplasm 1 1 4.37% 28575.43 6.41 R.SGMLNVSPIGR.D 1147.331 –0.93531 2 1 2.4934 0.5942 1042 24 K germin-like protein N. tabacum gi\|31711507 disease resistance Cytoplasm 1 1 2.86% 21968.49 5.83 K.LNPLIK.A 697.889 0.18904 1 1 2.1532 0.3057 1413 ribulose bisphosphate carboxylase small-subunit precursor N. tabacum gi\|30013663 photosynthesis and photorespiration Chloroplast 2 1 5.00% 20281.19 7.57 R.GFVYRENNK.S 1127.235 –0.58645 2 1 2.5666 0.539 R.GFVYRENNK.S 1127.235 0.29255 2 1 2.6403 0.4976 doi:10 1371/journal pone 0037944 t002 ne.org 5 May 201 Table 2. Differentially expressed proteins identified by DIGE and LTQ spectrometry. DIGE spot no. Protein Description Species GI no. PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 Gene Expression Levels of Some Differentially Expressed Proteins Gene Expression Levels of Some Differentially Expressed Proteins for the largest proportion of all categories of MF (Figure 5A); metabolic process, cellular process, and response to stimulus accounted for the largest proportion of all categories of BP (Figure 5B); and the ratio of cells and organelles was the largest category in CC (Figure 5C). Based on this, a directed acyclic graph (DAG) containing MF, BP, and CC data was also made using the Blast2GO software (Figure 6 and Figure S8). A node with a score of 3.0 for receptor activity was found in the MF- DAG and confirmation that the receptor was HrBP1 was done using proteomic data. A subsequent node indicating signal transduction functions and a score of 1.8 was also observed in the MF-DAG (Figure 6). In addition, an initial signal transduc- tion node with a score of 3.0 was found in the BP-DAG, and subsequent nodes represented signal transduction and regulation of cellular processes with node scores reaching 1.8 (Figure S8). These findings suggested to us that the antiviral effects of Dufulin are related to signal transduction and that the receptor protein HrBP1 could be its initial target due to the protein’s role in signal transduction. Finally, similar to the MF-DAG and BP- DAG results, the antiviral effects of Dufulin may be attributed to signal transduction triggered by HrBP1. In the analysis of CC- DAG, a node with a high score of 8.09 was found for ‘‘cell’’, and the next node with score reaching 7.37 indicated ‘‘intracellular’’; these results suggested that the antiviral responses are completed within the plant cell (Figure S9). Taken together, these data strongly suggest HrBP1 as a target protein of Dufulin and further confirmation should be done via molecular biology methods. For many proteins that appeared to be upregulated in proteomic assays, expression at the mRNA level was also evaluated. Primers for these genes were designed using the Beacon Designer software (Table 4). Gene expression levels were examined using semi-quantitative RT-PCR (18 Cycles) and electrophoresis on 2.2% agarose gel. Many genes were found to be upregulated in tobacco leaves infected with TMV and subsequently treated with Dufulin. These changes in expression at the mRNA level detected by RT-PCR were in accord with changes at the protein level as indicated by DIGE and 2-DE analyses (Figure 7). Upregulation of the SA-mediated Signaling Pathway Identified Using Bioinformatic Analysis of Proteomic Dat Function Subcellular location Peptide count Unique peptide count CoverPercent MW PI XC DeltaCn Peptide Sequence MH+ Diff(MH+) Charge Rank 422 ribulose-bisphosphate carboxylase activase N. tabacum gi\|19992 photosynthesis and photorespiration Chloroplast 4 2 8.19% 25929.41 5.01 R.TDNVPEEAVVK.I 1201.309 0.39451 2 1 2.4651 0.6007 R.TDNVPEEAVVK.I 1201.309 0.48751 2 1 2.4786 0.5422 R.VYDDEVRK.W 1024.109 –0.41359 2 1 2.6064 0.5159 R.VYDDEVRK.W 1024.109 0.18941 2 1 2.2002 0.4997 701 Phosphomannomutase N. tabacum gi\|90762161 carbon metabolism Cytoplasm 1 1 4.37% 28575.43 6.41 R.SGMLNVSPIGR.D 1147.331 –0.93531 2 1 2.4934 0.5942 1042 24 K germin-like protein N. tabacum gi\|31711507 disease resistance Cytoplasm 1 1 2.86% 21968.49 5.83 K.LNPLIK.A 697.889 0.18904 1 1 2.1532 0.3057 1413 ribulose bisphosphate carboxylase small-subunit precursor N. tabacum gi\|30013663 photosynthesis and photorespiration Chloroplast 2 1 5.00% 20281.19 7.57 R.GFVYRENNK.S 1127.235 –0.58645 2 1 2.5666 0.539 R.GFVYRENNK.S 1127.235 0.29255 2 1 2.6403 0.4976 PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 PLoS ONE \| www.plosone.org 5 Enhanced Disease Resistance Figure 4. 2-DE image analysis of total proteins from the tobacco leaves of Groups E. The spots were visualized by silver staining and detected using the DeCyder software. Quantitative image analysis revealed the protein spots with significantly different intensity (p,0.05) (by more than 1.5-fold) when compared with the B group. doi:10.1371/journal.pone.0037944.g004 Figure 4. 2-DE image analysis of total proteins from the tobacco leaves of Groups E. The spots were visualized by silver staining and detected using the DeCyder software. Quantitative image analysis revealed the protein spots with significantly different intensity (p,0.05) (by more than 1.5-fold) when compared with the B group. doi:10.1371/journal.pone.0037944.g004 PLoS ONE \| www.plosone.org Enhanced Expression of Crucial Genes and Proteins in the SA-mediated Signaling Pathway HrBP1 was identified as the first protein in the EDS/SA/ NPR1/AOX/PR signaling pathway [1]. Activation of HrBP1 leads plants to exhibit antiviral activity by generating SAR [17,18]. In addition, some molecules in downstream of the SA signaling pathway, such as the gene of NPR1, EDS1, lipid associated protein, PRs and phytoalexins, are regarded as markers of antiviral responses [19,20]. Therefore, NPR1, EDS1, PR-1a and PR-5 were examined as putative target genes in the activated state, and the degree of SA signaling and the level of SAR were evaluated [19]. Data obtained from semi-quantitative RT-PCR indicated that these genes were also upregulated after Dufulin was sprayed on the leaves of N. tabacum K326 (Figure 8), revealing that Dufulin PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 6 Enhanced Disease Resistance Spot no. Protein description Species GI no. Function Subcellular location Theoretical Mw (Da)/pI Experimental Mw (kDa)/pI Peptide count Protein Score Protein Score C.I % Best Ion Score ratio Regulation tendency 5942 ribulose bisphosphate carboxylase/oxygenase-2 (RuBisCO activase) N. tabacum gi\|12643758 photosynthesis and photorespiration chloroplast 48312.5/8.14 39.14/4.40 9 217 100 76 1.28723 down 6071 lignin-forming anionic peroxidase N. tabacum gi\|129837 disease resistance cytoplasm 34652.1/4.69 36.24/4.25 8 260 100 142 2.31966 down 6138 Photosystem-II stability/ assembly factor HCF 136 Ricinus communis gi\|255559812photosynthesis and photorespiration chloroplast 43414.9/7.11 36.01/5.40 9 209 100 99 1.28514 down 6087 ribulose-bisphosphate carboxylase activase N. tabacum gi\|100380 photosynthesis and photorespiration chloroplast 25913/5.01 36.67/6.25 8 302 100 117 1.31604 up 6095 NAD-dependent epimerase/dehydratase Ricinus communis gi\|255542956redox metabolism cytoplasm 42547/8.52 66.35/6.75 8 364 100 110 1.6325 up 6257 plastid-lipid-associated protein N. tabacum gi\|2632088 Lipid metabolism stress response chloroplast 29385.4/4.83 32.00/4.55 8 377 100 89 1.98999 up 6288 oxygen-evolving complex 33-kDa photosystem-II protein N. tabacum gi\|30013657 photosynthesis and photorespiration chloroplast 35176.9/5.63 30.67/4.90 15 653 100 170 1.29081 up 6265 predicted protein Micromonas pusilla gi\|226461019No annotation No annotation 272410.5/5.67 32.67/5.44 32 75 97.645 1.49341 down 6287 protein-binding protein Ricinus communis gi\|255564826No annotation No annotation 37785.1/7.56 31.33/5.48 8 136 100 60 1.31604 down 6246 predicted protein Physcomitrella patens subsp. Patens gi\|168050023No annotation/ No annotation 70926.8/9.06 33.34/5.78 19 78 98.972 1.49111 down 6291 chain A of ribulose-1,5- Bisphosphate carboxylase oxygenase N. tabacum gi\|515239 photosynthesis and photorespiration chloroplast 49526.1/6.19 31.15/7.84 11 85 99.78 27 1.24821 up 6388 HrBP1 N. tabacum gi\|38679323 disease resistance cell membrane 29980/8.8 34.4/4.66 8 197 100 73 1.36946 6527 oxygen-evolving enhancer protein 2-1 N. Enhanced Expression of Crucial Genes and Proteins in the SA-mediated Signaling Pathway tabacum gi\|52000814 photosynthesis and photorespiration chloroplast 28634.4/6.84 26.16/4.75 7 315 100 109 1.44689 up 6563 superoxide dismutase Nicotiana plumbaginifolia gi\|134642 disease resistance chloroplast 23027.5/5.53 25.40/5.66 9 323 100 78 1.35317 down 6545 predicted protein Populus trichocarpa gi\|224104053No annotation No annotation 70270.2/7.85 25.81/5.74 14 64 72.963 6 1.27646 down 6449 cytosolic isoform-like triose phosphate isomerase Solanum tuberosum gi\|77745458 energy metabolism cytoplasm 26995/5.73 27.75/6.40 9 376 100 102 1.77561 up 6556 light-harvesting complex I protein Lhca1 Populus trichocarpa gi\|224109746photosynthesis and photorespiration chloroplast 26319.5/5.83 25.46/6.90 2 58 0 50 1.28703 up 6675 PSI-D1 precursor Nicotiana sylvestris gi\|407769 photosynthesis and photorespiration thylakoid membrane 23441.2/9.84 23.05/7.46 9 106 99.998 45 1.50195 down PLoS ONE \| www.plosone.org 7 May 2012 \| Volume 7 \| Issue 5 \| e3 May 2012 \| Volume 7 \| Issue 5 \| e37944 7 Enhanced Disease Resistance effectively activates the SA signaling pathway and SAR. In addition, trend analysis of expression by western blotting indicated that the lipid associated protein was also upregulated by Dufulin (Figure 9). Spot no. Protein description Species GI no. Function Subcellular location Theoretical Mw (Da)/pI Experimental Mw (kDa)/pI Peptide count Protein Score Protein Score C.I % Best Ion Score ratio Regulation tendency 6395 HrBP1 N. tabacum gi\|38679323 disease resistance cell membrane 29980/8.8 28.55/5.66 9 201 100 66 2.05393 down 6818 chain ‘s’ of ribulose-1,5- bisphosphate carboxylase oxygenase N. tabacum gi\|230922 photosynthesis and photorespiration chloroplast 14550.2/5.19 19.95/4.15 7 213 100 68 1.6636 up 6883 putative ribulose bisphosphate carboxylase small subunit precursor N. tabacum gi\|30013663 photosynthesis and photorespiration chloroplast 20268.1/7.57 18.75/3.55 7 162 100 27 1.38414 up Induction of PR-1a Gene Expression by Dufulin and Other Inducers of SAR BTH and exogenous SA were known as inducers of SAR [21– 23]. BTH was the first successful commercial plant activator, whose target of action has been shown to fall between NPR1 and the SA molecules [21,22]. Exogenous administration of SA on plants can increase the levels of SA, promote NPR1 expression and ultimately increase the amount of PR protein [23]. In order to better understand the antiviral activity and application of Dufulin, we decided to compare the effect of Dufulin with that of BTH and exogenous SA on the expression level of PR-1a. The three compounds were sprayed onto the leaves of N. tabacum K326 at 500 mg/mL, respectively, and the expression status of PR-1a was determined at different time points by real time PCR (Figure 10 and Figure S10). Interestingly, expression of PR-1a was found to commence the fastest in the Dufulin treatment group, followed by the BTH and the exogenous SA treatment groups. Maximal expression of PR-1a occurred at 9, 9 and 6 days in the BTH, Dufulin and exogenous SA treatment groups, respectively. Over the course of 12 days, PR-1a expression was maintained the longest in the BTH treatment group, followed by the Dufulin treatment group and the exogenous SA treatment group (Figure 11). Protective Activity of Dufulin Against TMV-induced Lesions TMV, the most common pathogen in China affecting tobacco, tomato, cucumber, and potato, was selected for evaluation of antiviral bioactivity of Dufulin [24]. The ability of the antiviral agent to protect plants against local lesions caused by TMV was examined in our assays [25]. Varying concentrations of Dufulin were sprayed onto the tobacco leaves of 8-week-old N. glutinosa plants, and TMV inoculation was performed 5 days later. Dimethyl sulfoxide (DMSO; 0.05%) was used as the solvent control (the CK treatment group) (Figure 12A, Figure 12B). The average numbers of local lesions were 3.260.14, 2.660.14, 2.160.12, 1.660.11, and 1.160.09 lesions/cm2 for 100, 200, 300, 400, and 500 mg/mL administration of Dufulin, respectively (Figure 12A, Figure 12B). These values were significantly lower than those in the CK treatment group (3.660.13 lesions/cm2) (Figure 12C). Our results demonstrate that Dufulin inhibits lesions caused by TMV in a dose-dependent manner. Discussion HrBP1 and the harpin-like binding protein are both found in plant cell walls and belong to a family of complex receptors. Activated HrBP1 can act upstream of the SA, JA, and ethylene (ETH) signaling pathways in plant cells [1]. Activation of HrBP1 can thus induce resistance against viruses, bacteria, fungi and pests in plants, and improve plant growth and development via those signaling pathways [5]. Here, we show that Dufulin can upregulate the expression of HrBP1 and activate the SA signaling pathway, leading to the upregulation of PR-1a and PR-5. Other studies have shown that the SA signaling pathway is closely associated with antiviral resistance of host plants and that its downstream molecules are PRs [21] and PAL protective enzymes [26], among which PR-1a and PR-5 are closely related to antiviral resistance [27]. Earlier PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 8 Enhanced Disease Resistance Figure 5. Pie graphs illustrating the distribution of target sequences based on their annotation in KEGG. (A) Based on their annotated molecular function (MF), the genes were classified as ‘‘transporter activity’’, ‘‘molecular transducer activity’’, ‘‘nutrient reservoir activity’’, or ‘‘catalytic activity’’. (B) Based on their annotated involvement in various biological processes (BP), genes were classified as ‘‘response to stimulus’’, ‘‘localization’’, ‘‘biological regulation’’, ‘‘cellular process’’, and ‘‘metabolic process’’. (C) Based on their annotated localization in various cellular components (CC), genes were classified as ‘‘cell’’, ‘‘organelle’’, and ‘‘macromolecular complex’’. doi:10.1371/journal.pone.0037944.g005 Figure 5. Pie graphs illustrating the distribution of target sequences based on their annotation in KEGG. (A) Based on their annotated molecular function (MF), the genes were classified as ‘‘transporter activity’’, ‘‘molecular transducer activity’’, ‘‘nutrient reservoir activity’’, or ‘‘catalytic activity’’. (B) Based on their annotated involvement in various biological processes (BP), genes were classified as ‘‘response to stimulus’’, ‘‘localization’’, ‘‘biological regulation’’, ‘‘cellular process’’, and ‘‘metabolic process’’. (C) Based on their annotated localization in various cellular components (CC), genes were classified as ‘‘cell’’, ‘‘organelle’’, and ‘‘macromolecular complex’’. doi:10.1371/journal.pone.0037944.g005 and enhancement of antiviral activity by Dufulin were mediated via HrBP1 activation. In addition, a protective assay based on counting of lesions also indicated that Dufulin had protective effects against TMV. Taken together, these results show that Dufulin is an immunoactivator of SAR that confers antiviral activity to cells via regulation of the SA signaling pathway. Discussion The findings of this research lay the groundwork for developing a molecular model for selection of antiviral drugs based on the SA signaling pathway. studies demonstrated that Dufulin can induce resistance in tobacco by activating PAL, peroxidase (POD), and superoxide dismutase (SOD). The results of the current study extend those findings by revealing that Dufulin induces antiviral resistance in the host plant by activating HrBP1 to trigger the SA signaling pathway, thereby producing SAR [9]. We further analyzed the activation of the SA signaling pathway by Dufulin and other known immunoactivators. We found that the time of induction of PR-1a expression after Dufulin treatment was quicker than that after exogenous SA application, and the maximal level of PR-1a gene expression in the Dufulin treatment group was higher than that in the exogenous SA treatment group. In addition, PR-1a gene expression persisted longer in the Dufulin treatment group than that in the SA and BTH treatment groups. Moreover, a full set of methods that combine proteome technology and bioinformatics was created to identify the target of the drug [28–30]. To our knowledge, this is the first example of a study combining aspects of modern biotechnology, namely proteomics and bioinformatics, and molecular biology to find a drug’s target of action against a plant viral disease. Plant Materials and Growth Conditions Nicotiana tabacum K-326 and N. glutinosa seeds were purchased from the Laboratory of Germplasm Genetic Resources of the Tobacco Research Institute, Chinese Academy of Agricultural Sciences. TMV was obtained from the Wuhan Institute of Virology at the Chinese Academy of Sciences, and its source was preserved in an N. tabacum K-326 plant over an extended period. The extraction and purification of TMV were carried out as previously described by Gooding [31]. N. tabacum K-326 and N. glutinosa were grown in a disease-free greenhouse, and 12-week-old plants were used for the study. The temperature and humidity levels in the greenhouse were maintained close to 25uC and 60%, respectively. In summary, this study used DIGE and 2-DE in combination with MS techniques and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to identify HrBP1 as a possible target protein of Dufulin. Our result was verified by western blotting assays against lipid associated protein, and the downstream signaling pathway was identified by RT-PCR analysis of the related genes NPR1, EDS1, PR-1a and PR-5. All results obtained using our proteomics and molecular biology methods indicated that acquisition of SAR Materials and Methods Plant Materials and Growth Conditions Protein Sample Preparation for Proteomic Analysis The leaf samples were divided into six groups: group A (control), healthy tobacco leaves; group B, tobacco leaves infected with PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 9 Figure 6. Pathway analysis of the target of Dufulin in mediating anti-viral responses using MF-DAG. Nodes are colored according to their score values. The DAGs of BP and CC are shown in Figures S7 and S8. doi:10.1371/journal.pone.0037944.g006 Enhanced Disease Resistance Enhanced Disease Resistance Figure 6. Pathway analysis of the target of Dufulin in mediating anti-viral responses using MF-DAG. Nodes are colored according to their score values. The DAGs of BP and CC are shown in Figures S7 and S8. doi:10.1371/journal.pone.0037944.g006 TMV for 24 h; group C, tobacco leaves infected with TMV for 24 h and then treated with Dufulin for 24 h; group D, tobacco leaves infected with TMV and subsequently treated with Dufulin for 72 h; group E, tobacco leaves infected with TMV and then treated with Dufulin for 120 h; and group F, tobacco leaves infected with TMV for 120 h. The tobacco leaves were first washed with sterile water at room temperature and then inoculated with TMV. Powdered Dufulin was dissolved in an appropriate amount of dimethyl formamide (DMF) and mixed in phosphate-buffered saline (PBS) with 1% Tween-20 to obtain 500 mg/L solutions. The dried leaves were sprayed with Dufulin at a concentration of 500 mg/L at 24 h intervals. Samples of tobacco leaves were removed from the main leaf vein, frozen in liquid nitrogen, and stored at 280uC before use. Total proteins were extracted from groups A–E using a modified trichloroacetic acid/acetone procedure as follows [10– 12]. The samples were ground into a fine powder in liquid nitrogen, transferred into a tube containing 25 mL of trichlor- oacetic acid/acetone (1:9, v/v) as well as 65 mM dithiothreitol, and then precipitated at –20uC for more than 1 h. The samples were centrifuged at 10,000 rpm at 4uC for 45 min, and the supernatants were discarded. Twenty-five milliliters of acetone was added to the precipitate, and the solutions were stored at 220uC for 1 h and centrifuged at 10,000 rpm at 4uC for 45 min, after which the supernatant was removed. The precipitate was vacuum-dried, weighed, and stored at 280uC. 2D-DIGE and Image Analysis conditions using 50 mg of protein and 400 pmol of fluorescent dyes. The reaction was terminated by adding 1 mL of 10 mM lysine after 10 min. The internal standard was labeled with Cy2, half the number of samples from each group was labeled with Cy3, and the remaining half was labeled with Cy5. The multiplexing of all samples was also randomized separately to keep the results unbiased, as shown in Table 4. IPG strips (pH 3–10, 13 cm) (http://www.gelifesciences.com/) were cup-loaded with 50 mg of protein of each of the Cy2-, Cy3-, and Cy5-labeled samples in a buffer containing 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 65 mM dithiothreitol, and 1% (v/v) IPG buffer. Isoelectric focusing was carried out under the following conditions: 12 h at 30 v, 1 h at 500 v, 1 h at 1000 v, 8 h at 8000 v, and 4 h at 500 v. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS- PAGE) was carried out on a 12.5% gel using Hoefer SE600 (Hoefer-GE Healthcare Apparatuses). Second-dimension SDS- PAGE was run by overlaying the strips on 12.5% isocratic Laemmli gel (13613 cm) cast in low-fluorescence glass plates on an Ettan DALT VI system. Gels were run at 20uC at a constant power of 2.5 W/gel for 30 min, followed by constant power of 17 W/gel until the bromophenol blue tracking front ran off the gel. Fluorescence images of the gels were acquired on a Typhoon 9400 scanner. Cy2, Cy3, and Cy5 images for each gel were scanned at excitation/emission wavelengths of 488/520, 532/580, and 633/670 nm, respectively, at 100 mm resolution, yielding 27 images. Protein spots in both the analytical and preparative gels were observed using the silver staining procedure as described previously [13]. Spot identification, background elimination, point matching, and differential analysis of the protein spots were completed using a 2D DeCyder software (Differential In-Gel Analysis and Biological Variance Analysis Version 4.0; Amersham conditions using 50 mg of protein and 400 pmol of fluorescent dyes. The reaction was terminated by adding 1 mL of 10 mM lysine after 10 min. The internal standard was labeled with Cy2, half the number of samples from each group was labeled with Cy3, and the remaining half was labeled with Cy5. The multiplexing of all samples was also randomized separately to keep the results unbiased, as shown in Table 4. 2D-DIGE and Image Analysis IPG strips (pH 3–10, 13 cm) (http://www.gelifesciences.com/) were cup-loaded with 50 mg of protein of each of the Cy2-, Cy3-, and Cy5-labeled samples in a buffer containing 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 65 mM dithiothreitol, and 1% (v/v) IPG buffer. Isoelectric focusing was carried out under the following conditions: 12 h at 30 v, 1 h at 500 v, 1 h at 1000 v, 8 h at 8000 v, and 4 h at 500 v. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS- PAGE) was carried out on a 12.5% gel using Hoefer SE600 (Hoefer-GE Healthcare Apparatuses). Second-dimension SDS- PAGE was run by overlaying the strips on 12.5% isocratic Laemmli gel (13613 cm) cast in low-fluorescence glass plates on an Ettan DALT VI system. Gels were run at 20uC at a constant power of 2.5 W/gel for 30 min, followed by constant power of 17 W/gel until the bromophenol blue tracking front ran off the gel. Fluorescence images of the gels were acquired on a Typhoon 9400 scanner. Cy2, Cy3, and Cy5 images for each gel were scanned at excitation/emission wavelengths of 488/520, 532/580, and 633/670 nm, respectively, at 100 mm resolution, yielding 27 images. Protein spots in both the analytical and preparative gels were observed using the silver staining procedure as described previously [13]. Spot identification, background elimination, point matching, and differential analysis of the protein spots were completed using a 2D DeCyder software (Differential In-Gel Analysis and Biological Variance Analysis Version 4.0; Amersham According to the instructions provided with the Bio-Rad Protein Assay Kit (http://www.bio-rad.com/LifeScience/pdf/ Bulletin_9004.pdf), the concentrations of all the samples were adjusted to 5 mg/mL. An internal standard was prepared by mixing equal amounts of all samples to eliminate systemic and inherent biological variations. The samples and internal standards were labeled with DIGE dyes for 30 min in an ice bath under dark Figure 8. Gene expression analysis of crucial components of the SA signaling pathway by semi-quantitative RT-PCR. The genes encoding EDS1, NPR1, PR-1a and PR-5 were analyzed. Semi- quantitative RT-PCR was performed using gene-specific primers, and b- actin, EF-1a and 18S ribosomal RNA genes were selected as internal controls. doi:10.1371/journal.pone.0037944.g008 Figure 8. Gene expression analysis of crucial components of the SA signaling pathway by semi-quantitative RT-PCR. The genes encoding EDS1, NPR1, PR-1a and PR-5 were analyzed. Protein Sample Preparation for Proteomic Analysis A total of 500 mg of dry powder samples was added to 500 mL of DIGE lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS (3-[(3- cholamidopropyl) dimethylammonio]-1-propanesulfonate), and 0.2% immobilized pH gradient (IPG) buffer. The ratio of the volume of the lysis buffer and that of the enzyme inhibitors was maintained at 50:1 and the mixture was stirred to homogeneity before spinning at 12,000 rpm at 4uC for 1 h. The supernatant was collected and filtered using a 0.22 mm filter to obtain a clear solution. The pH of the supernatant was adjusted to 8.0 for the DIGE experiment. Table 4. 2D-DIGE experimental design. Number of the gel Cy2 Cy3/Cy5 Cy5/Cy3 1 Pool A2 B3 2 Pool B2 C3 3 Pool C2 A4 4 Pool B5 C4 5 Pool B4 A5 6 Pool C5 A3 Each gel was loaded with 50 mg of the Cy2-labeled protein pool from all samples (as an internal standard), 50 mg of the Cy3-labeled sample, and 50 mg of the Cy5-labeled sample, as indicated. doi:10.1371/journal.pone.0037944.t004 Table 4. 2D-DIGE experimental design. Each gel was loaded with 50 mg of the Cy2-labeled protein pool from all samples (as an internal standard), 50 mg of the Cy3-labeled sample, and 50 mg of the Cy5-labeled sample, as indicated. doi:10.1371/journal.pone.0037944.t004 May 2012 \| Volume 7 \| Issue 5 \| e37944 10 PLoS ONE \| www.plosone.org Enhanced Disease Resistance Figure 7. Gene expression analysis of differentially expressed proteins from the proteomics assay by semi-quantitative RT-PCR. Genes encoding the 26 different proteins listed in Table 5 were analyzed. Semi-quantitative RT-PCR was performed using gene-specific primers, and b-actin, EF-1a and 18S ribosomal RNA genes were used as internal controls. doi:10.1371/journal.pone.0037944.g007 Figure 7. Gene expression analysis of differentially expressed proteins from the proteomics assay by semi-quantitative RT-PCR. Genes encoding the 26 different proteins listed in Table 5 were analyzed. Semi-quantitative RT-PCR was performed using gene-specific primers, and b-actin, EF-1a and 18S ribosomal RNA genes were used as internal controls. doi:10.1371/journal.pone.0037944.g007 Figure 7. Gene expression analysis of differentially expressed proteins from the proteomics assay by semi-quantitative RT-PCR. Genes encoding the 26 different proteins listed in Table 5 were analyzed. Semi-quantitative RT-PCR was performed using gene-specific primers, and b-actin, EF-1a and 18S ribosomal RNA genes were used as internal controls. doi:10.1371/journal.pone.0037944.g007 2D-DIGE and Image Analysis Semi- quantitative RT-PCR was performed using gene-specific primers, and b- actin, EF-1a and 18S ribosomal RNA genes were selected as internal controls. doi:10.1371/journal.pone.0037944.g008 doi:10.1371/journal.pone.0037944.g008 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 11 Enhanced Disease Resistance Figure 9. Protein expression analysis of plastid-lipid-associated proteins by western blot. (A). Semi-quantitative western blot of plastid- lipid-associated proteins. The letters a, b, c, d, and e represent the treatment groups described previously in ‘‘Experimental Procedures’’. (B) Standard protein samples (5, 10 and 20 mg) were loaded on the well and analyzed by western blotting. (C) Optical density values obtained from the imaging software. The protein ratios were extrapolated using linear regression analysis and expressed as mean 6 SD (n = 4). Error bars represent standard deviation (SD). Asterisks indicate values significantly different from the mean at P,0.05. (D) Linear regression was used to quantify the differences between the bands in (B). doi:10.1371/journal.pone.0037944.g009 Figure 9. Protein expression analysis of plastid-lipid-associated proteins by western blot. (A). Semi-quantitative western blot of plastid- lipid-associated proteins. The letters a, b, c, d, and e represent the treatment groups described previously in ‘‘Experimental Procedures’’. (B) Standard protein samples (5, 10 and 20 mg) were loaded on the well and analyzed by western blotting. (C) Optical density values obtained from the imaging software. The protein ratios were extrapolated using linear regression analysis and expressed as mean 6 SD (n = 4). Error bars represent standard deviation (SD). Asterisks indicate values significantly different from the mean at P,0.05. (D) Linear regression was used to quantify the differences between the bands in (B). d i 10 1371/j l 0037944 009 2-DE and Image Analysis Biosciences, Piscataway, NJ). The biological variation analysis module was used for inter-gel matching of the internal standard, and all the gel samples were subjected to comparative cross-gel statistical analysis based on spot volumes with a t-test. P value ,0.01 was considered statistically significant. Importantly, spots corresponding to the same protein were identified in different gels and their trends of up-regulation and down-regulation were validated in all the gels in order to avoid false positives. At least three sets of gels were run for each sample. Although the DIGE experiments exhibited high sensitivity and good reproducibility, it was not effective in identifying low- abundance proteins as the loading volume in DIGE was significantly lower than that in traditional 2-DE. The total loading sample size was 150 mg for 3 samples labeled with Cy2, Cy3 and Cy5 in the DIGE experiments, but the total loading sample size was 800 mg for each sample in 2-DE. The traditional 2-DE test was introduced to overcome this shortcoming of lower loading Figure 10. Standard curves for the PR-1a gene obtained in real-time PCR assays. Ct values represent the mean of 10 measurements and are shown on the y-axis, whereas differential concentrations are shown on the x-axis. Error bars represent standard deviation (SD). doi:10.1371/journal.pone.0037944.g010 Figure 10. Standard curves for the PR-1a gene obtained in real-time PCR assays. Ct values represent the mean of 10 measurements and are shown on the y-axis, whereas differential concentrations are shown on the x-axis. Error bars represent standard deviation (SD). doi:10.1371/journal.pone.0037944.g010 PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 12 Enhanced Disease Resistance Figure 11. PR-1a gene expression analysis by qPCR. (A) Quantitative real-time PCR was performed using gene-specific primers (listed in Table 5) and the SYBR Green Realtime Master Mix. Expression ratios of the PR-1a gene in plants treated with BTH, Dufulin and exogenous SA at 500 mg/mL at 0, 3, 6, 9 and 12 days. Asterisks indicate statistically significant difference (P,0.05). The different lowercase letters indicate PR-1a expression values that are significantly different between the different treatment groups at the same treatment time at P,0.05. (B) Molecular structures of Dufulin, BTH and SA. doi:10 1371/journal pone 0037944 g011 Figure 11. PR-1a gene expression analysis by qPCR. (A) Quantitative real-time PCR was performed using gene-specific primers (listed in Table 5) and the SYBR Green Realtime Master Mix. 2-DE and Image Analysis Expression ratios of the PR-1a gene in plants treated with BTH, Dufulin and exogenous SA at 500 mg/mL at 0, 3, 6, 9 and 12 days. Asterisks indicate statistically significant difference (P,0.05). The different lowercase letters indicate PR-1a expression values that are significantly different between the different treatment groups at the same treatment time at P,0.05. (B) Molecular structures of Dufulin, BTH and SA. d i 10 1371/j l 0037944 011 doi:10.1371/journal.pone.0037944.g011 volume in DIGE. Expression profiles were acquired by comparing the results between groups B and E, and results were obtained using both 2-DE and DIGE. For the comparative analysis, samples from these groups were first separated by 2-DE according to the conventional method [13,32]. The gel was then stained via the silver staining method to identify protein spots with differential expression. The protein spots found by silver staining were excised from another SDS-PAGE gels dyed with coomassie brilliant blue, then identified by MS. Image analysis was done using the DeCyder differential analysis software (Version 4.0, Amersham Biosciences). modifications in terms of oxidation (of Met). A peptide tolerance level of 100 ppm, a fragment mass tolerance of 60.5 Da, and a peptide charge of 1+ were selected for the study. Only significant hits, as defined by the MASCOT probability analysis (P,0.05), were accepted. Thirty-one protein spots were identified using the AB SCIEX 4700 Proteomics Analyzer, which was operated in reflection mode and calibrated using AB SCIEX 4700 Calibration Mixture (Applied Biosystems, Foster City, CA). The data obtained from MALDI-TOF/TOF MS/MS was analyzed by GPS Explorer software (version 2.0, Applied Biosystems). Both MS and MS/MS data were acquired with a neodymium:yttrium–aluminum–garnet laser at a sampling rate of 200 Hz. The MS/MS mode was operated at 1 kV of collision energy. Collision-induced dissocia- tion was carried out using air as the collision gas. For MS spectra, the peaks were calibrated using trypsin auto-digestion peaks and subsequently smoothed. The signal-to-noise criterion was set at 25 or greater. Monoisotopic masses were processed for identification. For MS/MS spectra, the peaks were calibrated by default and smoothed. All the peaks were de-isotoped. Protein Digestion and Protein Identification by MS g y Protein spots were excised from several gels stained with coomassie brilliant blue, destained in 30 mM potassium ferricy- anide/100 mM sodium thiosulfate (1:1, v/v) for 20 min, and subsequently with Milli-Q water. The spots were incubated in 0.2 M NH4HCO3 for 20 min and then lyophilized. Each spot was digested overnight in 2 mL of 12.5 ng/mL trypsin in 0.1 M NH4HCO3. The peptides were extracted thrice with a mixture of 50% acetonitrile and 0.1% trifluoroacetic acid. The digests of the main protein spots were analyzed on a MALDI-TOF/TOF mass spectrometer (AB SCIEX 4700 Proteomics Analyzer, USA) and a Finnigan linear ion trap quadrupole mass spectrometer (LTQ; ThermoQuest, San Jose, CA, USA). Tryptic digests were prepared according to the manufacturer’s instructions (AB SCIEX 4700 Proteomics Analyzer, USA). Tandem MS (MS/MS) data were acquired with a nitrogen laser at a sampling rate of 25 Hz. The data obtained were submitted to the MASCOT software for protein identification. The NR Protein Database (6,122,577 sequences; 209,623,0148 residues) of the National Center for Biotechnology Information (NCBI-nr 20080221) was then searched with reference to green plants. The other parameters involved in the search were trypsin, one missed cleavage, fixed modifications of the carbamidomethyl (C) group, and variable Four additional protein spots were identified using the Finnigan LTQ mass spectrometer (ThermoQuest) coupled with a Surveyor HPLC system (ThermoQuest). Reversed-phase HPLC was con- ducted using a Surveyor LC system (Thermo Finnigan, San Jose, CA) on a C18 column (RP, 180 mm6150 mm; BioBasicH C18, 5 mm, Thermo Hypersil-Keystone). The pump flow rate was divided 1:120 to achieve a column flow rate of 1.5 mL/min. Mobile phase A consisted of 0.1% formic acid in water, whereas mobile phase B consisted of 0.1% formic acid in acetonitrile. The tryptic peptide mixtures were eluted using a gradient of 2%–98% B over 60 min. The temperature of the heated capillary was set at 170uC. A voltage of 3.4 kV applied to the electrospray ionization needle resulted in a distinct signal. The normalized collision energy was 35.0. The mass spectrometer was set up in such a way that one full scan was followed by three MS/MS scans on the PLoS ONE \| www.plosone.org P May 2012 \| Volume 7 \| Issue 5 \| e37944 PLoS ONE \| www.plosone.org 13 Enhanced Disease Resistance Figure 12. Dufulin-induced virus resistance in N. glutinosa. (A) Local lesions in the leaves of N. Protein Digestion and Protein Identification by MS glutinosa after inoculation of 0.05% DMSO solution (the CK treatment group). (B) Local lesions in the leaves of N. glutinosa after inoculation with Dufulin at 500 mg/mL. Small white needle-like points evenly distributed on the leaves represent silicon carbide. (C) Number of local lesions. Values are expressed as the mean of at least 5 replicates. Asterisks indicate statistically significant differences at P,0.05 () and P,0.01 () when compared with the controls. doi:10.1371/journal.pone.0037944.g012 Figure 12. Dufulin-induced virus resistance in N. glutinosa. (A) Local lesions in the leaves of N. glutinosa after inoculation of 0.05% DMSO solution (the CK treatment group). (B) Local lesions in the leaves of N. glutinosa after inoculation with Dufulin at 500 mg/mL. Small white needle-like points evenly distributed on the leaves represent silicon carbide. (C) Number of local lesions. Values are expressed as the mean of at least 5 replicates. Asterisks indicate statistically significant differences at P,0.05 () and P,0.01 (*) when compared with the controls. doi:10.1371/journal.pone.0037944.g012 Figure 12. Dufulin-induced virus resistance in N. glutinosa. (A) Local lesions in the leaves of N. glutinosa after inoculation of 0.05% DMSO solution (the CK treatment group). (B) Local lesions in the leaves of N. glutinosa after inoculation with Dufulin at 500 mg/mL. Small white needle-like points evenly distributed on the leaves represent silicon carbide. (C) Number of local lesions. Values are expressed as the mean of at least 5 replicates. Asterisks indicate statistically significant differences at P,0.05 () and P,0.01 (**) when compared with the controls. doi:10.1371/journal.pone.0037944.g012 three most intense ions in the spectrum. The Dynamic ExclusionTM settings were as follows: repeat count, 2; repeat duration, 0.5 min; and exclusion duration, 2.0 min. proteins was done based on their GO numbers using the BLAST software. Bioinformatic Analysis g y Leaves of N. tabacum K-326 plants were ground to a fine powder in liquid nitrogen and homogenized in an ice-cold mortar with homogenization buffer (200 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, and 100 mM Tris–HCl, pH 7.8) supplemented with Complete Protease Inhibitor Cocktail (Roche, Basel) with 4% b-mercaptoethanol (2-ME). The homogenate was centrifuged at 12,000 g for 10 min at 4uC. The supernatant containing the target protein was measured and determined using the Bradford assay. Extracted proteins of the same amount in each treatment group were analyzed by SDS-PAGE followed by western blotting. Proteins were electrotransferred to a Hybond-P membrane (Amersham 13 Biosciences, Little Chal- font, UK) and detected using antibodies against lipid-associated proteins (1:1500) as well as anti-rabbit IgG (alkaline phosphatase conjugate) antibody raised in goat (1:3000; SIGMA). Antibodies For the overall analysis of the data generated from the proteomic studies and the rapid identification of the target, a novel bioinformatics-based approach was used. The functional categories of the proteins were defined according to the GO database. The sequences were downloaded from GenBank based on their genInfo identifier (GI) number, and 13 redundant data were discarded. Furthermore, the sequence information for GI entries 76869447 and 51964984 were not acquired. The sequences were analyzed using the Blast2GO procedure [14– 16], and the NR database was searched using BLAST with the E- value fixed at 1e210. The GO numbers corresponding to the sequences were acquired through the abovementioned method, and the categorization (MF, BP and CC) of differentially expressed PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 14 Enhanced Disease Resistance Table 5. Primer sequences used in semi-quantitative RT-PCR and qPCR. Table 5. Primer sequences used in semi-quantitative RT-PCR and qPCR. Spot no. Bioinformatic Analysis GI Gene name Forward primer Reverse primer Product (bp) 178 U35619.1 bisphosphate carboxylase oxygenase 59-gtgcaaacattttggcaatg-39 59-attcagcaactgcatctgga-39 178 187 U15605.1 truncated N protein 59-ggggagtcggtaaaacaaca-39 59-cagagagaagggcattttgc-39 153 210 AJ866275.1 putative cell cycle protein 59-tggtaccgggaacttcagtc-39 59-tggtcacagagctccatttg-39 228 301 NC_001879 ATP synthase CF1 alpha subunit 59-aaaagggcttctgcttcctc-39 59-aaaagggcttctgcttcctc-39 153 422 Z14980.1 ribulose-bisphosphate carboxylase activase 59-cggagaattggaaagtggaa-39 59-acgtttgtcgggttatcagc-39 219 457 AJ250431.1 beta-galactosidase 59-agcctggaactgattgtgct-39 59-tggttgggactttttcttgg-39 240 598 gi\|223593 carboxylase/oxygenase 59-tcagatcaagggcattttcc-39 59-caagcgccatatcagcatta-39 240 701 DQ442995 Phosphomannomutase 59-agcacctcgcaaggaagtta-39 59-tccttgagcttctcgtctcc-39 240 769 AY383625.1 harpin binding protein 59-aatttgccagtcgggtattg-39 59-caatttccacctcccttgaa-39 247 990 AB449366 germin like protein 59-ggcttagtgcagctggaaac-39 59-cagggtgagtgtgaaatgga-39 153 1015 AY220076.1 oxygen evolving complex photosystem II 59-acctgcagttgaggtctgct-39 59-catttgctcctgagacgaca-39 180 1041 gi\|31711507 24 K germin-like protein 59-cgatttctgtgttggggact-39 59-ggtgtgactgcggctttaat-39 150 5942 gi\|12643758 ribulose bisphosphate carboxylase/oxygenase-2 (RuBisCO activase) 59-tgttcctgctgaagacgttg-39 59-agttggtggtccgtcaaaag-39 175 6071 J02979.1 lignin-forming peroxidase 59-agcccttgcatctgaaattg-39 59-ttccaaatgtgtgtgcacct-39 215 6095 gi\|255542955 NAD-dependent epimerase/dehydratase 59-gcaggcaaaaggagcactac-39 59-ggtctgattcacctggcaat-39 179 6138 XM_002520879 stability/assembly factor HCF 136 59-acatccaatgggggatacaa-39 59-cggcttgagacagcaacata-39 164 6257 Y15489 plastid-lipid-associated protein 59-agcagaggaagaaccaccaa-39 59-ctcggtaggagcaggagttg-39 178 6288 AY220076.1 complex 33-kDa photosystem-II protein 59-acctgcagttgaggtctgct-39 59-catttgctcctgagacgaca-39 180 6388 AM087457.1 chloroplast cysteine synthase 1 59-cttattgttgccgtggacct-39 59-gcaatgtttgcaacacaacc-39 157 6449 DQ222496.1 cytosolic isoform of triosephosphate isomerase 59-gttgtgagccctccatttgt-39 59-ccaaaattacccacggaatg-39 163 6527 gi\|52000814 oxygen-evolving enhancer protein 2-1 59-gtagcgatcgccttcttgac-39 59-ttggggttaataggggaagg-39 204 6545 AB120535 CDK-5 regulatory subunit associated 59-gtcaagcaaaggaggctcac-39 59-atgacaccctatggcctctg-39 169 6556 X64198 photosystem I light-harvesting chlorophyll a/b-binding protein 59-gtttccccctctgtcctctc-39 59-gggcagaaccatcaaggtaa-39 151 6563 AB093097 Superoxide dismutase 59-agctacatgacgccatttcc-39 59-ccctgtaaagcagcaccttc-39 238 6675 D13718.1 PSI-D1 precursor 59-ctcaaattggcccgtaaaga-39 59-tgaaattctgcccaactcct-39 181 6883 AY220079.1 carboxylase small subunit precursor protein 59-catggttgcacctttcactg-39 59-ttccaaacaaggaacccatc-39 238 AF480488 NPR1 59-tagcgtattgcgatgcaaag-39 59-ttccatcggatgtcagatca-39 180 AF480489 EDS1 59-aggccgaagcgttataggtt-39 59-aaaacatcatcgcccagaag-39 203 X05959.1 PR-1a 59-caatacggcgaaaacctagctga -39 59- cctagcacatccaacacgaa-39 170 X03913 PR-5 59-gcttccccttttatgccttc-39 59-cctgggttcacgttaatgct-39 150 reference gene GQ339768.1 b-actin 59-ttgtccgtgacatgaaggag-39 59-atcatggatggctggaagag-39 178 reference gene AF120093.1 EF-1a 59-ctctcaggctcccacttcag-39 59-aagagcttcgtggtgcatct-39 164 reference gene HQ384692.1 18S ribosomal RNA gene 59-atggccgttcttagttggtg-39 59-tgtcggccaaggctataaac-39 235 doi:10.1371/journal.pone.0037944.t005 were diluted in 5% non-fat milk in TBS-T buffer. The color reaction was terminated by adding nitroblue tetrazolium (NBT) (50 mg/mL) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (50 mg/mL), and the blotting membrane was analyzed using Bio-Rad GelDoc XR [33,34]. ribosomal RNA genes were designed with the Beacon Designer software using the gene sequences obtained from GenBank (Table 5). The primers were synthesized (Takara) and plasmid transformation and cloning were conducted using a pGEM-T Cloning Kit (Tiangen Biotech, Beijing, China). Plasmid standards were diluted 10-fold and calculated according to the following formulas: Supporting Information Additional supporting information may be found in the online version of this article. Figure S9 DAG of CC. (TIF) Figure S10 Absolute quantity of the PR-1a gene. (A) Log view of the amplification curve of PR-1a in the control group. (B) Log view of the amplification curves of PR-1a in the treatment groups. (C) Melt curve of PR-1a in the treatment groups. (D) Results of PCR and agarose gel electrophoresis of PR-1a. (TIF) Figure S4 Sequence distribution of cellular component (CC). (TIF) Figure S4 Sequence distribution of cellular component (CC). (TIF) Figure S5 Direct GO count of MF. (TIF) Figure S6 Direct GO count of BP. (TIF) Figure S7 Direct GO count of CC. (TIF) Figure S2 Sequence distribution of molecular function (MF). (TIF) Figure S2 Sequence distribution of molecular function (MF). (TIF) Induction of Protective Activity Against TMV Figure S3 Sequence distribution of biological processes (BP). (TIF) Dufulin was dissolved in 0.05% DMSO to concentrations of 100, 200, 300, 400, and 500 mg/mL, and the solution was sprayed onto 8-week-old N. glutinosa plant leaves. Tobacco leaves that were sprayed with standard 0.05% DMSO solution comprised the control group. Five days after spraying, the plants were inoculated with TMV, and then maintained in a growth chamber at 25uC and 80% humidity under a 16-h light/8-h dark cycle for 3 days. The numbers of local lesions and of mottled leaves were documented. Five leaves of each plant were inoculated with TMV in each treatment group of at least four replicates [25]. The experiment was repeated thrice with similar results. Figure S4 Sequence distribution of cellular component (CC). (TIF) RNA Extraction and RT-PCR Tobacco leaf samples were collected and frozen immediately in liquid nitrogen until use. Total RNA was extracted using the Takara Trizol Reagent Kit. DNase digestion followed by passing through adsorption columns was carried out to eliminate possible contamination with DNA. Two micrograms of total RNA was used as a template to synthesize first-strand cDNA with SuperScript VILO cDNA Synthesis Kit (Life Technologies). The primers for 26 differentially expressed genes (based on proteomics), EDS1, NPR1, PR-1a, PR-5, b-actin, EF-1a and 18S 1. Plasmid Sample (ng/mL) = OD260650-fold Dilution. 2. Sample MW = Base Number6324. 3. Copies of Test Sample (copies/mL) = Concentration of Test Sample/Sample MW6661014. 1. Plasmid Sample (ng/mL) = OD260650-fold Dilution. 2. Sample MW = Base Number6324. 3. Copies of Test Sample (copies/mL) = Concentration of Test Sample/Sample MW6661014. 1. Plasmid Sample (ng/mL) = OD260650-fold Dilution. 2. Sample MW = Base Number6324 3. Copies of Test Sample (copies/mL) = Concentration of Test Sample/Sample MW6661014. The copies of unknown samples (copies/mL) were calculated using a regression equation. Quantitative real-time PCR was performed using the SYBR Green reagent mix in an iCycler iQTM PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 15 Enhanced Disease Resistance spectra for protein spots no.6449. Part 34.The MS spectra for protein spots no.6556. Part 35.The MS spectra for protein spots no.6395. Part 36.The MS spectra for protein spots no.6883. (ZIP) spectra for protein spots no.6449. Part 34.The MS spectra for protein spots no.6556. Part 35.The MS spectra for protein spots no.6395. Part 36.The MS spectra for protein spots no.6883. (ZIP) instrument according to the manufacturer’s instructions (Bio-Rad, USA). Semi-quantitative RT-PCR was also performed on a Bio- Rad iCycler iQTM Real-Time PCR. qPCRs were run with a program consisting of 95uC denaturation for 30 second, followed by 58uC annealing for 30 second, and 72uC extension for 40 second for 40 cycles. Statistical Analysis ANOVA was conducted using the statistical software SPSS (version 11.5). Means (of opposite ratios in western blotting assays, of Ct values and gene copies in RT-PCR analysis, and of lesion number in the protective assay) were compared using the LSD test (homogeneity of variance, P.0.05) or Dunnett’s T3 test (hetero- geneity of variance, P,0.05). Figure S8 DAG of BP. (TIF) Figure S9 DAG of CC. (TIF) Supporting Information Table S1 Differentially expressed proteins identified by DIGE and MS. 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(2008) 3-acetonyl-3- hydroxyoxindole: a new inducer of systemic acquired resistance in plants. Plant Biotechnol J 6: 301–308. 13. Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, et al. (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21: 3666–3672. 31. Gooding GV, Jr, Hebert TT (1967) A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57(11): 1285. 32. Yan SP, Zhang QY, Tang ZC, Su WA, Sun WN (2005) Comparative proteomic analysis provides new insights into chilling stress responses in rice. Mol Cell Proteomics 53: 484–496. p y p 14. References 1. Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, et al. (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257: 85–88. 17. Metraux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, et al. (1990) Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250: 1004–1006. y 2. Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 284: 2148–2152. 18. Uknes S, Mauch-Mani B, Moyer M, Potter S, Williams S, et al. (1992) Acquired resistance in arabidopsis. Plant Cell 4: 645–656. 19. Van Loon LC (1997) Induced resistance in plants and the role of pathogenesis related proteins. Eur J Plant Pathol 103: 753–765. 3. Wang KL, Li H Ecker JR (2002) Ethylene biosynthesis and signaling networks. Plant Cell 14: S131–S151. 20. Edreva A (2005) Pathogenesis-related proteins: research progresssin the last 15 years. Gen Appl Plant Physiology 31(1–2): 105–124. 4. Camehl I, Sherameti I, Venus Y, Bethke G, Varma A, et al. (2010) Ethylene signalling and ethylene-targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis between the endophytic fungus Piriformospora indica and Arabidopsis thaliana. New Phytol 185: 1062–1073. 21. Kang BC, Yeam I, Jahn MM (2005) Genetics of plant virus resistance. Annu Rev Phytopathol 43: 581–621. 22. Friedrich L, Lawton K, Ruess W, Masner P, Specker N, et al. (1996) A benzothiadiazole derivative induces systemic acquired resistance in tobacco. Plant J 10(1): 61–70. 5. Liu FQ, Liu HX, Jia Q, Wu XJ, Guo XJ, et al. (2006) The internal glycine-rich motif and cysteine suppress several effects of the HpaGXooc protein in plants. Bacteriology, 96(10): 1052–1059. 23. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, et al. (1993) Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261: 754–756.24. 6. Lee J, Klessig DF, Nu¨rnberger T (2001) A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity. Plant Cell 13: 1079–1093. 24. Roger Hull, Richard Ellis Ford Matthews (2002) Matthews’ plant virology: Bei Jing: Academic Press. pp 56–86, 736–807. p y 7. Peng JL, Dong HS, Dong HP, Delaney TP, Bonaserab, JM, et al. (2003) Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes. Author Contributions Conceived and designed the experiments: BS SY. Analyzed the data: CZ BS. Contributed reagents/materials/analysis tools: CZ SY BS. Wrote the paper: CZ SY BS. Participated in discussions about the manuscript and offered necessary assistance to ZC in the experiments: LJ DH. Performed the antiviral tests: ZC MZ XL ZW HF LB JL DY. Conducted the proteomic tests: CH. Table S1 Differentially expressed proteins identified by DIGE and MS. (DOCX) (DOCX) PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 16 Enhanced Disease Resistance Enhanced Disease Resistance Table S8 KEGG categorization of differentially ex- pressed proteins based on MS data. Table S1 Differentially expressed proteins identified by DIGE and MS. (DOCX) Figure S1 The MS spectra for protein spots with different expression. Part 1.The MS spectra for protein spots no.301. Part 2.The MS spectra for protein spots no.178. Part 3.The MS spectra for protein spots no.457. Part 4.The MS spectra for protein spots no.598. Part 5.The MS spectra for protein spots no.769. Part 6.The MS spectra for protein spots no.990. Part 7.The MS spectra for protein spots no.1015. Part 8.The MS spectra for protein spots no.1041. Part 9.The MS spectra for protein spots no.187. Part 10.The MS spectra for protein spots no.210. Part 11.The MS spectra for protein spots no.422. Part 12.The MS spectra for protein spots no.701. Part 13.The MS spectra for protein spots no.1042. Part 14.The MS spectra for protein spots no.1413-1. Part 15.The MS spectra for protein spots no.1413-2. Part 16.The MS spectra for protein spots no.6388. Part 17.The MS spectra for protein spots no.6675. Part 18.The MS spectra for protein spots no.6818. Part 19.The MS spectra for protein spots no.5942. Part 20.The MS spectra for protein spots no.6071. Part 21.The MS spectra for protein spots no.6138. Part 22.The MS spectra for protein spots no.6087. Part 23.The MS spectra for protein spots no.6095. Part 24.The MS spectra for protein spots no.6257. Part 25.The MS spectra for protein spots no.6288. Part 26.The MS spectra for protein spots no.6265. Part 27.The MS spectra for protein spots no.6287. Part 28.The MS spectra for protein spots no.6246. Part 29.The MS spectra for protein spots no.6291. Part 30.The MS spectra for protein spots no.6527. Part 31.The MS spectra for protein spots no.6563. Part 32.The MS spectra for protein spots no.6545. Part 33.The MS Table S2 The ratio of up-regulation to differentially expressed protein in group E comparing with that in group B in 2-DE. (DOCX) Table S3 Differentially expressed proteins identified by 2-DE and MS. (DOCX) Table S4 Sequence data (FASTA) of the differentially expressed proteins obtained from MS. (DOCX) Table S5 Gene Ontology (GO) categorization of differ- entially expressed protein spots based on their molec- ular function. (DOCX) Table S6 GO categorization of differentially expressed proteins based on their localization in various cellular components. (DOCX) Table S7 GO categorization of differentially expressed proteins based on their involvement in various biolog- ical processes. Acknowledgments We thank the Shanghai Applied Protein Technology Co. Ltd. for help with the MS experiments. We also thank Prof. Chuan He for critically reading the manuscript and giving advice and support. p the manuscript and giving advice and support. References Go¨tz S, Garcı´a-Go´mez JM, Terol J, Williams TD, Nagaraj SH, et al. (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucl Acids Res 36: 3420–3435. 33. Schneppenheim R, Budde U, Dahlmann N, Rautenberg P (1991) Lumino- graphy-a new, highly sensitive visualization method for electrophoresis. Electrophoresis 12: 367–372. 15. Conesa A, Go¨tz S (2008) Blast2GO: A comprehensive suite for functional analysis in plant genomics. Int J Plant Genomics, 1–12. 16. Conesa A, Go¨tz S, Garcı´a-Go´mez JM, Terol J, Talo´n M, et al. (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3676. 34. Gillespie PG, Hudspeh AJ (1991) Chemiluminescence detection of proteins from single cells. Proc Natl Acad Sci USA 88: 2563–2567. PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37944 17
https://openalex.org/W4393062244	https://ojs.uma.ac.id/index.php/diversita/article/download/9314/5323	Indonesian	null	Pengaruh Achievement Goal Orientation terhadap Prokrastinasi pada Mahasiswa Tingkat Awal dan Mahasiswa Tingkat Akhir	Jurnal Diversita	2,023	cc-by	3,949	Jurnal Diversita, 9 (2) Desember (2023) ISSN 2461-1263 (Print) ISSN 2580-6793 (Online) DOI: 10.31289/diversita.v9i2.9314 Jurnal Diversita, 9 (2) Desember (2023) ISSN 2461-1263 (Print) ISSN 2580-6793 (Online) DOI: 10.31289/diversita.v9i2.9314 The Effect of Achievement Goal Orientation on Procrastination in First Year and Final Year Student Harnadia Firsya Difa(1) & Meilani Sandjaja(2) Universitas Ciputra Surabaya, Indonesia Harnadia Firsya Difa(1) & Meilani Sandjaja(2) Universitas Ciputra Surabaya, Indonesia 23; Diproses: 14 Oktober 2023; Diaccept: 23 November 2023; Dipublish: 25 Desember 2023 Disubmit: 27 Maret 2023; Diproses: 14 Oktober 2023; Diaccept: 23 November 2023; Dipublish: 25 Desember 2023 Corresponding author: hfirsyadifa@student.ciputra.ac.id Disubmit: 27 Maret 2023; Diproses: 14 Oktober 2023; Diaccept: 23 November 2023; Dipublish: 25 Desember 2023 Corresponding author: hfirsyadifa@student.ciputra.ac.id Disubmit: 27 Maret 2023; Diproses: 14 Oktober 2023; Diaccept: 23 November 2023; Dipublish: 25 D Corresponding author: hfirsyadifa@student.ciputra.ac.id Corresponding author: hfirsyadifa@student.ciputra.ac.id Abstrak Kewajiban mahasiswa adalah menyelesaikan tugas-tugas. Namun banyak ditemukan mahasiswa melakukan prokrastinasi dalam menyelesaikan tugas jika dilakukan secara terus menerus dapat merugikan mahasiswa seperti menurunnya ipk dan terhambat untuk mencapai tujuan. Penelitian ini menggunakan pendekatan kuantitatif menggunakan dengan desain regresi linear karena peneliti ingin mengetahui pengaruh achievement goal orientation terhadap prokrastinasi pada mahasiswa tingkat awal dan akhir. Responden dalam penelitian ini adalah mahasiswa tingkat awal dan mahasiswa tingkat akhir. Subjek penelitian sebanyak 133 mahasiswa dengan 67 mahasiswa tingkat akhir dan 66 mahasiswa tingkat awal. Hasil menyatakan bahwa achievement goal orientation berpengaruh secara signifikan terhadap prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir. Namun terdapat perbedaan besaran pengaruh achievement goal orientation terhadap prokrastinasi mahasiswa tingkat awal dan juga mahasiswa tingkat akhir. Dalam riset ini achievement goal orientation memberikan pengaruh yang lebih rendah terhadap prokrastinasi mahasiswa tingkat akhir dibandingkan mahasiswa tingkat awal dikarenakan terdapat faktor lain yang mempengaruhi prokrastinasi selain achievement goal orientation. a Kunci: Achievement Goal Orientation; Mahasiswa Tingkat Awal; Mahasiswa Tingkat Akhir krastinasi. How to Cite: Diva, H. F. & Sandjaja, M. (2023), Pengaruh Achievement Goal Orientation Terhadap Prokrastinasi pada Mahasiswa Tingkat Awal dan Mahasiswa Tingkat Akhir, Jurnal Diversita, 9 (2): 198- 206. Jurnal Diversita Available online https://ojs.uma.ac.id/index.php/diversita Abstract University students have an obligation to complete the tasks. However, it is found that many students do procrastinate in completing assignments if done continuously, it can harm students such as decreasing their gpa and being difficult to achieve their goals. This study uses a quantitative approach using a linear regression design because researchers want to know the effect of achievement goal orientation on procrastination in early and final year students. Respondents in this study were early and final year students. The participants in this study totaled 133 students with 67 final year students and 66 early years students. The results in this study indicate that achievement goal orientation has a significant effect on procrastination in early and final year students. However, there are differences in the magnitude of the effect of achievement goal orientation on procrastination in early and final year students. In this study, achievement goal orientation has a lower effect on the procrastination of final year students than early year students because there are other factors that can influence procrastination besides achievement goal orientation. f p g Keywords: Achievement Goal Orientation; Beginning Student; Final Year Student; Procrastination. How to Cite: Diva, H. F. & Sandjaja, M. (2023), Pengaruh Achievement Goal Orientation Terhadap Prokrastinasi pada Mahasiswa Tingkat Awal dan Mahasiswa Tingkat Akhir, Jurnal Diversita, 9 (2): 198- 206. 198 Diva, H. F. & Sandjaja, M, Pengaruh Achievement Goal Orientation Terhadap Prokrastinasi pada Mahasiswa Diva, H. F. & Sandjaja, M, Pengaruh Achievement Goal Orientation Terhadap Prokrastinasi pada Mahasiswa PENDAHULUAN berulang dapat memberikan dampak berupa ipk yang rendah jika dibandingkan dengan mahasiswa tingkat awal yang tidak melakukan prokrastinasi. Namun pada mahasiswa tingkat akhir perilaku prokrastinasi akan rendah jika mahasiswa merasakan takut gagal ketika tidak dapat menyelesaikan skripsi. Sebaliknya, perilaku prokrastinasi akan tinggi pada mahasiswa tahun terakhir jika tidak merasakan takut akan gagal dalam menyelesaikan skripsi (Mudjiran & Mujahidah, 2019). Hal ini disebabkan mahasiswa percaya bahwa kegagalan dalam menyelesaikan skripsi dapat merubah rencana yang telah disiapkan untuk masa depan selain itu mahasiswa yang gagal dalam menyelesaikan skripsi akan merasa rendah diri menganggap tidak pintar dan tidak kompeten. Mahasiswa memiliki kewajiban dalam menyelesaikan tugas-tugas. Namun, sering ditemukan mahasiswa mengalami kesulitan dalam mengatur waktu antara menyelesaikan tugas, bersenang-senang dan organisasi. Oleh karena itu mahasiswa mengerjakan tugas dengan rasa malas yang dapat menunda dan cenderung menghindari tugas. Menunda dalam mengerjakan tugas disebut juga prokrastinasi. Prokrastinasi merupakan mengundur waktu dalam menyelesaikan tugas oleh individu secara sadar dan cenderung mencari kegiatan yang lebih menyenangkan jika dihadapkan dengan tugas yang menyulitkan (Tuckman, 1990). Penelitian yang dilaku- kan Litbang data PK identitas (2018) menunjukkan bahwa mahasiswa pada Universitas Hasanudin melakukan pro- krastinasi dalam menyelesaikan kewa- jiban berupa , belajar sebelum ujian dan membaca. Selain itu riset yang dilakukan Jannah (2014) menyatakan 307 mahasis- wa Fakultas Ilmu Pendidikan Universitas Negeri Surabaya melaksanakan prokras- tinasi seperti membaca referensi dan buku mengenai tugas yang diberikan oleh dosen sebanyak 285 mahasiswa (93%). Menurut Conroy dan Elliot (2004) ketakutan akan kegagalan dapat mempengaruhi kinerja pada mahasiswa. Mahasiswa yang mengalami kegagalan akan merasa malu terhadap lingkungan sekitarnya dan takut karena tidak memiliki masa depan yang pasti. Menurut Barabanshchikova (2019) mahasiswa pada tahun pertama melakukan perilaku prokrastinasi karena lingkungan sekitar yang tidak dikenal sehingga mahasiswa butuh waktu untuk melakukan adaptasi dengan dunia perkuliahan, rasa malas, dan tidak dapat mengatur waktu dengan baik. Berbeda dengan mahasiswa tingkat akhir cenderung melakukan perilaku prokrastinasi karena sebagai coping negatif yang terbentuk karena ketakutan akan kegagalan dan konflik selama menjalani perkuliahan. Hasil penelitian yang dilakukan Nugroho (2018) terdapat perbedaan perilaku prokrastinasi pada mahasiswa yang baru memulai dunia perkuliahan dan mahasiswa tingkat akhir dengan penelitian tersebut ditemukan bahwa mahasiswa yang berada di tingkat awal cenderung melakukan perilaku prokras- tinasi yang rendah jika dibandingkan dengan mahasiswa tingkat akhir. Menurut Oematan (2013) mahasiswa tingkat awal yang melakukan prokrastinasi secara Perilaku prokrastinasi akademik yang dilakukan secara terus menerus pada 199 Jurnal Diversita, 9 (2) Desember 2023: 198-206. Jurnal Diversita, 9 (2) Desember 2023: 198-206. PENDAHULUAN & Sandjaja, M, Pengaruh Achievement Goal Orientation Terhadap Prokrastinasi pada Mahasiswa approach, Mastery avoidance, Performance approach dan Performance avoidance. salah satu dimensi achievement goal orientation memiliki peran penting dalam perilaku prokrastinasi karena dengan mastery approach dan performance approach individu akan berusaha mempelajari hal baru sehingga cenderung akan menggunakan waktu yang tepat untuk menyelesaikan tugas. PENDAHULUAN mahasiswa dapat merugikan mahasiswa. Menurut Lubis (2018) prokrastinasi dapat memberikan dampak yang serius berupa menurunnya prestasi pada mahasiswa jika berlangsung secara terus menerus dapat mengarah kegagalan. Hal ini disebabkan karena mahasiswa tidak dapat mengatur waktunya dengan baik dalam menyelesaikan tugas. Menurut Mortazavi et.al, (2015) dampak mahasiswa yang melakukan prokrastinasi secara terus menerus dan dilakukan secara sadar dapat menjadi sebuah hambatan dalam mencapai tujuan untuk berprestasi. goal orientation sangat penting bagi mahasiswa untuk mencapai tujuan dalam berprestasi dan mahasiswa akan merasa tertantang untuk melakukan prestasi. Mahasiswa yang memiliki achievement goal orientation melihat bahwa tujuan dalam berprestasi akan menjadi peluang dalam bertumbuh dan siap akan tugas yang akan datang. Menurut Prabowo & Setiyani (2019) dengan memiliki tujuan dalam berprestasi membuat individu menyiapkan dirinya untuk meningkatkan kemampuannya dalam menghadapi tugasnya. Individu yang memiliki strategi untuk mencapai tujuannya dalam berprestasi secara tidak langsung dapat mengatur waktu secara efektif maka terhindar perilaku prokrastinasi dalam mengerjakan tugas atau penundaan dalam menyelesaikan tugas (Santika, 2017). Sebaliknya, mahasiswa yang tidak memiliki achievement goal orientation akan merasa tidak tertantang dalam menyelesaikan tugas dan cenderung akan meninggalkan tugas. Oleh karena itu dengan tidak memiliki tujuan mahasiswa akan perilaku prokrastinasi yang tinggi. Hal ini didukung oleh penelitian Nur Wangid (2014) ketika mahasiswa tidak memiliki tujuan dalam berprestasi maka mahasiswa akan ragu- ragu dan cenderung untuk melakukan prokrastinasi dalam menyelesaikan tugas. Berdasarkan uraian diatas prokrastinasi merupakan masalah yang sering terjadi di kalangan mahasiswa. Walaupun perilaku prokrastinasi dilaku- kan secara sadar dan dampak yang merugikan mahasiswa, namun mahasiswa tetap mengulangi perilaku prokrastinasi. Prokrastinasi pada mahasiswa dapat dipengaruhi beberapa hal. Menurut Steel (2007) prokrastinasi dapat dipengaruhi 4 faktor yaitu self control, rendahnya self efficacy, keyakinan takut akan gagal dan self regulatory failure (kegagalan dalam pengaturan diri). Menurut Howwel & Watsons (2007) mahasiswa yang memiliki tujuan atau achievement goal orientation dalam berprestasi dapat membangun komitmen individu terhadap tujuannya, sehingga terhindar dari perilaku prokrastinasi. Hal ini didukung oleh Abe et.al (2014) dengan mempunyai tujuan dan target yang jelas individu akan fokus dalam menetapkan arahnya untuk mencapai tujuan tanpa ada- nya gangguan. Menurut Elliot & Murayama (2008) achievement goal orientation adalah tujuan kognitif yang dinamis dengan fokus kompetensi. Achievement Penelitian terdahulu yang dilakukan oleh Howwel & Watson (2007) menunjukkan bahwa salah satu dimensi achievement goal orientation yaitu mastery avoidance terdapat hubungan dengan prokrastinasi. Selain itu penelitian terdahulu Howwel & Burro (2009) menunjukkan mastery approach dan performance approach yang merupakan 200 Diva, H. F. Jurnal Diversita, 9 (2) Desember 2023: 198-206. Jurnal Diversita, 9 (2) Desember 2023: 198-206. Tabel 2 Tingkatan prokrastinasi berdasarkan jenis kelamin pada mahasiswa tingkat awal Jenis Kelamin Sedang Tinggi Sangat tinggi Total Laki-laki 12 1 2 15 Perempuan 24 20 7 67 Tabel 3 Tempat tinggal mahasiswa tingkat akhir Tinggal Rendah Sedang Tinggi Sangat Tinggi Total Bersama orang tua 6 20 9 2 37 Sendiri (kost) 1 23 4 2 30 Penelitian ini diperoleh 66 populasi dan terdapat 38 mahasiswa tingkat awal yang tinggal sendiri (kost) dan 28 mahasiswa lainnya tinggal bersama orang tua. Dimana diketahui mahasiswa tingkat awal yang tinggal sendiri melakukan prokrastinasi tergolong tinggi sebanyak 12 dan tergolong sedang sebanyak 22 mahasiswa, maka dapat disimpulkan mahasiswa yang tinggal sendiri lebih sering melakukan prokrastinasi. Menurut Khorunnisa et al (2021) mahasiswa yang tinggal sendiri lebih sering melakukan prokrastinasi dikarenakan mahasiswa bebas untuk melakukan aktivitas yang menyenangkan tanpa pengawasan orang tua dan mahasiswa merasa tidak diperhatikan saat mengerjakan tugasnya. Tabel 4 tempat tinggal mahasiswa tingkat awal Tinggal Sedang Tinggi Sangat Tinggi Total Bersama orang tua 14 9 5 28 Sendiri (kost) 22 12 4 38 Analisis data menggunakan JASP versi 0.16.4. Uji hipotesa menggunakan regresi linear dengan hasil p-value <0.01 h l i i j kk hi i di i HASIL DAN PEMBAHASAN Tabel 2 Tingkatan prokrastinasi berdasarkan jenis kelamin pada mahasiswa tingkat awal Tabel 2 Tingkatan prokrastinasi berdasarkan jenis kelamin pada mahasiswa tingkat awal Jenis Kelamin Sedang Tinggi Sangat tinggi Total Laki-laki 12 1 2 15 Perempuan 24 20 7 67 Penelitian ini dilakukan uji reliabilitas untuk menguji konsistensi kuesioner. Uji reliabilitas dilakukan pada 30 responden pertama sehingga hasil uji reliabilitas pada skala prokrastinasi cronbach α 0.883. Selanjutnya uji reliabilitas pada skala achievement goal orientation terbagi menjadi 4 dimensi pada dimensi mastery approach menunjukkan cronbach α 0.894 pada dimensi mastery avoidance menunjukkan cronbach α 0.275 pada dimensi performance approach menunjukkan cronbach α 0.899 dan pada dimensi performance avoidance cronbach α 0.830. Responden pada penelitian ini merupakan mahasiswa tingkat awal berjumlah 66 dan mahasiswa tingkat akhir 67 sehingga total keseluruhan 133 responden. Tabel 3 Tempat tinggal mahasiswa tingkat akhir Tinggal Rendah Sedang Tinggi Sangat Tinggi Total Bersama orang tua 6 20 9 2 37 Sendiri (kost) 1 23 4 2 30 Penelitian ini diperoleh 66 populasi dan terdapat 38 mahasiswa tingkat awal yang tinggal sendiri (kost) dan 28 mahasiswa lainnya tinggal bersama orang tua. Dimana diketahui mahasiswa tingkat awal yang tinggal sendiri melakukan prokrastinasi tergolong tinggi sebanyak 12 dan tergolong sedang sebanyak 22 mahasiswa, maka dapat disimpulkan mahasiswa yang tinggal sendiri lebih sering melakukan prokrastinasi. Menurut Khorunnisa et al (2021) mahasiswa yang tinggal sendiri lebih sering melakukan prokrastinasi dikarenakan mahasiswa bebas untuk melakukan aktivitas yang menyenangkan tanpa pengawasan orang tua dan mahasiswa merasa tidak diperhatikan saat mengerjakan tugasnya. Pada mahasiswa tingkat awal dan akhir tingkatan prokrastinasi dipengaruhi oleh jenis kelamin. Bedasarkan penelitian pada mahasiswa tingkat akhir menunjukkan 30 perempuan melakukan prokrastinasi dengan kategori tinggi. Tabel 1 Tingkatan prokrastinasi bedasarkan jenis kelamin pada mahasiswa tingkat akhir Jenis Kelamin Rendah Sedang Tinggi Sangat tinggi Laki-Laki 1 13 6 2 Perempuan 6 30 7 30 Tabel 4 tempat tinggal mahasiswa tingkat awal Tinggal Sedang Tinggi Sangat Tinggi Total Bersama orang tua 14 9 5 28 Sendiri (kost) 22 12 4 38 Sedangkan pada mahasiswa tingkat awal terdapat 51 perempuan dan 15 laki- laki, dari 51 mahasiswa tingkat awal yang berjenis kelamin perempuan terdapat 20 mahasiswa perempuan mengalami prokrastinasi dengan kategori tinggi sedangkan pada mahasiswa tingkat awal yang berjenis kelamin laki-laki hanya 1 mahasiswa mengalami prokrastinasi dengan kategori tinggi. Analisis data menggunakan JASP versi 0.16.4. METODE PENELITIAN Penelitian ini menggunakan pendekatan kuantitatif desain regresi linear karena peneliti ingin mengetahui pengaruh achievement goal orientation terhadap prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir dengan teknik sampling berupa purposive sampling. Kriteria dalam penelitian ini merupakan mahasiswa tingkat awal dan mahasiswa tingkat akhir dengan usia 18- 24 tahun. Pengumpulan data akan dilakukan menggunakan survei secara online menggunakan google form. Peneliti tertarik untuk melakukan penelitian pengaruh achievement goal orientation dengan prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir. Hal ini karena mayoritas penelitian sebelumnya hanya melakukan penelitian di satu universitas dan jurusan tertentu. Penelitian yang dilakukan Howwel & Watsons (2007) hanya melakukan penelitian di satu universitas dan hanya terpaku pada jurusan psikologi. Adapun tujuan dari penelitian ini adalah untuk mengetahui pengaruh achievement goal orientation terhadap prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir. Prokrastinasi diukur menggunakan skala yang dikembangkan oleh Tuckman (1990) yaitu Tuckman Procrastination Scale terdapat 35 butir yang terdiri dari 3 aspek Tendency to delay or put off doing things, Tendency to have difficulty doing unpleasant thing, Tendency to blame others. Tuckman (1990) prokrastinasi adalah tingkah laku untuk menghindari atau menunda pekerjaan oleh individu dengan sadar dan cenderung mencari hal- hal yang menyenangkan dibandingkan dengan mengerjakan tugas yang sulit. Tuckman (1990) prokrastinasi terbagi menjadi 3 aspek tendency to delay or put off doing things, tendency to have difficulty doing unpleasant thing dan tendency to blame others. Achievement goal orientation diukur menggunakan skala achievement Goal Questionnaire–Revised (AGQ-R) yang dikembangkan oleh Eliot & Murayama (2008) yang berjumlah 12 butir yang terbagi menjadi 4 dimensi performance approach, performance avoidance, mastery approach dan mastery avoidance Kuesioner telah di uji validitas isi dengan pendapat ahli sehingga hasil dari uji validitas terdapat beberapa butir yang telah disesuaikan sehingga bunyi butir menjadi lebih sederhana dan nantinya dapat dipahami oleh responden selanjutnya kuesioner telah dilakukan uji bahasa terhadap 8 responden. Menurut Elliot & Murayama (2008) Achievement goal orientation merupakan tujuan dengan proses kognitif yang bersifat dinamis dan fokus kepada kompetensi. Terdapat 4 dimensi achievement goal orientation menurut Elliot dan McGregor (2001) yaitu Mastery 201 HASIL DAN PEMBAHASAN Uji hipotesa menggunakan regresi linear dengan hasil p-value <0.01 hal ini menunjukkan hipotesis diterima sehingga achievement goal orientation berpengaruh secara signifikan terhadap prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir. Hal ini sejalan dengan penelitian yang dilakukan oleh Howell & Buro (2009) bahwa 202 Diva, H. F. & Sandjaja, M, Pengaruh Achievement Goal Orientation Terhadap Prokrastinasi pada Mahasiswa tujuan untuk berprestasi akan menyelesaikan tugas-tugasnya dengan baik untuk mencapai kesuksesan sehingga mahasiswa tidak akan melakukan prokrastinasi. Menurut Rohmaniyah (2018) mahasiswa yang dapat melakukan perencanaan untuk mencapai tujuannya akan terhindar dari perilaku prokrastinasi. terdapat pengaruh yang signifikan achievement goal orientation terhadap prokrastinasi dimana di dalam penelitian ini ditemukan bahwa achievement goal orientation mampu berpengaruh terhadap tingkatan prokrastinasi mahasiswa. Hal ini disebabkan mahasiswa yang mempunyai tujuan untuk mencapai kesuksesan dan mempunyai prestasi yang lebih baik dari orang lain akan tidak akan melakukan prokrastinasi jika dibandingkan dengan mahasiswa yang meningkatkan wawasan atau bakat untuk menolak kegagalan akan melakukan prokrastinasi. Achievement goal orientation pada mahasiswa tingkat akhir lebih rendah pengaruhnya dibandingkan mahasiswa tingkat awal. Hal ini disebabkan terdapat keadaan yang mampu mempengaruhi tingkatan prokrastinasi pada mahasiswa tingkat akhir selain achievement goal orientation seperti mahasiswa yang kuliah sambil bekerja. Sehingga mahasiswa mengalami masalah dalam membagi waktu antara bekerja dan menyelesaikan tugasnya. Jika tidak dapat membagi waktunya secara efektif maka mahasiswa cenderung melakukan penundaan terha- dap tugas-tugasnya(Arumsari ,2016). Pada mahasiswa tingkat akhir prokrastinasi dapat dipengaruhi faktor lingkungan (Fatima ullah et al, 2019). Lingkungan sekitar individu yang tidak mendukung dapat menjadi sebuah hambatan bagi mahasiswa untuk menyelesaikan tugas- tugasnya dikarenakan mahasiswa lebih banyak membuang waktunya dengan aktivitas yang menyenangkan jika dibandingkan dalam mengerjakan tugas. Menurut Fauziah (2016) faktor prokras- tinasi dapat dipengaruhi oleh saling menggantungkan teman jika mahasiswa dihadapkan dengan pekerjaan yang sulit dan waktu yang terbatas maka mahasiswa akan menggantungkan temannya untuk dijadikan contoh sehingga mahasiswa akan merasa terbantu dan cenderung tidak memiliki rasa tanggung jawab Tabel 5 Hasil uji regresi linear secara keseluruhan Model R R² RMSE P H₀ 0.000 0.000 13.879 H₁ 0.453 0.205 12.424 >0.001 Tabel 5 Hasil uji regresi linear secara keseluruhan Model R R² RMSE P H₀ 0.000 0.000 13.879 H₁ 0.453 0.205 12.424 >0.001 Namun terdapat perbedaan besaran pengaruh achievement goal orientation terhadap prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir. DAFTAR PUSTAKA Arumsari, A. D., & Muzaqi, S. (2016). Prokrastinasi Akademik pada Mahasiswa yang Bekerja. E Jurnal Spirit Pro Patria, 2(2). Asmawan, A. (2016). Analisis Kesulitan Mahasiswa Menyelesaikan Skripsi. Jurnal Pendidikan Ilmu Sosial, 26(2), 51–57 Aziz, A., & Rahardjo, P. (2013). Faktor-faktor Prokrastinasi pada Mahasiswa Tingkat Akhir yang Menyusun Skripsi di Universitas Muhammadiyah Purwekerto Tahun Akademik 2011/2012Psycho Idea, 12(1). HASIL DAN PEMBAHASAN Tabel 6 Hasil uji regresi linear pada mahasiswa tingkat akhir Model R R² RMSE P H₀ 0.000 0.000 13.626 H₁ 0.333 0.111 12.949 >0.001 Tabel 7 Hasil uji regresi linear pada mahasiswa tingkat awal Model R R² RMSE P H₀ 0.000 0.000 13.061 H₁ 0.591 0.349 10.621 >0.001 Pada penelitian ini menunjukkan bahwa achievement goal orientation berpengaruh sebesar 34,9% terhadap prokrastinasi pada mahasiswa tingkat awal. Sedangkan pada mahasiswa tingkat akhir pengaruh achievement goal orientation sebesar 11,1% terhadap prokrastinasi. Prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir dapat dipengaruhi achievement goal orientation. Mahasiswa yang mempunyai Namun terdapat perbedaan besaran pengaruh achievement goal orientation terhadap prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir. Tabel 6 Hasil uji regresi linear pada mahasiswa tingkat akhir Prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir dapat dipengaruhi achievement goal orientation. Mahasiswa yang mempunyai 203 Jurnal Diversita, 9 (2) Desember 2023: 198-206. Jurnal Diversita, 9 (2) Desember 2023: 198-206. terhadap pekerjannya. Selain itu mahasiswa akan melakukan prokrastinasi dikarenakan mengalami stress and fatigue dimana mahasiswa mengalami banyak tekanan dan tuntutan yang dihadapi sehingga dapat melemahkan sikap maha- siswa semester akhir dalam memecahkan masalah sehingga menjadi individu yang melakukan prokrastinasi (Aziz,2013) orientation memberikan pengaruh yang lebih rendah terhadap prokrastinasi mahasiswa tingkat akhir dibandingkan mahasiswa tingkat awal. Diketahui bahwa terdapat faktor-faktor lain mempengaruhi prokrastinasi selain achievement goal orientation. Penelitian ini terdapat batasan yaitu peneliti hanya melakukan uji regresi untuk melihat pengaruh achievement goal orientation terhadap prokrastinasi pada mahasiswa tingkat awal dan tingkat akhir. Oleh karena itu penelitian selanjutnya peneliti menyarankan untuk melakukan uji beda terhadap prokrastinasi pada mahasiswa tingkat awal dan mahasiswa tingkat akhir. Saran kedua selanjutnya peneliti dapat mencari variabel-variabel lain yang dapat menjadi moderator antara achievement goal orientation dan prokrastinasi. Pada Mahasiswa tingkat awal prokrastinasi dapat dipengaruhi faktor- faktor lain selain achievement goal orientation. Menurut Lee & Hall (2020) faktor yang dapat mempengaruhi prokrastinasi pada mahasiswa yang baru memulai perkuliahan adalah adaptasi. Dikarenakan mahasiswa tingkat awal identik dengan masa peralihan dari sekolah menengah atas ke jenjang kuliah sehingga mahasiswa akan cenderung melakukan prokrastinasi dikarenakan perbedaan sistem belajar. Mahasiswa tingkat awal akan mengalami kesulitan saat melakukan tugas akademik di awal perkuliahan dikarenakan mahasiswa dituntut untuk menulis makalah, praktikum dan membuat laporan. Hal ini sangat berbeda dari tugas saat sekolah menengah sehingga mahasiswa tingkat awal cenderung melakukan prokrastinasi SIMPULAN Barabanshchikova, V. V., Boyarinov, D. M., Sultanova, F. R., & Ognyannikova, E. I. (2019). Academic Procrastination in Students at Different Age. The European Proceedings of Social and Behavioural Sciences, 43, 318–328. Berdasarkan hasil penelitian menunjukkan bahwa achievement goal orientation berpengaruh secara signifikan terhadap prokrastinasi mahasiswa tingkat awal dan mahasiswa tingkat akhir. Namun ditemukan perbandingan besaran pengaruh achievement goal orientation terhadap prokrastinasi mahasiswa tingkat awal dan juga mahasiswa tingkat akhir. Pada penelitian ini, achievement goal Burka, J., & Yuen, L. M. (2008). Procrastination: Why You Do It, What to Do About It Now (2nd ed.). Da Capo Lifelong Books. Conroy, D. E., & Elliot, A. J. (2004). Fear of Failure and Achievement Goals in Sport: Addressing the Issue of the Chicken and the egg. Anxiety, Stress & Coping, 17(3), 271– 285. 204 Diva, H. F. & Sandjaja, M, Pengaruh Achievement Goal Orientation Terhadap Prokrastinasi pada Mahasiswa Diva, H. F. & Sandjaja, M, Pengaruh Achievement Goal Orientation Terhadap Prokrastinasi pada Mahasiswa Attribution Theory. Social Sciences, 9(8), 136. Dluha, M. S., & Solicha, S. (2019). Pengaruh Perfeksionisme, Achievement Goal Orientation dan Jenis Kelamin terhadap Prokrastinasi Akademik Mahasiswa. TAZKIYA: Journal of Psychology, 6(1), 53– 68. Lubis, I. S. L. (2018). Hubungan Regulasi Diri dalam Belajar dan Efikasi Diri dengan Prokrastinasi Akademik Mahasiswa. Jurnal Diversita, 4(2), 90. Elliot, A. J., & McGregor, H. A. (2001). A 2 × 2 Achievement Goal Framework. Journal of Personality and Social Psychology, 80(3), 501–519. Mudjiran, M., & Mujahidah, A. (2019). Hubungan antara Ketakutan Akan Kegagalan dengan Prokrastinasi pada Mahasiswa Tingkat Akhir. Jurnal Riset Psikologi, 2(2019). Nugroho, I. H. (2018). Perbedaan Prokrastinasi Akademik pada Mahasiswa tingkat awal dan Tingkat Akhir. Skripsi Universitas Brawijaya. Fatimaullah, Jahada, & Silondae, S. (2019). Faktor- Faktor Penyebab Prokrastinasi Akademik terhadap Penyelesaian Tugas Akhir Mahasiswa Jurusan Bimbingan dan Konseling FKIP Universitas Halu Oleo Kendari, Jurnal Ilmiah Bening (Belajar Bimbingan Dan Konseling), Nur Wangid, M. (2014). Prokrastinasi Akademik: Perilaku yang harus dihilangkan. TAZKIYA: Journal of Psychology, 2(2), 235–248. Fauziah, H. H. (2016). 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https://openalex.org/W3035718362	https://www.aclweb.org/anthology/2020.acl-main.177.pdf	English	null	Probing Linguistic Systematicity	null	2,020	cc-by	8,128	Abstract (1988) provided a famous example: If a compe- tent speaker of English knows the meaning of the sentence John loves the girl, they also know the meaning of The girl loves John. This is because for speakers of English knowing the meaning of the ﬁrst sentence implies knowing the meaning of the individual words the, loves, girl, and John as well as grammatical principles such as how transi- tive verbs take their arguments. But knowing these words and principles of grammar implies know- ing how to compose the meaning of the second sentence. Recently, there has been much interest in the question of whether deep natural language understanding models exhibit systematicity— generalizing such that units like words make consistent contributions to the meaning of the sentences in which they appear. There is ac- cumulating evidence that neural models often generalize non-systematically. We examined the notion of systematicity from a linguistic perspective, deﬁning a set of probes and a set of metrics to measure systematic behaviour. We also identiﬁed ways in which network ar- chitectures can generalize non-systematically, and discuss why such forms of generalization may be unsatisfying. As a case study, we per- formed a series of experiments in the setting of natural language inference (NLI), demonstrat- ing that some NLU systems achieve high over- all performance despite being non-systematic. Deep learning systems now regularly exhibit very high performance on a large variety of natural language tasks, including machine translation (Wu et al., 2016; Vaswani et al., 2017), question answer- ing (Wang et al., 2018; Henaff et al., 2016), visual question answering (Hudson and Manning, 2018), and natural language inference (Devlin et al., 2018; Storks et al., 2019). Recently, however, researchers have asked whether such systems generalize sys- tematically (see §4). ∗Corresponding author Proceedings of the 58th Annual Meeting of the Association for Computational Linguistics, pages 1958–1969 July 5 - 10, 2020. c⃝2020 Association for Computational Linguistics Probing Linguistic Systematicity Emily Goodwin,∗1,5 Koustuv Sinha,2,3,5 Timothy J. O’Donnell1,4,5 1Department of Linguistics, 2School of Computer Science, McGill University, Canada 3Facebook AI Research (FAIR), Montreal 4Canada CIFAR AI Chair, Mila 5Quebec Artiﬁcial Intelligence Institute (Mila) {emily.goodwin, koustuv.sinha}@mail.mcgill.ca timothy.odonnell@mcgill.ca ∗Corresponding author 3 Compositional Structure in Natural Language Compositionality is often stated as the principle that the meaning of an utterance is determined by the meanings of its parts and the way those parts are combined (see, e.g., Heim and Kratzer, 2000). Natural language distinguishes between content or open-class lexical units and function or closed- class lexical units. The former refers to categories, such a nouns and verbs, which carry the major- ity of contentful meaning in a sentence and which permit new coinages. Closed-class units, by con- trast, carry most of the grammatical structure of the sentence and consist of things like inﬂectional mor- phemes (like pluralizing -s in English) and words like determiners, quantiﬁers, and negation (e.g., all, some, the in English). These are mostly ﬁxed; adult speakers do not coin new quantiﬁers, for example, the way that they coin new nouns. Systematicity, the property that words mean the same thing in different contexts, is closely related to compositionality; nevertheless, compositional systems can vary in their degree of systematic- ity. At one end of the spectrum are systems in which primitive units contribute exactly one identi- cal meaning across all contexts. This high degree of systematicity is approached by artiﬁcial formal systems including programming languages and log- ics, though even these systems don’t fully achieve this ideal (Cantwell Smith, 1996; Dutilh Novaes, 2012). Leveraging this distinction gives rise to the possi- bility of constructing probes based on jabberwocky- type sentences. This term references the poem Jabberwocky by Lewis Carroll, which combines nonsense open-class words with familiar closed- class words in a way that allows speakers to recog- nize the expression as well formed. For example, English speakers identify a contradiction in the sen- tence All Jabberwocks ﬂug, but some Jabberwocks don’t ﬂug, without a meaning for jabberwock and ﬂug. This is possible because we expect the words all, some, but, and don’t to contribute the same meaning as they do when combined with famil- iar words, like All pigs sleep, but some pigs don’t sleep. The opposite of systematicity is the phenomenon of contextually conditioned variation in meaning where the contribution of individual words varies according to the sentential contexts in which they appear. Natural languages exhibit such context dependence in phenomena like homophony, poly- semy, multi-word idioms, and co-compositionality. Nevertheless, there are many words in natural language—especially closed-class words like quan- tiﬁers (see below)—which exhibit very little vari- ability in meaning across sentences. 1 Introduction Language allows us to express and comprehend a vast variety of novel thoughts and ideas. This creativity is made possible by compositionality— the linguistic system builds utterances by combin- ing an inventory of primitive units such as mor- phemes, words, or idioms (the lexicon), using a small set of structure-building operations (the gram- mar; Camap, 1947; Fodor and Pylyshyn, 1988; Hodges, 2012; Janssen et al., 2012; Lake et al., 2017b; Szabó, 2012; Zadrozny, 1994; Lake et al., 2017a). Systematicity is the property whereby words have consistent contributions to composed mean- ing; the alternative is the situation where words have a high degree of contextually conditioned meaning variation. In such cases, generalization may be based on local heuristics (McCoy et al., 2019b; Niven and Kao, 2019), variegated similar- ity (Albright and Hayes, 2003), or local approx- imations (Veldhoen and Zuidema, 2017), where the contribution of individual units to the meaning of the sentence can vary greatly across sentences, interacting with other units in highly inconsistent and complex ways. One property of compositional systems, widely studied in the cognitive sciences, is the phe- nomenon of systematicity. Systematicity refers to the fact that lexical units such as words make consistent contributions to the meaning of the sen- tences in which they appear. Fodor and Pylyshyn This paper introduces several novel probes for testing systematic generalization. We employ an artiﬁcial language to have control over systematic- ity and contextual meaning variation. Applying our probes to this language in an NLI setting reveals 1958 The logical extreme—a system where each word has a different and unrelated meaning every time it occurs—is clearly of limited usefulness since it would make generalization impossible. Neverthe- less, learners with sufﬁcient memory capacity and ﬂexibility of representation, such as deep learning models, can learn systems with very high degrees of contextual conditioning—in particular, higher than human language learners. An important goal for building systems that learn and generalize like people is to engineer systems with inductive biases for the right degree of systematicity. In §8, we give evidence that some neural systems are likely too biased toward allowing contextually conditioned meaning variability for words, such as quantiﬁers, which do not vary greatly in natural language. 1 Introduction that some deep learning systems which achieve very high accuracy on standard holdout evaluations do so in ways which are non-systematic: the net- works do not consistently capture the basic notion that certain classes of words have meanings which are consistent across the contexts in which they appear. The rest of the paper is organized as follows. §2 discusses degrees of systematicity and contextually conditioned variation; §3 introduces the distinction between open- and closed-class words, which we use in our probes. §5 introduces the NLI task and describes the artiﬁcial language we use; §6 dis- cusses the models that we tested and the details of our training setup; §7 introduces our probes of systematicity and results are presented in §8.1 1Code for datasets and models can be found here: https://github.com/emilygoodwin/systematicity 3 Compositional Structure in Natural Language At the other end of the spectrum from program- ming languages and logics are systems where many or most meanings are highly context dependent. Using jabberwocky-type sentences, we tested the generalizability of certain closed-class word representations learned by neural networks. Giving the networks many examples of each construction 1959 with a large variety of different content words— that is, large amounts of highly varied evidence about the meaning of the closed-class words—we asked during the test phase how fragile this knowl- edge is when transferred to new open-class words. That is, our probes combine novel open-class words with familiar closed-class words, to test whether the closed-class words are treated systematically by the network. For example, we might train the networks to identify contradictions in pairs like All pigs sleep; some pigs don’t sleep, and test whether the network can identify the contradiction in a pair like All Jabberwocks ﬂug; some Jabberwocks don’t ﬂug. A systematic learner would reliably identify the contradiction, whereas a non-systematic learner may allow the closed-class words (all, some, don’t) to take on contextually conditioned meanings that depend on the novel context words. investigates learned representations, rather than de- veloping probes of model behavior. This is done ei- ther through visualization (Veldhoen and Zuidema, 2017), training a second network to approximate learned representations using a symbolic structure (Soulos et al., 2019) or as a diagnostic classiﬁer (Giulianelli et al., 2018), or reconstructing the se- mantic space through similarity measurements over representations (Prasad et al., 2019). 4 Related Work There has been much interest in the problem of sys- tematic generalization in recent years (Bahdanau et al., 2019; Bentivogli et al., 2016; Lake et al., 2017a,b; Gershman and Tenenbaum, 2015; McCoy et al., 2019a; Veldhoen and Zuidema, 2017; Soulos et al., 2019; Prasad et al., 2019; Richardson et al., 2019; Johnson et al., 2017, inter alia). In contrast to our approach (testing novel words in familiar combinations), many of these studies probe systematicity by testing familiar words in novel combinations. Lake and Baroni (2018) adopt this approach in semantic parsing with an artiﬁcial language known as SCAN. Dasgupta et al. (2018, 2019) introduce a naturalistic NLI dataset, with test items that shufﬂe the argument structure of nat- ural language utterances. In the in the inductive logic programming domain, Sinha et al. (2019) in- troduced the CLUTTR relational-reasoning bench- mark. The novel-combinations-of-familiar-words approach was formalized in the CFQ dataset and as- sociated distribution metric of Keysers et al. (2019). Ettinger et al. (2018) introduced a semantic-role- labeling and negation-scope labeling dataset, which tests compositional generalization with novel com- binations of familiar words and makes use of syn- tactic constructions like relative clauses. Finally, Kim et al. (2019) explore pre-training schemes’ abilities to learn prepositions and wh-words with syntactic transformations (two kinds of closed- class words which our work does not address). 5.1 Natural Language Inference We make use of the Natural language inference (NLI) task to study the question of systematicity. The NLI task is to infer the relation between two sentences (the premise and the hypothesis). Sen- tence pairs must be classiﬁed into one of a set of predeﬁned logical relations such as entailment or contradiction. For example, the sentence All mam- mals growl entails the sentence All pigs growl. A rapidly growing number of studies have shown that deep learning models can achieve very high perfor- mance in this setting (Evans et al., 2018; Conneau et al., 2017; Bowman et al., 2014; Yoon et al., 2018; Kiela et al., 2018; Munkhdalai and Yu, 2017; Rock- täschel et al., 2015; Peters et al., 2018; Parikh et al., 2016; Zhang et al., 2018; Radford et al., 2018; De- vlin et al., 2018; Storks et al., 2019). 5.2 Natural Logic We adopt the formulation of NLI known as natu- ral logic (MacCartney and Manning, 2014, 2009; Lakoff, 1970). Natural logic makes use of seven logical relations between pairs of sentences. These are shown in Table 1. These relations can be in- terpreted as the set theoretic relationship between the extensions of the two expressions. For instance, if the expressions are the simple nouns warthog and pig, then the entailment relation (⊏) holds be- tween these extensions (warthog ⊏pig) since every warthog is a kind of pig. For higher-order operators such as quantiﬁers, relations can be deﬁned between sets of possible worlds. For instance, the set of possible worlds consistent with the expression All blickets wug is a subset of the set of possible worlds consistent with the logically weaker expression All red blick- ets wug. Critically, the relationship between com- posed expressions such as All X Y and All P Q is determined entirely by the relations between X/Y and P/Q, respectively. Thus, natural logic allows A different type of systematicity analysis directly 1960 erarchies (e.g., lizards/animals, run/move). Nouns and verbs from different blocks have no taxonomic relationship (e.g., lizards and screwdrivers or run and read) and do not co-occur in the same sentence pair. Because each block includes a six verbs and six nouns in a linear taxonomic hierarchy, no single block is intrinsically harder to learn than any other block. us to compute the relation between the whole ex- pressions using the relations between parts. We deﬁne an artiﬁcial language in which such align- ments are easy to compute, and use this language to probe deep learning systems’ ability to generalize systematically. Symbol Name Example Set-theoretic deﬁnition x ≡y equivalence pig ≡pig x = y x ⊏y forward entailment pig ⊏mammal x ⊂y x ⊐y reverse entailment mammal ⊐pig x ⊃y x ∧y negation pig ∧not pig x ∩y = ∅∧x ∪y = U x \| y alternation pig \| cat x ∩y = ∅∧x ∪y ̸= U x ⌣y cover mammal ⌣not pig x ∩y ̸= ∅∧x ∪y = U x#y independence hungry # warthog (all other cases) Table 1: MacCartney and Manning (2009)’s implemen- tation of natural logic relations The same set of closed-class words appear with all blocks of open-class words, and their meanings are systematic regardless of the open-class words (nouns and verbs) they are combined with. 5.5 Test and Train Structure We now describe the structure of our training blocks, holdout test set, and jabberwocky blocks. We also discuss our two test conditions, and sev- eral other issues that arise in the construction of our dataset. We compute the relation between position- aligned pairs of sentences in our language using the natural logic system (described in §5.2). Quanti- ﬁers and negation have their usual natural-language semantics in our artiﬁcial language; pre- and post- modiﬁers are treated intersectively. Open-class items (nouns and verbs) are organized into linear hierarchical taxonomies, where each open-class word is the sub- or super-set of exactly one other open-class item in the same taxonomy. For exam- ple, since dogs are all mammals, and all mammals animals, they form the entailment hierarchy dogs ⊏mammals ⊏animals. We vary the number of distinct noun and verb taxonomies according to an approach we refer to as block structure, described in the next section. Training set: For each training block, we sam- pled (without replacement) one sentence pair for every possible combination of open-class words, that is, every combination of nouns and verbs ⟨noun1, noun2, verb1, verb2⟩. Closed-class words were sampled uniformly to ﬁll each remain- ing positions in the sentence (see Table 2). A ran- dom subset of 20% of training items were reserved for validation (early stopping) and not used during training. Holdout test set: For each training block, we sampled a holdout set of forms using the same nouns and verbs, but disjoint from the training set just described. The sampling procedure was iden- tical to that for the training blocks. These holdout items allow us to test the generalization of the mod- els with known words in novel conﬁgurations (see §8.1). 5.2 Natural Logic For ex- ample, the quantiﬁer some has a consistent meaning when it is applied to some screwdrivers or some ani- mals. Because closed-class words are shared across blocks, models are trained on extensive and varied evidence of their behaviour. We present closed- class words in a wide variety of sentential contexts, with a wide variety of different open-class words, to provide maximal pressure against overﬁtting and maximal evidence of their consistent meaning. Table 1: MacCartney and Manning (2009)’s implemen- tation of natural logic relations 5.3 The Artiﬁcial Language In our artiﬁcial language, sentences are generated according to the six-position template shown in Ta- ble 2, and include a quantiﬁer (position 1), noun (position 3), and verb (position 6), with optional pre- and post-modiﬁers (position 2 and 4) and op- tional negation (position 5). For readability, all examples in this paper use real English words; however, simulations can use uniquely identiﬁed abstract symbols (i.e., generated by gensym). 5.4 Block Structure In natural language, most open-class words do not appear with equal probability with every other word. Instead, their distribution is biased and clumpy, with words in similar topics occurring to- gether. To mimic such topic structure, we group nouns and verbs into blocks. Each block consists of six nouns and six verbs, which form taxonomic hi- Jabberwocky test set: Each jabberwocky block consisted of novel open-class items (i.e., nouns and verbs) that did not appear in training blocks. For each jabberwocky block, we began by following a 1961 Position 1 2 3 4 5 6 Category quantifier nominal premodifier noun nominal postmodifier negation verb Status Obligatory Optional Obligatory Optional Optional Obligatory Class Closed Closed Open Closed Closed Open Example All brown dogs that bark don’t run Table 2: A template for sentences in the artiﬁcial language. Each sentence ﬁlls the obligatory positions 1, 3, and 6 with a word: a quantiﬁer, noun, and verb. Optional positions (2, 4 and 5) are ﬁlled by either a word (adjective, postmodiﬁer or negation) or by the empty string. Closed-class categories (Quantiﬁers, adjectives, post modiﬁers and negation) do not include novel words, while open-class categories (nouns and verbs) includes novel words that are only exposed in the test set. Table 2: A template for sentences in the artiﬁcial language. Each sentence ﬁlls the obligatory positions 1, 3, and 6 with a word: a quantiﬁer, noun, and verb. Optional positions (2, 4 and 5) are ﬁlled by either a word (adjective, postmodiﬁer or negation) or by the empty string. Closed-class categories (Quantiﬁers, adjectives, post modiﬁers and negation) do not include novel words, while open-class categories (nouns and verbs) includes novel words that are only exposed in the test set. premise v are individually encoded by neural se- quence encoders such as a long short-term mem- ory (LSTM; Hochreiter and Schmidhuber, 1997) or gated recurrent unit (GRU; Cho et al., 2014). These vectors, together with their element-wise product u ∗v and element-wise difference u −v are fed into a fully connected multilayer perceptron layer to predict the relation. The encodings u and v are produced from an input sentence of M words, w1, . . . , wM, using a recurrent neural network, which produces a set of a set of M hidden repre- sentations h1, . . . , ht, where ht = f(w1, . . . , wM). 5.4 Block Structure The sequence encoding is represented by its last hidden vector hT . sampling procedure identical to that for the train- ing/holdout sets with these new words. Several of our systematicity probes are based on the behavior of neighboring pairs of test sentences (see §7). To ensure that all such necessary pairs were in the jab- berwocky test set, we extended the initial sample with any missing test items. Training conditions: Since a single set of closed-class words is used across all blocks, adding more blocks increases evidence of the meaning of these words without encouraging overﬁtting. To study the effect of increasing evidence in this man- ner, we use two training conditions: small with 20 training blocks and large with 185 training blocks. Both conditions contained 20 jabberwocky blocks. The small condition consisted of 51, 743 training, 10, 399 validation, and 3, 694, 005 test (holdout and jabberwocky) pairs. The large condition con- sisted of 478, 649 training, 96, 005 validation, and 3, 694, 455 test items. The simplest of four models sets f to be a bidi- rectional gated recurrent unit (BGRU). This model concatenates the last hidden state of a GRU run forwards over the sequence and the last hidden state of GRU run backwards over the sequence, for example, u = [←− hM, −→ hM]. Our second embedding system is the Infersent model reported by Conneau et al. (2017), a bidirec- tional LSTM with max pooling (INFS). This is a model where f is an LSTM. Each word is repre- sented by the concatenation of a forward and back- ward representation: ht = [←− ht, −→ ht]. We constructed a ﬁxed vector representation of the sequence ht by selecting the maximum value over each dimension of the hidden units of the words in the sentence. Balancing: One consequence of the sampling method is that logical relations will not be equally represented in training. In fact, it is impossible to simultaneously balance the distributions of syntac- tic constructions, logical relations, and instances of words. In this trade-off, we chose to balance the distribution of open-class words in the vocab- ulary, as we are focused primarily on the ability of neural networks to generalize closed-class word meaning. Balancing instances of open-class words provided the greatest variety of learning contexts for the meanings of the closed-class items. 5.4 Block Structure Our third model is a self-attentive sentence en- coder (SATT) which uses an attention mechanism over the hidden states of a BiLSTM to generate the sentence representation (Lin et al., 2017). This attention mechanism is a weighted linear combi- nation of the word representations, denoted by u = P M αihi, where the weights are calculated as follows: 7.2 Identical Open-class Words Probe Some sentence pairs are classiﬁable without any knowledge of the novel words’ meaning; for ex- ample, pairs where premise and hypothesis have identical open-class words. An instance is shown in Example 3: the two sentences must stand in con- tradiction, regardless of the meaning of blicket or wug. 7 Probing Systematicity In this section, we study the systematicity of the models described in §6.1. Recall that systematicity refers to the degree to which words have consistent meaning across different contexts, and is contrasted with contextually conditioned variation in meaning. We describe three novel probes of systematicity which we call the known word perturbation probe, the identical open-class words probe, and the con- sistency probe. In order to rule out trivially correct behavior where the model simply ignores the perturbation, we consider only perturbations which result in a change of class (e.g., ⊏7→⊐) for the sentence pair. In addition to accuracy on these perturbed items, we also examine the variance of model accuracy on probes across different blocks. If a model’s accu- racy varies depending only on the novel open-class items in a particular block, this provides further evidence that it does not treat word meaning sys- tematically. All probes take advantage of the distinction be- tween closed-class and open-class words reﬂected in the design of our artiﬁcial language, and are performed on sentence pairs with novel open-class words (jabberwocky-type sentences; see §5.5 ). We now describe the logic of each probe. (3) All blickets wug. Some blickets don’t wug. The closed-class items and compositional struc- ture of the language is sufﬁcient for a learner to deduce the relationships between such sentences, even with unfamiliar nouns and verbs. Our second probe, the identical open-class words probe, tests the models’ ability to correctly classify such pairs. (2) Some blickets wug. All blockets wug. (2) Some blickets wug. All blockets wug. where, uw is a learned context query vector and (W, bw) are the weights of an afﬁne transformation. This self-attentive network also has multiple views of the sentence, so the model can attend to multiple parts of the given sentence at the same time. There are two critical features of this probe. First, because we start from a correctly-classiﬁed jabberwocky pair, we can conclude that the novel words (e.g., wug and blickets above) were assigned appropriate meanings. Finally, we test the Hierarchical Convolution- alNetwork (CONV) architecture from (Conneau et al., 2017) which is itself inspired from the model AdaSent (Zhao et al., 2015). This model has four convolution layers; at each layer the intermediate representation ui is computed by a max-pooling operation over feature maps. The ﬁnal representa- tion is a concatenation u = [u1, ..., ul] where l is the number of layers. Second, since the perturbation only involves closed-class items which do not vary in mean- ing and have been highly trained, the perturbation should not affect the models ability to correctly classify the resulting sentence pair. If the model does misclassify the resulting pair, it can only be because a perturbed closed-class word (e.g., some) interacts with the open-class items (e.g., wug), in a way that is different from the pre-perturbation closed-class item (i.e., all). This is non-systematic behavior. 6.1 Models We analyze performance on four simple baseline models known to perform well on standard NLI tasks, such as the Stanford Natural Language In- ference datasets, (Bowman et al., 2015). Follow- ing Conneau et al. (2017), the hypothesis u and ¯hi = tanh(Whi + bw) αi = e ¯hi ⊤uw P i e ¯hi ⊤uw 1962 (1) All blickets wug. All blockets wug. 7.1 Known Word Perturbation Probe We test whether the models treat the meaning of closed-class words systematically by perturbing correctly classiﬁed jabberwocky sentence pairs with a closed-class word. More precisely, for a pair of closed-class words w and w′, we consider test items which can be formed by substitution of w by w′ in a correctly classiﬁed test item. We allow both w and w′ to be any of the closed-class items, includ- ing quantiﬁers, negation, nominal post-modiﬁers, or the the empty string ϵ (thus modeling insertions and deletions of these known, closed-class items). Suppose that Example 1 was correctly classiﬁed. Substituting some for all in the premise of yields Example 2, and changes the relation from entail- ment (⊏) to reverse entailment (⊐). (4) All blickets wug. All red blickets wug. (4) All blickets wug. All red blickets wug. (4) All blickets wug. All red blickets wug. (5) All red blickets wug. All blickets wug. Table 3: Accuracy on holdout evaluations (training con- ditions and holdout evaluation are explained in §5.5) In Example 4, the two sentences stand in an en- tailment (⊏) relation. In Example 5, by contrast, the two sentences stand in a reverse entailment (⊐) relation. This is a logically necessary consequence of the way the relations are deﬁned. Reversing the order of sentences has predictable effects for all seven natural logic relations: in particular, such reversals map ⊏7→⊐and ⊐7→⊏, leaving all other relations intact. Based on this observation, we de- velop a consistency probe of systematicity. We ask for each correctly classiﬁed jabberwocky block test item, whether the corresponding reversed item is also correctly classiﬁed. The intuition behind this probe is that whatever meaning a model assumes for the novel open-class words, it should assume the same meaning when the sentence order is re- versed. If the reverse is not correctly classiﬁed, then this is strong evidence of contextual depen- dence in meaning. Figure 1: Visualization of trained and novel open-class word embeddings. Figure 1: Visualization of trained and novel open-class word embeddings. trained on these novel words, it is important to establish that they are from the same distribution as the trained words and, thus, that the models’ performance is not driven by some pathological feature of the novel word embeddings. Trained word embeddings were initialized ran- domly from N(0, 1) and then updated during train- ing. Novel word embeddings were simply drawn from N(0, 1) and never updated. Figure 1 plots visualizations of the trained and novel open-class word embeddings in two dimensions, using t-SNE parameters computed over all open-class words (Maaten and Hinton, 2008). Trained words are plotted as +, novel words as •. Color indicates the proportion of test items containing that word that were classiﬁed correctly. As the plot shows, the two sets of embeddings overlap considerably. Moreover, there does not appear to be a systematic relationship between rates of correct classiﬁcation for items containing novel words and their prox- imity to trained words. We also performed a re- sampling analysis, determining that novel vectors did not differ signiﬁcantly in length from trained vectors (p = 0.85). 8.2 Analysis II: Distribution of Novel Words 8.2 Analysis II: Distribution of Novel Words Our three systematicity probes employ jabberwocky-type sentences—novel open- class words in sentential frames built from known closed-class words. Since models are not 8 Results In this section, we report the results of two control analyses, and that of our three systematicity probes described above. (4) All blickets wug. All red blickets wug. Finally, we observed mean and standard deviation of the pairwise cosine similarity between trained and novel words to be 0.999 and 0.058 respectively, conﬁrming that there is little evidence the distributions are different. 8.1 Analysis I: Holdout Evaluations We ﬁrst establish that the models perform well on novel conﬁgurations of known words. Table 3 re- ports accuracy on heldout sentence pairs, described in §5.5. The table reports average accuracies across training blocks together with the standard devia- tions of these statistics. As can be seen in the table, all models perform quite well on holdout forms across training blocks, with very little variance. Be- cause these items use the same sampling scheme and vocabulary as the trained blocks, these simula- tions serve as a kind of upper bound on the perfor- mance and a lower bound on the variance that we can expect from the more challenging jabberwocky- block-based evaluations below. 7.3 Consistency Probe (1) All blickets wug. All blockets wug. Consider Examples 4 and 5, which present the same two sentences in opposite orders. 1963 1963 Condition BGRU CONV SATT INFS mean (sd) mean (sd) mean (sd) mean (sd) small 95.1 ±0.21 95.43 ±0.12 93.14 ±0.94 96.02 ±0.51 large 95.09 ±1.03 95.22 ±0.55 94.89 ±1.09 96.17 ±0.74 Table 3: Accuracy on holdout evaluations (training con- ditions and holdout evaluation are explained in §5.5) Figure 1: Visualization of trained and novel open-class word embeddings. Condition BGRU CONV SATT INFS mean (sd) mean (sd) mean (sd) mean (sd) small 95.1 ±0.21 95.43 ±0.12 93.14 ±0.94 96.02 ±0.51 large 95.09 ±1.03 95.22 ±0.55 94.89 ±1.09 96.17 ±0.74 8.4 Analysis IV: Identical Open-Class Words Probe Recall that the identical open-class words probe consist of sentence pairs where all open-class lexi- cal items were identical. Table 4 shows the accura- cies for these probes, trained on the small language. Average accuracies across jabberwocky blocks are reported together with standard deviations. Figure 2: Performance on the known word perturbation probe, small and large training conditions (see §5.5). Relation BGRU CONV SATT INFS mean (sd) mean (sd) mean (sd) mean (sd) # 100 ±0 100 ±0 99.94 ±0.26 99.67 ±0.98 ∧ 55.68 ±20.29 73.29 ±10.8 23.71 ±11.45 90.67 ±10.98 ⊏ 90.78 ±4.99 82.84 ±6.51 75.22 ±5.98 95.53 ±2.64 ≡ 90.43 ±17.1 38.12 ±15.56 71.94 ±24.1 95.93 ±6.5 ⊐ 90.34 ±4.18 77.11 ±5.9 81.4 ±6.67 93.81 ±2.96 \| 93.08 ±3.58 85.34 ±5.47 74.05 ±8.03 92.23 ±4.6 ⌣ 88.01 ±3.55 71.5 ±7.32 78.4 ±7.91 95.22 ±3.58 Table 4: Identical open-class words probe performance, trained on the small language condition (trained on 51, 743 sentence pairs, see §5.5) Figure 2: Performance on the known word perturbation probe, small and large training conditions (see §5.5). of a trained closed-class word in a correctly classi- ﬁed jabberwocky-type sentence pair. Figure 2 plots the results of this probe. Each point represents a perturbation type—a group of perturbed test items that share their before/after target perturbed closed- class words and before/after relation pairs. The upper plot displays the mean accuracy of all pertur- bations, averaged across blocks, and the lower plot displays the standard deviations across blocks. Table 4: Identical open-class words probe performance, trained on the small language condition (trained on 51, 743 sentence pairs, see §5.5) Table 4: Identical open-class words probe performance, trained on the small language condition (trained on 51, 743 sentence pairs, see §5.5) Accuracy on the probe pairs fails to reach the holdout test levels for most models and most rela- tions besides #, and variance between blocks is much higher than in the holdout evaluation. Of special interest is negation (∧), for which accuracy is dramatically lower and variance dramatically higher than the holdout evaluation. All models perform substantially worse than the holdout-evaluation on at least some of the perturba- tions. In addition, the standard deviation of accu- racy between blocks is higher than the holdout tests. As discussed in §7.1, low accuracy on this probe indicates that closed-class words do not maintain a consistent interpretation when paired with different open-class words. 8.3 Analysis III: Known Word Perturbation Probe Recall from §7.1 that the known word perturbation probe involves insertion, deletion, or substitution 1964 Figure 2: Performance on the known word perturbation probe, small and large training conditions (see §5.5). tence pair (84.79% ± 7.50%) or another forward- entailing pair (92.32%, ±3.60%) or an equality pair (100% ± 0.00%). All the possible perturba- tions we studied exhibit similarly inconsistent per- formance. 8.4 Analysis IV: Identical Open-Class Words Probe Variance across blocks shows that under all models the behavior of closed-class words is highly sensitive to the novel words they appear with. The results are similar for the large language condition, shown in Table 5. Although model accu- racies improve somewhat, variance remains higher than the heldout level and accuracy lower. Recall that these probe-items can be classiﬁed while ignor- ing the speciﬁc identity of their open-class words. Thus, the models inability to leverage this fact, and high variance across different sets novel open-class words, illustrates their sensitivity to context. Performance is also susceptible to interference from sentence-level features. For example, con- sider the perturbation which deletes a post-modiﬁer from a sentence pair in negation, yielding a pair in cover relation. The self-attentive encoder per- forms perfectly when this perturbation is applied to a premise (100% ± 0.00%), but not when applied to a hypothesis (86.60% ± 18.08%). Similarly, deleting the adjective red from the hypothesis of a forward-entailing pair results in an unrelated sen- 8.5 Analysis V: Consistency Probe The consistency probe tests abstract knowledge of relationships between logical relations, such as the fact that two sentences that stand in a contradiction still stand in a contradiction after reversing their or- der. Results of this probe in the small-language con- dition are in Table 6: For each type of relation, we show the average percentage of correctly-labeled 1965 Relation BGRU CONV SATT INFS mean (sd) mean (sd) mean (sd) mean (sd) # 99.82 ±0.45 99.57 ±0.73 98.67 ±1.81 100 ±0 ∧ 84.18 ±12.29 73.73 ±18.31 79.97 ±16.58 85.54 ±14.11 ⊏ 96.13 ±2.59 93.88 ±2.67 97.3 ±2.36 97.02 ±2.39 ≡ 89.33 ±12.5 77.84 ±12.08 94.44 ±11.23 94.59 ±7.02 ⊐ 95.4 ±2.48 94.55 ±2.04 98.05 ±1.51 97.6 ±2.08 \| 89.97 ±6.73 92.36 ±6.29 84.52 ±7.07 98.72 ±2.08 ⌣ 90.78 ±6.33 93.18 ±2.95 87.85 ±6.46 97.48 ±2.56 Table 5: Identical open-class words probe performance when trained on the large language training condition (trained on 478, 649 sentence pairs, see §5.5) Relation BGRU CONV SATT INFS mean (sd) mean (sd) mean (sd) mean (sd) # 98.45 ±0.65 98.69 ±0.54 98.83 ±0.6 98.38 ±0.74 ∧ 70.46 ±33.72 77.82 ±26 84.27 ±23.89 65.64 ±35.13 ⊏ 96.02 ±2.96 96.6 ±3.26 96.78 ±4.23 95.01 ±5.38 = 100 ±0 100 ±0 100 ±0 100 ±0 ⊐ 93.5 ±4.51 95.76 ±4.23 94.23 ±5.86 90.11 ±8.5 \| 96.31 ±2.73 97.25 ±2.05 97.17 ±2.23 94.46 ±4.24 ⌣ 96.25 ±2.49 96.98 ±2.66 97.18 ±2.17 93.88 ±4.78 Table 7: Consistency probe performance, trained on the large langauge condition (478, 649 sentence pairs). Table 5: Identical open-class words probe performance when trained on the large language training condition (trained on 478, 649 sentence pairs, see §5.5) Table 5: Identical open-class words probe performance when trained on the large language training condition (trained on 478, 649 sentence pairs, see §5.5) Table 7: Consistency probe performance, trained on the large langauge condition (478, 649 sentence pairs). Table 7: Consistency probe performance, trained on the large langauge condition (478, 649 sentence pairs). guage inference task. Our results indicate that de- spite their high overall performance, these models tend to generalize in ways that allow the meanings of individual words to vary in different contexts, even in an artiﬁcial language where a totally sys- tematic solution is available. This suggests the networks lack a sufﬁcient inductive bias to learn systematic representations of words like quantiﬁers, which even in natural language exhibit very little meaning variation. 8.5 Analysis V: Consistency Probe sentence pairs that, when presented in reverse order, were also correctly labeled. The best-performing model on negation reversal is SATT, which correctly labeled reversed items 66.92% of the time. Although performance on negation is notably more difﬁcult than the other relations, every model, on every relation, exhibited inter-block variance higher than that of the hold-out evaluations. Relation BGRU CONV SATT INFS mean (sd) mean (sd) mean (sd) mean (sd) # 97.4 ±0.86 97.8 ±0.93 98.58 ±0.74 97.03 ±0.87 ∧ 63.03 ±36.19 63.42 ±35.91 66.92 ±31.45 57.16 ±38.24 ⊏ 92.45 ±6.26 88.1 ±8.16 93.16 ±5.42 90.64 ±6.76 ≡ 100 ±0 100 ±0 100 ±0 100 ±0 ⊐ 91.37 ±6.23 94.73 ±6.51 96.42 ±3.22 87.02 ±9.61 \| 96.02 ±2.6 96.29 ±2.51 96.95 ±2.14 94.2 ±3.48 ⌣ 93.57 ±3.56 95 ±2.97 96.4 ±2.83 93.1 ±3.77 Table 6: Consistency probe performance, trained on the small language condition (51, 743 sentence pairs, see §5.5). Our analyses contain two ideas that may be use- ful for future studies of systematicity. First, two of our probes (known word perturbation and con- sistency) are based on the idea of starting from a test item that is classiﬁed correctly, and applying a transformation that should result in a classiﬁable item (for a model that represents word meaning systematically). Second, our analyses made criti- cal use of differential sensitivity (i.e., variance) of the models across test blocks with different novel words but otherwise identical information content. We believe these are a novel ideas that can be em- ployed in future studies. Table 6: Consistency probe performance, trained on the small language condition (51, 743 sentence pairs, see §5.5). Table 6: Consistency probe performance, trained on the small language condition (51, 743 sentence pairs, see §5.5). Furthermore, as can be seen in Table 7, the large language condition yields little improvement. Negation pairs are still well below the hold-out test threshold, still with a high degree of variation. Variation remains high for many relations, which is surprising because the means report accuracy on test items that were chosen speciﬁcally because the same item, in a reverse order, was already correctly labeled. 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https://openalex.org/W2998425101	https://iiste.org/Journals/index.php/JBAH/article/download/50672/52365	English	null	Phytochemical Screening of Tannins in Selected Indigenous Plants Species Used as Chewing Sticks in Ekiti State, Nigeria	null	2,019	cc-by	3,245	Introduction The role of plants in maintaining effective oral hygiene cannot be undermined. A number of plant species are used as chewing sticks, most of which have substances in them that can keep the buccal cavity healthy. The actions of certain bacteria in the mouth are known to cause several oral infections. The most common oral infections are periodontal diseases that are mainly caused by bacterial plaque accumulation around the teeth as a result of poor oral hygiene (Kemboi, 2016). This infection affects all humans without regard for race or gender or better still the origin of a person or his/her nationality. For example, The World Health Organization (WHO) ranks oral disease among the top five causes of health burden in the world (WHO, 2004). Some plants possess antimicrobial activity against oral microbial flora. This indicates that some chewing sticks (plant), in addition to providing mechanical stimulation to the gums and teeth also destroy microbes present in the mouth (Homer et al., 1990). Dental care needs of the majority of Nigerians remains largely unmet because there are a few professionals offering care, as a result services are often too costly, scarce or totally absent especially in rural areas (Kayode, 2005). Chewing sticks are said to be twigs or roots of certain plants that are chewed until one end is frayed and brush-like. This end can be used to brush against the teeth (Price and Butter, 2017). ) Chewing sticks constitute an important non-wood forest product (NWFP) in Ekiti State, Nigeria where tooth cleaning with botanicals has formed part of the cultural norms). They are commonly used as close substitutes or alternatives to modern tooth pastes and brush by people in many developing countries like Nigeria. In Nigeria, the use of chewing stick is peculiar to the countryside; however, semi-urban and urban residents also use chewing sticks. The choice of stick depends on the known or perceived cleansing potentials, taste (peppery or bitter), therapeutic values, simplicity in being chewed into brush, and the ability to froth (Akande and Ajao, 2011). Isawunmi (1978) further asserted that in Nigeria, prior to civilization, cleaning of the teeth were initially done with chewing sticks before the prevalence of the use of modern tooth paste and brush. Previous studies by Isawemi (1978 a, b,c, and 1979) have revealed that the common chewing stick in Nigeria consist of 23 botanical species that belong to 14 species. Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.9, No.24, 2019 Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.9, No.24, 2019 Phytochemical Screening of Tannins in Selected Indigenous Plants Species Used as Chewing Sticks in Ekiti State, Nigeria Oluwafoise, B. G. Biology Department, College of Education, Ikere-Ekiti Adaramodu, A. A. Chemistry Department, College of Education, Ikere-Ekiti Abstract Chewing sticks form an alternative to modern tooth pastes and brush and their use in brushing the teeth predates the later. The astringent and antimicrobial properties of tannins in chewing sticks give it one of its therapeutic values. This paper present a critical look at the analysis of tannins in selected indigenous plant species used as chewing sticks in Ekiti State, Nigeria. Forty eight (48) plant species belonging to 28 different families observed to be used for dental and oral health care in the study area were screened for the presence of tannins. Result of tannin phytochemical screening revealed that 34 (70.83%) contained tannins, while the remaining 14 (29.17%) do not contain tannin. In conclusion, the results showed that most of the Nigerian chewing sticks do contain tannins which have antibacterial activities which may contribute to the reported anticaries effect of chewing sticks. These sticks may be sources for new lead antibacterial agents for therapeutic or preventive applications. Based on the result obtained, it was recommended that users of chewing sticks should be sensitized on the type of chewing sticks that are of potential therapeutic benefit (anti-microbial) to the mouth health especially those containing tannins. DOI: 10.7176/JBAH/9-24-05 Publication date: December 31st 2019 Publication date: December 31st 2019 Sample Preparation Samples of roots and stems of forty-eight different indigenous plants commonly used as chewing sticks were collected in Ekiti State between November and January period. This period of the year fall within the dry season month where adequate sunlight is present with little or no disturbance of rain. The samples were air-dried in a cool area twelve weeks after which dried samples were ground into powder. 300g each of dried powder of the stem and root samples were extracted into 80% ethanol, Hexane and Dichloromethane respectively for two days at room temperature. The solutions are filtered. The solvent-containing extracts was then decanted and filtered in a 500 mL beaker through cotton wool to remove course particles and lastly through filter paper (Whatmann No.1) to obtain crude extract. Test for Tannins One milliliter of the filtrate was mixed with 3 mL of iron III chloride (FeCl3), A dark green color indicated a positive test for the tannins. Introduction It has been reported that plants most commonly used as chewing sticks are those that have a high content of tannins (astringent and antibacterial) or other compounds that benefit the health of gums and teeth (Wolf and Rohan, 2009). Tannins (or tannoids) are a class of astringent poly phenolic biomolecules that bind to and precipitate proteins and various other organic compounds including ammonic acids and alkaloids (Akande and Hayashai, 2008).The tannin compound are widely distributed in many species of plants, where they play a role in protection from, predation (including as pesticides) and might help in regulating plant growth (Adekunle and Odunkoya, 2006). The importance of chewing sticks in maintaining oral or buccal hygiene has been well reported. Adekunle and Odunkoya, 2006 reported the importance of chewing stick, particularly to the relatively low cases of dental 33 Journal of Biology, Agriculture and Healthcare Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) V l 9 N 24 2019 www.iiste.org caries and maintenance of strong teeth. In chewing sticks, tannin forms a coat over the enamel and thus protect against tooth decay (Gills, 1992). In most part of Africa, chewing sticks are often cut into pieces to make concoction thereby extracting tannins from the sticks for healing open wounds, stomach ache, diarrhea, gunn diseases and fever (Odukoyaet al, 2001). According to Sofowora (2008), secondary compounds in acacia contain tannins are important for a variety of functions, chief among these are anti-microbial, anti-cancer diveric, natriuretic, important nutraceutical, anti-digestive disorder, anti-oxidant, antiplasmodia. caries and maintenance of strong teeth. In chewing sticks, tannin forms a coat over the enamel and thus protect against tooth decay (Gills, 1992). In most part of Africa, chewing sticks are often cut into pieces to make concoction thereby extracting tannins from the sticks for healing open wounds, stomach ache, diarrhea, gunn diseases and fever (Odukoyaet al, 2001). According to Sofowora (2008), secondary compounds in acacia contain tannins are important for a variety of functions, chief among these are anti-microbial, anti-cancer diveric, natriuretic, important nutraceutical, anti-digestive disorder, anti-oxidant, antiplasmodia. The aim of this study therefore is to screen the selected indigenous plants used as chewing sticks in Ekiti State, Nigeria for the presence of tannins using different organic solvents – etanol, hexane and dichloromethane (DCM) and compare the potency of the various extractants for tannin. Results and Discussion and Discussion ts of phytochemical screening are presented in the table below of the various plant samples are presented le below. The results of phytochemical screening are presented in the table below of the various plant samples are presented in the table below. The results of phytochemical screening are presented in the table below of the various plant samples are presented in the table below. Table 1. Results and Discussion Result of tannin screening S/N Botanical Name Stem Extract Root Extract Ethanol Extract Hexane Extract DCM Extract Ethanol Extract Hexane Extract DCM Extract 1 Alchornea cordifolia + + + + + + 2 Alchornea laxiflora + + + + + + 3 Alstonia boonei + + + + + + 4 Anacardium occidentale + + + + + + 5 Anogeissus leiocarpus + + + + + + 6 Azadirachta India - - - - - - 7 Blighia sapida + + + + + + 8 Bridelia ferruginea + + + + + + 9 Bridelia micrantha + + + + + + 10 Buchholzia coriacea + + + + + + 11 Calliandra portoricensus - - - - - - 12 Capsicum annuum - - - - - - 13 Carpotobia lutea + + + + + + 14 Citrus aurantifollia - - - - - - 15 Clerodondron buchholziii + + + + + + 16 Combretum racemosum - - - - - - 17 Croton zambesicus - - - - - - 18 Cynometra vogellii + + + + + + 19 Diospyros monbuttensis + + + + + + 20 Duranta erecta - - - - - - 21 Garcinia kola + + + + + + 22 Garcinia mannii - - - - - - 23 Gliricidia sepium + + + + + + 24 Glyphaea brevis + + + + + + 25 Hibsicusrosa-sinensis + + + + + + 26 Harungana madagascariensis + + + + + + 34 ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.9, No.24, 2019 S/N Botanical Name Stem Extract Root Extract Ethanol Extract Hexane Extract DCM Extract Ethanol Extract Hexane Extract DCM Extract 27 Jatropha curcas + + + + + + 28 Jatropha gossypiifolia - - - - - - 29 Jatropha multifida + + + + + + 30 Mallotus oppositifolius + + + + + + 31 Magnifera indica + + + + + + 32 Masscularia acuminate + + + + + + 33 Milletia thonningii + + + + + + 34 Ocimum gratissimum - - - - - - 35 Olaxsubscorpoidea - - - - - - 36 Prerseagratissima + + + + + + 37 Phaulopsis imbracata + + + + + + 38 Psidium guajava - - - - - - 39 Pycnanthus angolensis + + + + + + 40 Sarcocephalusl atifolius + + + + + + 41 Senna siamea + + + + + + 42 Sida acute + + + + + + 43 Sida corymbosa + + + + + + 44 Terminalia schimperiana + + + + + + 45 Theobroma cacao + + + + + + 46 Trema orientals - - - - - - 47 Vernonia amygdalina + + + + + + 48 Zanthoxylum xanthoxyloides - - - - - - Forty-eight (48) plant species belonging to 28 different families were observed to be in use for dental and oral health care in the study area. Results and Discussion The various similarity measures related that similar plant species were used and most of these species were indigenous. Forty-eight (48) plant species belonging to 28 different families were observed to be in use for dental and oral health care in the study area. The various similarity measures related that similar plant species were used and most of these species were indigenous. Result of phytochemical screening for tannin revealed that of the 48 plant samples screened, 34 (70.83%) of them contained tannins while 14 (29.17%) of the plants do not contain tannins. The result showed that tannins have been identified to be the most abundant chemical composition extraction from the 48 plant species used in making chewing stick. Presence of tannins in the ethanol, hexane and dichloromethane extracts have also shown to be absolutely correlated as the different extract revealed the same presence of tannins in specific plant. This might be due to the fairly high polarity of the various solvents used as extractants. Results and Discussion The folk medicinal value and chemical composition of the plant species identified were also itemized below: Table 2: Folk-Medicinal Values of selected plants S/No Botanical Name Folk-Medicinal Value 1 Alchornea cordifolia Healing of Teeth ache and mouth 2 Alchornea laxiflora Posses antimicrobial gents 3 Alstonia boonei Curing of fever, asthma, rheumatic pains, dizziness 4 Anacardium occidentale Curing of tooth ache, sore gums and cough 5 Anogeissus leiocarpus Used for oral hygiene 6 Azadirachta India Teeth and gum maintenance and cure of fever 7 Blighia sapida Curing of mouth wounds 8 Bridelia ferruginea Healing of mouth wounds 9 Bridelia micrantha Curing of headache 10 Buchholzia coriacea Used as anti-fungal and 11 Calliandra portoricensus Curing of headache, gonorrhea, lumbago, and pain 12 Capsicum annuum Curing of fever, cold and dysentery, act as stimulant 13 Carpotobia lutea Curing of stomach ache and used for oral hygiene 14 Citrus aurantifollia Used for oral hygiene 15 Clerodondron buchholziii Curing of fever 16 Combretum racemosum Curing of fever and cough 17 Croton zambesicus Curing of malaria 18 Cynometra vogellii Used for oral hygiene 19 Diospyros monbuttensis Used as anti-fungal and anti-bacteria agents 20 Duranta erecta Used for oral hygiene 35 Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.9, No.24, 2019 S/No Botanical Name Folk-Medicinal Value 21 Garcinia kola Curing of cough, tooth ache, fever and diabetes 22 Garcinia mannii Curing of cough 23 Gliricidia sepium Clearing microbes in mouth 24 Glyphaea brevis Curing of fever 25 Hibsicusrosa-sinensis Curing of cough 26 Harungana madagascariensis Curing of fever, cough, dysentery, asthma, jaundice, diahhoea, sore throat 27 Jatropha curcas Curing of gum bleeding 28 Jatropha gossypiifolia Healing of mouth cancer 29 Jatropha multifida Healing of mouth wounds 30 Mallotus oppositifolius Healing of mouth cancer 31 Magnifera indica Curing of malaria and sore gum 32 Masscularia acuminate Used for oral hygiene, curing of cough and lumbago 33 Milletia thonningii Curing of fever and sore throat 34 Ocimum gratissimum Curing of cough, cold, fever, chest pain, diarrhea 35 Olax subscorpoidea Curing of tooth ache 36 Prersea gratissima Curing of hypertension 37 Phaulopsis imbracata Used for mouth hygiene 38 Psidium guajava Curing of stomach ache and cough 39 Pycnanthus angolensis Clearing of 'white tongue' 40 Sarcocephalus latifolius Curing of malaria fever 41 Senna siamea Curing of fever 42 Sida acute Used for oral hygiene 43 Sida corymbosa Used for oral hygiene 44 Terminalia schimperiana Curing of sickle cell anemia 45 Theobroma cacao Acts as stimulant 46 Trema orientals Provides remedy for fever, black tongue, cough, bronchitis, dysentery 47 Vernonia amygdalina Curing of cough, diabetes 48 Zanthoxylum xanthoxyloides Curing of tooth ache and sickle cell anemia Discussion, Conclusion and Recommendation The long and variable history of the use of plants to improve dental health and promote oral hygiene has been known since antiquity. Results and Discussion Cutting of roots tem or twings of tree and shrub have been used severally as traditional tooth brush commonly called chewing sticks. The use medical plants is an importance pant of traditional medicine in many culture and is the basis of ongoing efforts to develop new drugs in conventional medicine. Tannins have been reported to have anti-microbial properties especially around the mouth region. From the folk-medicinal values itemized in table 2, it has been revealed that the plant samples used for this research not only contain tannins but also other chemical compounds that could be useful to improving oral health or well being in general. The result showed that tannins bas been identified to be the most abundance chemical ton extractors from the 48 plant species used in making showing stick.In conclusion, the results showed that most of the Nigerian chewing sticks do contain tannins which have antibacterial activities which may contribute to the reported anticaries effect of chewing sticks. These sticks may be sources for new lead antibacterial agents for therapeutic or preventive applications. Based on the result obtained, it was recommended that users of chewing sticks should be sensitized on the type of chewing sticks that are of potential therapeutic benefit (anti-microbial) to the mouth health especially those containing tannins. Adekunle, A.A. and Odunkoya, K.A. (2006). Antifungal activities of ethanol and aqeous crude extrats of four Nigerian chewing sticks. Ethnobotanical leaflets http/www.stu/-eb/leaflets. ( ) Akande, J.A. and Hayashai, Y. (2008). Potency extract constants from selected tropical sticks against staphylococcus areus and Etaphylococcusauricularis wordJournal of Microbiology and Bacteriology Adekunle, A.A. and Odunkoya, K.A. (2006). Antifungal activities of ethanol and aqeous crude extrats of four Nigerian chewing sticks. Ethnobotanical leaflets http/www.stu/-eb/leaflets. Ahmed, M., P. Khirtova and G. Icho, (2005). Comparative study of tannins of Acacia Nilotica and indigenous tannins materials in sudan with acacia mearnsiisuranaree, J.SCI.Technol, 12:259-265. Akande TA and Ajao AT, 2011. Chemotherapeutic values of four Nigerian chewing sticks on bacterial isolates of dental infection. Global Journal of Science Frontier Research 11(8): 91-95 Akande, J.A. and Hayashai, Y. (2008). Potency extract constants from selected tropical sticks against staphylococcus areus and Etaphylococcusauricularis wordJournal of Microbiology and Bacteriology de TA and Ajao AT, 2011. Chemotherapeutic values of four Nigerian chewing sticks on bacterial isolates dental infection. Global Journal of Science Frontier Research 11(8): 91-95 ed, M., P. Khirtova and G. Icho, (2005). Comparative study of tannins of Acacia Nilotica and indigeno annins materials in sudan with acacia mearnsiisuranaree, J.SCI.Technol, 12:259-265. References References Adekunle, A.A. and Odunkoya, K.A. (2006). Antifungal activities of ethanol and aqeous crude extrats of four Nigerian chewing sticks. Ethnobotanical leaflets http/www.stu/-eb/leaflets. Adekunle, A.A. and Odunkoya, K.A. (2006). Antifungal activities of ethanol and aqeous crude extrats of four Nigerian chewing sticks. Ethnobotanical leaflets http/www.stu/-eb/leaflets. Ahmed, M., P. Khirtova and G. Icho, (2005). Comparative study of tannins of Acacia Nilotica and in tannins materials in sudan with acacia mearnsiisuranaree, J.SCI.Technol, 12:259-265. Akande TA and Ajao AT, 2011. Chemotherapeutic values of four Nigerian chewing sticks on bacterial i dental infection. Global Journal of Science Frontier Research 11(8): 91-95 Akande, J.A. and Hayashai, Y. (2008). Potency extract constants from selected tropical sticks against staphylococcus areus and Etaphylococcusauricularis wordJournal of Microbiology and Bacteriology 36 Journal of Biology, Agriculture and Healthcare gy, g ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) G.ill, L.S. (1992). Ethnomedicinal uses of plants in nigeriauniben press, bennin city, Nigeria, pp.279. Hann, D.H, L. W. Rooney and C.F. Earp (1984). Tannins and phenols of sorgunCer. foods world 29:77 , , y p ( ) p g f Homer KA, Manji F, Beighton D, 1990. Inhibition of protease activities of periondontopathic bacteria by extracts of plants used in Kenya as chewing sticks (mswaki). Archives of Oral Biology 35: 421-424. 8). Nigerian chewing sticks. Nigeria field43(4): 61-17 Isawumi, M.A. (1978a). Nigeria chewing sticks. Nigeria field43 (2) 50-58. Isawumi, M.A. (1978b). Nigeria chewing sticks. Nigeria field43(3): 111-121. Isawumi, M.A. (1979). Nigeria chewing sticks. Nigeria field44(4): 21-28 Kayode, J. (2005). Ethnobotanical survey and conservation of medicinal composite species in Benin Kigdome, Nigeria composite new letter 42. 48 -45. KemboiDoglas (2016). Ethnobotanical Medicinal Plants Used as Chewing Sticks among the Kenyan Communities. British Journal of Pharmaceutical Research 13(1): 1-8, 2016, Article no.BJPR.28833 ISSN: 2231-2919, NLM ID: 101631759 Odebiyi, O.O. and A, Sofowora (20009). Antimicrobial alkaloids from Nigeria chewing sticks. Planta med, 36:204-204 Odukoya, O.A., A.E. Thomas and A. Adepoju- Bello (2001). Tannic acid equivalent and cytotoxic activity of selected medicinal plants. West Afr. J. pharm. 15:43-45. p f p Price, M.L. and L.G. Butter (2017). Rapid visual estimation and spectrophotometer determinations of tannins content of sorghum. J. Agric. Food Chem., 25-1268 – 1273. Sofowora A, 2008. Medicinal plants and traditional medicine in Africa. Spectrum Book Ltd., Ibadan, Nigeria. p. 289. Sofowora, A. (2003). Medicinal plants and traditional medicine if Africa. Spectrum Books, Ibadan Nigeria World Health Organization (WHO). The World Health Report, 2004, Geneva; 2004. 37
https://openalex.org/W4295008181	https://zenodo.org/records/7981624/files/D72901111422.pdf	English	null	RF and Shunt Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems	International journal of recent technology and engineering	2,022	cc-by	6,503	Nor Farahaida Abdul Rahman Nor Farahaida Abdul Rahman The linear loads require a sinusoidal supply current waveform for their operation. However, inductive and capacitive linear loads can cause the phase angle of the supply current to lag or lead to the phase angle of the supply voltage waveform. Hence, it reduces the input Power Factor (PF). The low PF increases the current in power systems and conduction losses in wirings and transformers [1]. As a result, it may damage or shorten the equipment’s lifespan. Abstract: This work investigates the effects of RL filters and single-phase Shunt Active Power Filters (SAPFs) on the supply and load current waveforms and their properties. The parameters involved are rms, input Power Factor (PF) and Total Harmonic Distortion (THD). These parameters can describe the quality of any electrical power system, especially PF and THD. This work focuses on implementing both filters in an electrical radial system due to the limited research work. Hence, the effects of utilising both filters in the radial system are studied. In this work, both filters connect at different Points of Common Couplings (PCCs) of a single-phase radial electrical system. The PCCs are located before composite loads (Case 1), before all nonlinear loads (Case 1) and before individual nonlinear loads (Case 3). Both Cases 1 and 2 apply a centralised SAPF, and Case 3 employs individual SAPFs. Matlab/ Simulink simulates all case studies under four operating conditions: (1) without any filters, (2) with SAPFs only, (3) with RL filters only, and (4) with the RL filters and SAPFs. According to the simulation results, the SAPFs require the RF filters to compensate harmonic components effectively. Otherwise, the SAPF’s injection current consists of high current spikes. However, the RF filters may slightly alter the load current waveforms, rms and THD values; the changes are insignificant. Moreover, based on the THD values of the compensated supply current waveform, the centralised SAPFs seem more suitable to be employed in the radial system. By utilising the centralised SAPFs, the THD values of the supply current are lower than using the individual SAPFs. Thus, it can be stated that the centralised SAPFs exhibit better performance. Nevertheless, connecting both filters on the PCCs of all nonlinear loads (Case 2) is suitable to avoid any linear load current waveform deterioration. In contrast, the nonlinear loads need a nonsinusoidal supply current waveform. International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 Nor Farahaida Abdul Rahman Thus, other than lowering the input PF, the loads increase the supply current’s total harmonic distortion (THD) value. It is due to the switching operation of the semiconductor power converters; they generate high harmonic components into the supply current waveform. Consequently, the high THD value can also increase the power system’s current. Furthermore, the high-frequency harmonic components generated can contribute to an additional core loss in motors and interfere with the frequency of nearby communication lines [2]. Generally, a radial electrical system has multiple linear and nonlinear loads connected to the same Point of Common Coupling (PCC). The combination of both loads is known as composite loads [3]. However, since the nonlinear loads cause the adverse quality of the supply current waveshape, it may affect the operation of the linear loads. The effect becomes severe to loads highly sensitive to the change of the supply current waveform. Among all mitigation tools, a Shunt Active Power Factor (SAPF) can mitigate both high THD and low PF simultaneously [4]. The filter injects harmonic current and reactive power to compensate for harmonic components and reactive power supplied by an AC source. Keywords: Composite Loads, Linear Load, Nonlinear Load, Shunt Active Power Filters (SAPFs), Power Factor (PF), Total Harmonic Distortion (THD). Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems Other than that, for the last three years (2019 – 2021), many scholars have been conducting research to enhance SAPF performance. There are five categories of those research work: (i) control algorithms/ controllers [6][7][9][12][14][16][17][18], (ii) SAPF topologies/ converters [5][16][19][20][21], (iii) SAPF for specific loads/ applications and performance analysis [8][10][22][23][24], (iv) Optimisation techniques [5][15][19][25], and (v) extraction algorithms [7][11][13]. current controller that operates as a part of a SAPF control system. Lastly, few researchers tried to improve SAPF extraction techniques. In [7] and [13], the authors introduced an extraction algorithm based on the modified power balance theory [7] and the adaptive linear neural network approach (a) (b) Fig. 1. Flowcharts of phases (a) and (b) procedures. (b) g In the first category, authors in [6], [7], [16] and [17] focus on enhancing SAPFs’ current controllers. They introduced a three-level hysteresis controller [6], a Proportional Integral (PI) controller with integral windup action, a neuro-fuzzy controller based on the Adaptive Neuro-Fuzzy Inference System (ANFIS) [16], and a one-cycle digital controller for minimising the integral error [17]. Instead, authors in [9], [12], [14] and [18] focus on improving the control algorithm itself. The authors in [9] introduced a Finite Impulse Response (FIR) predictor controller to a digital microcontroller. It causes the microcontroller to have one sampling period delay only. Meanwhile, the authors in [12] presented a modulated model based on Pareto-based MO-M2 predictive control that can be adapted to any converter topology. In comparison, the authors in [16] presented an improved Predictive Direct Power Control (PDPC) algorithm. Finally, the authors in [18] developed a double closed looped current control algorithm to adapt weak grid application. Several inverter topologies based on SAPF operation have been introduced in the second category. There are three-phase Flying Capacitor Inverter (FCI) [5], neutral point clamped (NPC) [16], five-level cascaded H-bridge (CHB) inverter [19], 13-level cascaded H-bridge (CHB) converter [20], and shunt isolated active power filter with common dc link integrating braking energy recovery [21]. Some topologies were introduced to satisfy particular standards, such as the NRS 048-2:2003 grid code standard [19], and to minimise the THD value [20]. (b) Fig. 1. Flowcharts of phases (a) and (b) procedures. Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org I. INTRODUCTION El Electrical loads can be either linear or nonlinear loads. The elements of linear loads are resistors, inductors and capacitors only. Examples of linear loads are incandescent light bulbs and electric motors. Instead, nonlinear loads consist of a combination of semiconductor power converters and passive elements. Examples of nonlinear loads are computers, Adjustable Speed Drives (ASD) and LED lamps. An RL filter is commonly employed to filter the SAPF’s injection current. It is connected to the PCC on the SAPF’s AC side [5][6][7]. Moreover, to escalate compensation performance, other RL filters can be connected to the PCC on the AC source side only [8][9][10], the load side only [11][12][13] or the AC source and load sides [2][7][8]. However, no publication has highlighted the RL filters’ significance and consequences before and after the SAPF installation. Especially the effect of RL filters connected to the PCC on the AC source or load side on the load current waveform and its properties. Nevertheless, this paper is interested in only studying the effect of the RL filter connected to the load side. It focuses on the SAPF performance without and with the RL filter, the effect of the RL filter on the load current waveform, and its properties. Manuscript received on 05 September 2022 \| Revised Manuscript received on 07 September 2022 \| Manuscript Accepted on 15 November 2022 \| Manuscript published on 30 November 2022. * Correspondence Author (s) Nor Farahaaida Abdul Rahman*, School of Electrical Engineering, College of Engineering, Universiti Teknologi MARA, Shah Alam, Selangor, Malaysia. Email: farahaida@uitm.edu.my Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. © The Authors. Published by Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP). This is an open access article under the CC-BY-NC-ND license http://creativecommons.org/licenses/by-nc-nd/4.0/ Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org 6 II. METHODOLOGY Fig. 1 shows the procedures of the research work. It has two phases: (a) constructing and tuning the SAPF’s circuit and control algorithm and (b) investigating the effect of the RL filter and the single-phase centralised and individual SAPFs on the supply and load current waveforms and their properties. (a) International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 Even for the past ten years, there have been very few publications related to SAPFs for a system consisting of a combination of nonlinear loads or composite loads [3][26][27][28]. Nevertheless, all of them utilised a single SAPF (centralised) to compensate for harmonic components generated by the loads. The centre of attention of their results is the effectiveness of the SAPF for compensating the harmonic components and reactive power. Despite that, the effect of using the centralised SAPF on the linear load in the system is not subjective studied. Therefore, the authors of [29] published a work that studies the effect of using centralised and individual SAPFs on the supply current and linear load current waveforms in the radial electrical system. However, they only consider the waveshape of both current waveforms, THD values and harmonic components; there is no study on the rms current and input PF. Thus, this paper will continue the study by considering the above parameters. Additionally, unlike in [29], this work will study the effect of installing single-phase SAPFs at different PCCs. (a) (b) Fig. 2. Diagrams of (a) the RL filter and SAPF circuit and (b) SAPF control algorithm. (a) (a) ( ) (b) The paper has four sections: the introduction presents the background study, literature review, problem statements and objectives, the methodology explains all procedures executed to achieve the objectives, the results and discussions verify the centralised and individual SAPFs operation regarding three case studies, and the conclusion summaries the results according to the objectives. (b) Fig. 2. Diagrams of (a) the RL filter and SAPF circuit and (b) SAPF control algorithm. Fig. 2. Diagrams of (a) the RL filter and SAPF circuit and (b) SAPF control algorithm. (a) (b) (c) Fig. 3. PCCs in the primary radial circuits for Cases (a) 1, (b) 2 and (c) 3. (a) Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org 7 International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems Table- I: Parameters of components used in the simulation work Description Component Value Main power supply AC source 220 V & 50 Hz RL LL Resistor Inductor 80  50 mH RL NL Rectifier Resistor Inductor 80  50 mH RLC NL Rectifier Resistor Inductor Capacitor 80  50 mH 450 F RC NL Rectifier Resistor Capacitor 80  450 F RL filter Resistor, R Inductor, L 1  5 mH SAPF Resistor, RF Inductor, LF DC-link capacitor, C 0.1  8 mH 2200 F [13]. Meanwhile, the authors in [11] implemented an Adaptive Notch Filter (ANF) to extract the reference signal Table- I: Parameters of components used in the simulation work Despite converter topologies, in category three, some SAPFs were developed for specific applications, systems or loads. Such as an unbalance load system [8], medium and high power brushless DC (BLDC) motor drives [10], Induction Heating (IH) system [22], and varied power supply source and load parameters [23]. Meanwhile, the authors in [24] introduced a singular perturbation technique, and averaging theory is used for a complete and rigorous formal analysis to describe the control system performances. In recent years, some researchers introduced several optimisation techniques (in category four) to enhance the existing control algorithms or controllers. For example, the authors in [5], [15] and [19] designed an Artificial Neural Network based on Particle Swarm Optimisation (ANN-PSO) to improve a SAPF’s current control algorithm. They utilised it to obtain optimal control parameters to satisfy power quality requirements and high dynamic responses [15]. Meanwhile, the authors in [19] used the optimisation technique to perform the Selective Harmonic Minimisation-Pulse Amplitude Modulation (SHM-PAM) technique utilising the least number of switching based on the optimised waveform pattern. Instead, the authors in [25] developed an algorithm for finding the optimal control for a [13]. Meanwhile, the authors in [11] implemented an Adaptive Notch Filter (ANF) to extract the reference signal. p Adaptive Notch Filter (ANF) to extract the reference signal. However, most researchers focus on applying a SAPF for a single nonlinear load except the authors of the following papers: [6][8][13][21] and [23]. Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. ingle nonlinear load except the authors of the following apers: [6][8][13][21] and [23]. A. Phase A (b) This work considers four types of loads: Inductive Linear Load (RL LL) and Inductive Nonlinear Load (RL NL), Inductive-Capacitive Nonlinear Load (RCL NL), and Capacitive Nonlinear Load (RC NL). All nonlinear loads consist of single-phase uncontrollable rectifiers with passive elements. Table I presents the parameters of those loads. Initially, Matlab/ Simulink simulates each load separately with a single-phase AC source for measuring the rms and THD values of the instantaneous supply current iS(t). Then, a single-phase SAPF is constructed and tuned according to the circuit with the highest iS(t) THD value. (b) (c) Fig. 2 depicts the RL filter and the SAPF’s circuit and control algorithm. Fig. 2(a) indicates that the RL filter connects at the PCC of the load side, and the SAPF connects in parallel with the AC source. The current control algorithm utilises a bandless hysteresis controller, and the voltage control algorithm applies a PI controller. Both controllers regulate the instantaneous compensation current iF(t) and the dc-link voltage Vdc. The gains of the PI controller are tuned according to two indicators: (a) the Vdc value equals the (c) Fig. 3. PCCs in the primary radial circuits for Cases (a) 1, (b) 2 and (c) 3. Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. 8 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org 8 Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org 8 RF and Shunt Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems s of Common Couplings of Radial Electrical Systems Table- II: RMS and THD values of all current waveforms Parameters iS(t) = iL(t) iL1(t) iL2(t) iL3(t) iL4(t) RMS, A 19.93 2.697 2.688 8.38 8.402 THD, % 92.55 0 8.09 139.09 139.43 Fig. 4. Compensated is(t) waveforms when the SAPFs operate without the RL filters in Cases (a) 1, (b) 2 and (c) 3. Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems RF and Shunt Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems RF and Shunt Active Power Filters at Multiple Points of Common Couplings of Radial Ele of Common Couplings of Radial Electrical Systems Table- II: RMS and THD values of all current waveforms Parameters iS(t) = iL(t) iL1(t) iL2(t) iL3(t) iL4(t) RMS, A 19.93 2.697 2.688 8.38 8.402 THD, % 92.55 0 8.09 139.09 139.43 Fig. 4. Compensated is(t) waveforms when the SAPFs operate without the RL filters in Cases (a) 1, (b) 2 and (c) 3. Fig. 5. Compensated is(t) waveforms when the SAPFs operate without the RL filters. The primary circuit connects to (a) Load 4 (high THD value) or (b) Load 2 (low THD value) only. reference dc-link voltage Vdc, ref, and (b) the iS(t) THD value below 5% (according to the IEEE Standard 519-2014). Table I tabulates the parameter value of the RF filter and the SAPF. III. RESULTS AND DISCUSSIONS This section refers to Fig. 1(a) in arranging and discussing all results obtained from the simulation work. It considers four operating conditions for each study case: all primary radial circuits operate (1) without the RL filters and SAPFs, (2) with the SAPFs only, (3) with the RF filters only, and (4) with the RL filters and SAPFs. Fig. 5. Compensated is(t) waveforms when the SAPFs operate without the RL filters. The primary circuit connects to (a) Load 4 (high THD value) or (b) Load 2 (low THD value) only. B. Phase B In this phase, three primary radial circuits are constructed using the loads in Table I. Each circuit has different PCCs for installing the RF filter and the SAPF. All circuits are utilised for three case studies. Case 1 focuses on the effect of the RL filter and a centralised SAPF installed at the PCC of the composite loads. Other than the effect of the RL filter, Case 2 still considers the centralised SAPF implementation. However, the PCC is now at the connection point of all nonlinear loads. Meanwhile, Case 3 focuses on the individual SAPFs connected to each nonlinear load. Fig. 3 shows the configuration and PCCs of each primary circuit. This work applies the same SAPF in the previous phase as the centralised and individual SAPFs, regardless of the case studies. Firstly, all primary circuits only apply the SAPFs for investigating the necessity of the RL filter. The parameter involved in this investigation is iS(t) only. Then, the primary circuits only utilise the RF filter. The filter is installed on each PCC and in series with the dedicated loads. In this stage, the analysis involves the measurement of the rms and THD values of the instantaneous composite iL(t), RL LL iL1(t), RL NL iL2(t), RCL NL iL3(t), and RC NL iL4(t) current waveforms. The study also considers the input PF, the rms and THD values of iS(t). Lastly, the primary circuits use both the RF filters and SAPFs. In the last stage, the analysis focuses on the same parameter as the previous stage, except for the load currents. Fig. 4. Compensated is(t) waveforms when the SAPFs operate without the RL filters in Cases (a) 1, (b) 2 and (c) 3. iS(t) = iL(t). Table II indicated that each load drew a different rms current value; RCL NL and RC NL drew a higher rms current value than RL LL and RL NL. Hence, it increased the rms supply current value. In addition, according to the THD values, the presented data depicted that the capacitive nonlinear loads generated higher harmonic components in the load current waveform than the inductive nonlinear load. In contrast, the inductive linear load did not generate any harmonic component. Hence, it justified that the iL2(t), iL3(t) and iL4(t) waveforms were distorted from the sinusoidal waveshape, and the iL1(t) waveform was purely sinusoidal. Therefore, it has been proven that the primary source of the harmonic problem was the nonlinear loads. C. Operating Condition 3 (RF filters implementation) The study focuses on the effect of using the RF filters on the rms and THD values of iL(t), iL1(t), iL2(t), iL3(t), and iL4(t). Table III and IV present the rms and THD values of all current waveforms for all case studies. The discussion started with the effects of the RL filters on the composite load current (iL(t)), linear load current (iL1(t)) and nonlinear load currents (iL2(t), iL3(t), and iL4(t)). A. Operating Condition 1 (no filters implementation) of the study cases. However, the current waveforms had high current spikes in both positive and negative half-cycles. It happened due to the compensation of very high harmonic components. Fig. 5 shows the compensated iS(t) waveforms when the AC source supplies an individual load that generates either the highest or lowest harmonic components. The waveforms verified that the current spike positively correlated with the harmonic components. Therefore, the RF filter is necessary when installing SAPFs. Table II presents the rms and THD values of both supply and loads current waveforms. Regardless of the study cases, iS(t) = iL(t). () Table II presents the rms and THD values of both supply and loads current waveforms. Regardless of the study cases, iS(t) = iL(t). () B. Operating Condition 2 (SAPFs implementation) A di t T bl III th i (t) l d d Table- III: Percentage different of rms load current values without and with the RL filter Table- III: Percentage different of rms load current values without and with the RL filter (a) Case 1 Case 2 Case 3 iL(t) - 0.27% - 0.25% - 0.16% iL1(t) - 0.08% 0 0 iL2(t) - 0.08% - 0.06% - 0.02% iL3(t) - 0.38% - 0.38% - 0.26% iL4(t) - 0.38% - 0.37% - 0.26% Table- IV: Percentage different of load current THD values without and with the RL Table- IV: Percentage different of load current THD values without and with the RL Case 1 Case 2 Case 3 iL(t) - 0.56% - 0.55% - 0.42% iL1(t) 1211 % 0 0 iL2(t) 0.79 % 0.82% - 0.35% iL3(t) - 0.51% - 0.51% - 0.40% iL4(t) - 0.50% - 0.51% - 0.40% (a) (b) Fig. 6. Effects of the RL filters and SAPFs on the input PF. (b) Fig. 7. Effects of the RL filters and SAPFs on the iS(t) (a) RMS and (b) THD values. dropped slightly from 0.02% to 0.08% for iL2(t), and 0.26% to 0.38% for iL3(t) and iL4(t). Hence, it demonstrated that the RL filter caused a significant effect on the capacitive nonlinear loads (RLC LL and RC LL) instead of the inductive nonlinear load (RL LL). The effect was noticeable since RLC LL and RC LL drew higher currents than RL LL. Nonetheless, as shown in Table IV, the filters affected the THD values of all nonlinear loads’ currents differently. For RL LL, the iL2(t) THD values increased around 0.8% in Cases 1 and 2 and decreased to 0.35% in Case 3. It happened due to multiple connections of RL filters at three PCCs, and the load drew a small current. For RCL LL and RC LL, the iL3(t) and iL4(t) THD values decreased from 0.4% to 0.5%, regardless of case studies. Nonetheless, the changes in the THD values for both inductive and capacitive nonlinears’ currents are small and insignificant. Fig. 6. Effects of the RL filters and SAPFs on the input PF. According to Table III, the iL(t) rms values reduced between 0.16% to 0.27%. Similarly, based on Table IV, the iL(t) THD values decreased between 0.42% to 0.56%. B. Operating Condition 2 (SAPFs implementation) However, due to the slight drop in the rms and THD values, thus, the RF filters did not affect the current draws by the composite load significantly. In contrast, Table III indicated that the iL1(t) rms values remain unchanged in Cases 2 and 3 but in Case 1. Nevertheless, the change in Case 1 was minimal, which was 0.08% and can be negligible. In addition, Table IV revealed that the RF filters tremendously afflicted the iL1(t) THD value in Case 1. The value increased by 1211%. Hence, it has been proven that the RL filters have changed the iL1(t) waveform from a purely sinusoidal to a distorted sinusoidal wave shape. However, the THD values remind unchanged for Cases 2 and 3. Unlike Cases 2 and 3, the RL filter in Case 1 (Fig. 3(a)) connected to the PCC before RL LL. Thus, it affected the shape of the current drawn by the linear load. Therefore, the RF filters are not suitable to be connected before the linear load because they can adversely distort the wave shape of the current waveform. B. Operating Condition 2 (SAPFs implementation) Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. ( ( ) ( ) ( )) Fig. 4 depicts all case studies’ compensated iS(t) waveforms. The waveforms in all figures demonstrated that the SAPFs could perform harmonic compensation regardless Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org 9 International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 erent of rms load current with the RL filter Case 2 Case 3 - 0.25% - 0.16% 0 0 - 0.06% - 0.02% - 0.38% - 0.26% - 0.37% - 0.26% rent of load current THD nd with the RL Case 2 Case 3 - 0.55% - 0.42% 0 0 0.82% - 0.35% - 0.51% - 0.40% - 0.51% - 0.40% (a) (b) Fig. 7. Effects of the RL filters and SAPFs on the iS(t) (a) RMS and (b) THD values. l Journal of Recent Technology and Engineering (IJRTE) N: 2277-3878 (Online), Volume-11 Issue-4, November 2022 (a) (b) Fig. 7. Effects of the RL filters and SAPFs on the iS(t) (a) RMS and (b) THD values. International Journal of Recent Technology and Engineering (IJRTE) ISSN: 2277-3878 (Online), Volume-11 Issue-4, November 2022 Table- III: Percentage different of rms load current values without and with the RL filter Case 1 Case 2 Case 3 iL(t) - 0.27% - 0.25% - 0.16% iL1(t) - 0.08% 0 0 iL2(t) - 0.08% - 0.06% - 0.02% iL3(t) - 0.38% - 0.38% - 0.26% iL4(t) - 0.38% - 0.37% - 0.26% Table- IV: Percentage different of load current THD values without and with the RL Case 1 Case 2 Case 3 iL(t) - 0.56% - 0.55% - 0.42% iL1(t) 1211 % 0 0 iL2(t) 0.79 % 0.82% - 0.35% iL3(t) - 0.51% - 0.51% - 0.40% iL4(t) - 0.50% - 0.51% - 0.40% Fig. 6. Effects of the RL filters and SAPFs on the input PF. IV. CONCLUSION According to the simulation results, SAPFs require RL filters to operate effectively. However, the filters could alter the load current waveform and its properties. Nonetheless, the changes are small, and they can be considered negligible. Additionally, the simulation results verify that the centralised SAPFs are more suitable for the radial electrical system. The SAPFs must only be installed at the PCC of nonlinear loads to avoid linear load current waveform distortion. Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems Based on the relationship between iS(t) and iL(t) discussed in (1) and Fig. 6, the RF filter installation could also reduce the iS(t) rms value. Nevertheless, Fig. 7(a) revealed that the combination of the RF filters and SAPFs had further reduced the iS(t) rms values. The values dropped around 10% to 13% since the impedance catered by the AC source increased due to the composite loads, RF filters and SAPFs. he iS(t) rms value. Nevertheless, Fig. 7(a) revealed that the (a) (a) Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org (a) (b) (c) Fig. 10. Waveforms of all instantaneous currents after the RL filters and SAPFs installation in Case (a) 1, (b) 2, and (c) 3. Additionally, Fig. 7(b) showed that the RF filters reduced the iS(t) THD values from 92.55% (Table II for iL(t)) to 40% to 53%. Subsequently, the value decreased to below 5% after the SAPFs implementation. Hence, it exhibited that the function of the RF filters was to reduce the iS(t) THD values before the SAPFs could perform extensive harmonic compensation. Therefore, all iS(t) THD values presented have complied with the IEEE Standard 519-2014. Moreover, Fig. 7(b) indicated that both filters compensated harmonic currents more effective in Cases 1 and 2 than in Case 3. Thus, it justified that the centralised SAPFs could perform better than the individual SAPFs. The factors on why the individual SAPFs cannot perform better were related to the increase in the number of the SAPFs’ impedance and control algorithms. Nonetheless, as the continuation of the effect of the RL filters in Operating Condition 3, the installation of the SAPF in Case 1 could not rectify the distorted linear current waveform. Therefore, installing a centralised SAPF at the PCC that has composite loads is not recommended. Instead, it is suitable to group all nonlinear loads at one PCC for the filter installation to avoid any linear load current waveform deterioration, such as in Case 2. Fig. 8 shows the waveforms of all instantaneous currents in all case studies after the filter installation. The figures validate all the previous discussions. (a) ( ) ACKNOWLEDGMENT (b) (b) (c) The research was supported by Universiti Teknologi MARA (UiTM) under Lestari Grant 600-RMC/MYRA 5/3/ LESTARI (037/2020) & the College of Engineering UiTM. D. Operating Condition 4 (RF filters and SAPFs implementation) Figs. 6 to 8 present the value of the input PF, the rms and THD of iS(t) when the primary radial circuits apply the individual RF filters and the combination of the filter and SAPFs. According to the simulation work, the input PF of all primary circuits without both filters was 0.719. However, Fig. 6 indicated that both filters could increase the PF. Nonetheless, the circuits required both filters to achieve the unity PF. Hence, it has been proven that combining the RF filters and SAPFs could improve the input PF. Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Similar to the iL(t) and iL1(t) waveforms, Table III exhibited that the rms values of all nonlinear load currents Published By: Blue Eyes Intelligence Engineering and Sciences Publication (BEIESP) © Copyright: All rights reserved. Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org Retrieval Number: 100.1/ijrte.D72901111422 DOI: 10.35940/ijrte.D7290.1111422 Journal Website: www.ijrte.org 10 RF and Shunt Active Power Filters at Multiple Points of Common Couplings of Radial Electrical Systems 4. P. Salmerón Revuelta, S. Pérez Litrán, J. Prieto Thomas, “Active power line conditioners,” design, simulation and implementation for improving power quality, (), 231–284. doi:10.1016/b978-0-12-803216-9.00007-9. [CrossRef] Power Electronics, vol. 13, no. 11, pp. 2350 – 2360, August 2020, doi: 10.1049/iet-pel.2019.1295. [CrossRef] p [ ] 20. V. Narasimhulu, D. V. Ashok Kumar, and Ch. Sai Babu, “Recital analysis of multilevel cascade H‑bridge based active power filter under load variation,” SN Applied Sciences, vol. 1, no. 12, December 2019, doi: 10.1007/s42452-019-1669-8. [CrossRef] p g p q y () doi:10.1016/b978-0-12-803216-9.00007-9. [CrossRef] 5. D. Khaled, A. 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https://openalex.org/W1990342289	https://digitalcommons.mtu.edu/cgi/viewcontent.cgi?article=3479&context=michigantech-p	English	null	Projected effect of 2000–2050 changes in climate and emissions on aerosol levels in China and associated transboundary transport	Atmospheric chemistry and physics	2,013	cc-by	18,353	9-2-2013 See next page for additional authors Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p See next page for additional authors Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p Part of the Geological Engineering Commons, and the Mining Engineering Commons Part of the Geological Engineering Commons, and the Mining Engineering Commons Michigan Technological University Michigan Technological University Digital Commons @ Michigan Tech Digital Commons @ Michigan Tech Michigan Tech Publications 9-2-2013 Projected effect of 2000-2050 changes in climate and emissions Projected effect of 2000-2050 changes in climate and emissions on aerosol levels in China and associated transboundary on aerosol levels in China and associated transboundary transport transport H. Jiang Institute of Atmospheric Physics Chinese Academy of Sciences H. Liao Institute of Atmospheric Physics Chinese Academy of Sciences H. O.T. Pye United States Environmental Protection Agency S. Wu Michigan Technological University, slwu@mtu.edu L. J. Mickley Harvard University See next page for additional authors Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p Part of the Geological Engineering Commons, and the Mining Engineering Commons Recommended Citation Recommended Citation Jiang, H., Liao, H., Pye, H., Wu, S., Mickley, L., Seinfeld, J., & Zhang, X. (2013). Projected effect of 2000-2050 changes in climate and emissions on aerosol levels in China and associated transboundary transport. Atmospheric Chemistry and Physics, 13(16), 7937-7960. http://doi.org/10.5194/ acp-13-7937-2013 Retrieved from: https://digitalcommons.mtu.edu/michigantech-p/2489 Michigan Technological University Michigan Technological University Digital Commons @ Michigan Tech Digital Commons @ Michigan Tech Michigan Tech Publications 9-2-2013 Projected effect of 2000-2050 changes in climate and emissions Projected effect of 2000-2050 changes in climate and emissions on aerosol levels in China and associated transboundary on aerosol levels in China and associated transboundary transport transport H. Jiang Institute of Atmospheric Physics Chinese Academy of Sciences H. Liao Institute of Atmospheric Physics Chinese Academy of Sciences H. O.T. Pye United States Environmental Protection Agency S. Wu Michigan Technological University, slwu@mtu.edu L. J. Mickley Harvard University See next page for additional authors Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p Part of the Geological Engineering Commons, and the Mining Engineering Commons Recommended Citation Recommended Citation Jiang, H., Liao, H., Pye, H., Wu, S., Mickley, L., Seinfeld, J., & Zhang, X. (2013). Projected effect of 2000-2050 changes in climate and emissions on aerosol levels in China and associated transboundary transport. Atmospheric Chemistry and Physics, 13(16), 7937-7960. http://doi.org/10.5194/ acp-13-7937-2013 Retrieved from: https://digitalcommons.mtu.edu/michigantech-p/2489 Michigan Technological University Michigan Technological University Digital Commons @ Michigan Tech Digital Commons @ Michigan Tech Authors Authors H. Jiang, H. Liao, H. O.T. Pye, S. Wu, L. J. Mickley, J. H. Seinfeld, and X. Y. Zhang Authors Authors H. Jiang, H. Liao, H. O.T. Pye, S. Wu, L. J. Mickley, J. H. Seinfeld, and X. Y. Zhang Recommended Citation Recommended Citation Jiang, H., Liao, H., Pye, H., Wu, S., Mickley, L., Seinfeld, J., & Zhang, X. (2013). Projected effect of 2000-2050 changes in climate and emissions on aerosol levels in China and associated transboundary transport. Atmospheric Chemistry and Physics, 13(16), 7937-7960. http://doi.org/10.5194/ acp-13-7937-2013 Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p Part of the Geological Engineering Commons, and the Mining Engineering Commons This article is available at Digital Commons @ Michigan Tech: https://digitalcommons.mtu.edu/mich This article is available at Digital Commons @ Michigan Tech: https://digitalcommons.mtu.edu/michigantech-p/2489 Biogeosciences Open A Projected effect of 2000–2050 changes in climate and emissions on aerosol levels in China and associated transboundary transport . Jiang1,2, H. Liao1, H. O. T. Pye3, S. Wu4, L. J. Mickley5, J. H. Seinfeld6, and X. Y. Zhang7 1State Key Laboratory of Atmospheric Boundary Layer Physics and Atmospheric Chemistry Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing, China Climate of the Past n Access y y j g 3National Exposure Research Laboratory, Environmental Protection Agency, Research Triangle Park, North Carolina, USA 4Department of Geological and Mining Engineering and Sciences and Department of Civil and Environmental Engineering, Michigan Technological University, Houghton, Michigan, USA g g y g g 5School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA 6Departments of Chemical Engineering and Environmental Science and Engineering, California Institute of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA Earth System Open 5School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA 6Departments of Chemical Engineering and Environmental Science and Engineering, California Institute of Technology, Pasadena, California, USA Earth System Open 6Departments of Chemical Engineering and Environmental Science and Engineering, California Institute of Technology, Pasadena, California, USA Earth D 7Key Laboratory for Atmospheric Chemistry, Chinese Academy of Meteorological Sciences, CMA Correspondence to: H. Liao (hongliao@mail.iap.ac.cn) In Received: 13 February 2013 – Published in Atmos. Chem. Phys. Discuss.: 11 March 2013 Revised: 17 June 2013 – Accepted: 5 July 2013 – Published: 16 August 2013 In Received: 13 February 2013 – Published in Atmos. Chem. Phys. Discuss.: 11 March 2013 Revised: 17 June 2013 – Accepted: 5 July 2013 – Published: 16 August 2013 Abstract. We investigate projected 2000–2050 changes in concentrations of aerosols in China and the associated trans- boundary aerosol transport by using the chemical transport model GEOS-Chem driven by the Goddard Institute for Space Studies (GISS) general circulation model (GCM) 3 at 4◦× 5◦resolution. Future changes in climate and emissions projected by the IPCC A1B scenario are imposed separately and together through sensitivity simulations. Accounting for sulfate, nitrate, ammonium, black carbon (BC), and organic carbon (OC) aerosols, concentrations of individual aerosol species change by −1.5 to +0.8 µg m−3, and PM2.5 levels are projected to change by about 10–20 % in eastern China as a result of 2000–2050 change in climate alone. Biogeosciences Open A Projected effect of 2000–2050 changes in climate and emissions on aerosol levels in China and associated transboundary transport The ﬂuxes of nitrate and am- monium aerosols from Europe and Central Asia into west- ern China increase over 2000–2050 in response to projected changes in emissions, leading to a 10.5 % increase in an- nual inﬂow of PM2.5 to western China with future changes in both emissions and climate. Fluxes of BC and OC from South Asia to China in spring contribute a large fraction of the annual inﬂow of PM2.5. The annual inﬂow of PM2.5 from South Asia and Southeast Asia to China is estimated to change by −8 %, +281 %, and +227 % over 2000–2050 ow- ing to climate change alone, changes in emissions alone, and changes in both climate and emissions, respectively. While the 4◦× 5◦spatial resolution is a limitation of the present study, the direction of predicted changes in aerosol levels and transboundary ﬂuxes still provides valuable insight into fu- ture air quality. Biogeosciences Open A Projected effect of 2000–2050 changes in climate and emissions on aerosol levels in China and associated transboundary transport With fu- ture changes in anthropogenic emissions alone, concentra- tions of sulfate, BC, and OC are simulated to decrease be- cause of assumed reductions in emissions, and those of ni- trate are predicted to increase because of higher NOx emis- sions combined with decreases in sulfate. The net result is a predicted reduction of seasonal mean PM2.5 concentrations in eastern China by 1–8 µg m−3 (or 10–40 %) over 2000– 2050. It is noted that current emission inventories for BC and OC over China are judged to be inadequate at present. Trans- boundary ﬂuxes of different aerosol species show different sensitivities to future changes in climate and emissions. The et ods a d Data Systems cess Geoscientific Model Development Open Access Hydrology and Earth System Sciences Open Access Ocean Science Open Access annual outﬂow of PM2.5 from eastern China to the west- ern Paciﬁc is estimated to change by −7.0 %, −0.7 %, and −9.0 % over 2000–2050 owing to climate change alone, changes in emissions alone, and changes in both climate and emissions, respectively. The ﬂuxes of nitrate and am- monium aerosols from Europe and Central Asia into west- ern China increase over 2000–2050 in response to projected changes in emissions, leading to a 10.5 % increase in an- nual inﬂow of PM2.5 to western China with future changes in both emissions and climate. Fluxes of BC and OC from South Asia to China in spring contribute a large fraction of the annual inﬂow of PM2.5. The annual inﬂow of PM2.5 from South Asia and Southeast Asia to China is estimated to change by −8 %, +281 %, and +227 % over 2000–2050 ow- ing to climate change alone, changes in emissions alone, and changes in both climate and emissions, respectively. While the 4◦× 5◦spatial resolution is a limitation of the present study, the direction of predicted changes in aerosol levels and transboundary ﬂuxes still provides valuable insight into fu- ture air quality. Data Systems ess Geoscientific Model Development Open Access Hydrology and Earth System Sciences Open Access Ocean Science Open Access annual outﬂow of PM2.5 from eastern China to the west- ern Paciﬁc is estimated to change by −7.0 %, −0.7 %, and −9.0 % over 2000–2050 owing to climate change alone, changes in emissions alone, and changes in both climate and emissions, respectively. Authors Authors Atmospheric Chemistry and Physics Open Access 1 Introduction Aerosols are important air pollutants that lead to negative health impacts, reductions in visibility, and changes in cli- mate (Intergovernmental Panel on Climate Change (IPCC), 2007). Concentrations of major atmospheric aerosol species (sulfate, nitrate, ammonium, black carbon, organic carbon, and mineral dust) are especially high in China (Matsui et al., 2009; Tie and Cao, 2010; Qu et al., 2010; Cao et al., 2012), driven by a combination of direct and precursor emis- sions (Streets et al., 2003) and regional meteorological con- ditions (Zhang et al., 2010a; Zhu et al., 2012). Estimating fu- ture aerosol levels in China is essential in considerations of air quality both over China itself and in the Northern Hemi- sphere. p In the absence of changes in emissions of primary aerosols as well as aerosol precursors, climate change itself will inﬂuence future aerosol levels. For example, coupled climate–chemical transport modeling studies show that cli- mate change alone can lead to increased surface ozone in anthropogenically impacted regions by 1–10 ppbv in sum- mertime over the coming decades, based on the IPCC fu- ture scenarios (Jacob and Winner, 2009). This increase is a result of slower transport, enhanced biogenic hydrocarbon emissions, and accelerated decomposition of peroxyacetyl nitrate (PAN) at higher temperatures (Hogrefe et al., 2004; Liao et al., 2006; Murazaki and Hess, 2006; Steiner et al., 2006; Racherla and Adams, 2008; Wu et al., 2008; Jacob and Winner, 2009; Andersson and Engardt, 2010; Chang et al., 2010; Lam et al., 2011; Katragkou et al., 2011; Langner et al., 2012; Reuten et al., 2012; Wang et al., 2013a). A warmer future climate is also predicted to inﬂuence aerosol levels over the United States and Europe by as much as 1 µg m−3 through altered concentrations of atmospheric oxidants, by changed precipitation and boundary layer height, and by shifting gas-particle equilibria (Liao et al., 2006; Unger et al., 2006; Bauer et al., 2007; Jacob and Winner, 2009; Pye et al., 2009; Day and Pandis, 2011; Lam et al., 2011; Tai et al., 2012a, b; Juda-Rezler et al., 2012). Because of the enor- mous importance of China as a source of aerosols, a study that addresses how aerosol levels in China may change over the coming decades is called for. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7938 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol leve background surface-layer concentrations of black carbon over the United States. By using satellite measurements of aerosol optical depth over the North Paciﬁc together with GEOS-Chem simulation, Heald et al. (2006) showed that transport from Asia led to a seasonal mean increase of surface-layer sulfate concentration of 0.16 µg m−3 (with 50 % uncertainty) in the northwestern United States in spring of 2001. Chin et al. (2007) predicted an enhancement of sim- ilar magnitude in surface-layer sulfate aerosol in the west- ern United States in 2001 by long-range aerosol transport; the annual mean contribution to sulfate concentration was estimated to be 0.1–0.2 µg m−3 using the global model GO- CART. Yu et al. (2008) performed a satellite-based assess- ment of transpaciﬁc transport of anthropogenic and biomass burning aerosols based on 2002–2005 aerosol optical depths from the Moderate Resolution Imaging Spectroradiometer (MODIS). They estimated that about 25 % of aerosol mass exported from East Asia to the northwestern Paciﬁc Ocean can reach the west coast of North America. Two studies ex- amined aerosol transport from Europe and South Asia to China. Chin et al. (2007) estimated that European emissions can increase the surface ammonium sulfate concentrations over eastern Asia by 0.2–0.5 µg m−3. Using the GEOS-Chem model, Zhang et al. (2010b) estimated that organic carbon aerosol from South Asia contributed 50–70 % of organic car- bon (OC) mass over southern China and 20–50 % of OC over the western North Paciﬁc in the middle troposphere in sum- mer of 1998. Th Published by Copernicus Publications on behalf of the European Geosciences Union. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 2.1 GEOS-Chem/GISS models The atmospheric chemical transport model, GEOS-Chem (v.7-4-11, http://acmg.seas.harvard.edu/geos/), is driven by the GISS Model 3 meteorological data (Rind et al., 2007). Both the GISS Model 3 and the GEOS-Chem models have a horizontal resolution of 4◦latitude by 5◦longitude with 23 vertical layers. The interface between GEOS-Chem and the GISS meteorological ﬁelds was described by Wu et al. (2007) and Pye et al. (2009), and the same meteo- rology from the work of Wu et al. (2008) is used here. The GEOS-Chem model includes coupled ozone–NOx– hydrocarbon (∼80 species, ∼300 chemical reactions) (Bey et al., 2001) and aerosol chemistry. Aerosol species simu- lated in the GEOS-Chem model include sulfate (SO2− 4 ) (Park et al., 2004), nitrate (NO− 3 ) (Pye et al., 2009), ammonium (NH+ 4 ), primary organic carbon (OC) and black carbon (BC) (Park et al., 2003), secondary organic aerosol (SOA), sea salt (Alexander et al., 2005), and mineral dust (Fairlie et al., 2007). SOA formation considers the oxidation of iso- prene (Henze and Seinfeld, 2006), monoterpenes and other reactive volatile organic compounds (ORVOCs) (Liao et al., 2007), and aromatics (Henze et al., 2008). We focus on future changes in anthropogenic aerosols in this work; the assess- ment on mineral dust and sea salt aerosols will be our future work. Present-day and year 2050 natural emissions of ozone and aerosol precursors include NOx from lightning and soil, and biogenic hydrocarbons (Table 2), which are calculated based on the GISS Model 3 meteorological parameters. Lightning NOx emissions are parameterized based on convective cloud- top height (Price and Rind, 1992; Wang et al., 1998). Soil NOx emissions are calculated as a function of temperature, wind speed, and precipitation (Yienger and Levy, 1995). Representation of biogenic emissions follows the algorithm of Guenther et al. (1995), which considers light and temper- ature dependence but does not account for the suppression of isoprene emissions under elevated ambient CO2 concentra- tions (Rosenstiel et al., 2003) and climate-induced changes in land cover. Simulated natural emissions of NOx and biogenic hydrocarbons in eastern China are estimated to increase, re- spectively, by +20 % and +22 % over 2000–2050, with the increases in biogenic emissions resulting mainly from the fu- ture increases in temperature. 2.2 Emissions the direction of projected changes in aerosol levels should be correctly predicted. Present-day and year 2050 assumed anthropogenic emis- sions of aerosol precursor and aerosols are listed in Ta- ble 1. Emissions of O3 precursors (including NOx, CO, and non-methane volatile organic compounds (NMVOCs)) fol- low those in Wu et al. (2008), and those of NH3 and SO2 are taken from Pye et al. (2009). The base year for present-day anthropogenic emissions is 1999 for the United States (Wu et al., 2008) and 1998 elsewhere (Pye et al., 2009). Year 2050 anthropogenic emissions of ozone precursors, aerosol pre- cursors, and aerosols from the IPCC A1B scenario were gen- erated by the Integrated Model to Assess the Greenhouse Ef- fect (IMAGE) socioeconomic model using prescribed growth factors for different regions, species, and sources (Streets et al., 2004). Ammonia emissions have an imposed seasonality that was determined as a function of temperature for one base year in this model. In present day, anthropogenic emissions of NOx, CO, NMVOCs, SO2, NH3, OC, and BC in eastern China are estimated to account for 12 %, 16 %, 12 %, 20 %, 18 %, 10 %, and 19 %, respectively, of the total global emis- sions. Relative to the assumed present day, year 2050 anthro- pogenic emissions of NOx, CO, NMVOCs, SO2, NH3, OC, and BC in eastern China (20◦–55◦N, 98◦–125◦E) are esti- mated to change by +72 %, −7 %, +86 %, −29 %, −12 %, −40 %, and −59 %, respectively. The methods and model setup used to simulate present- day and year 2050 aerosols are described in Sect. 2. Section 3 evaluates simulated present-day concentrations of aerosols in China. Section 4 shows predictions of future aerosol levels over China due to changes in climate alone, emissions alone, and combined climate and emissions changes, and Sect. 5 es- timates future changes in transboundary transport of aerosols to examine inﬂow to and outﬂow from China. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7939 . Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 2.1 GEOS-Chem/GISS models We perform simulations for four cases: (1) year 2000 climate and emissions, (2) 2050 climate and 2000 an- thropogenic emissions of aerosol precursor and aerosols, (3) 2000 climate and 2050 anthropogenic emissions of aerosol precursor and aerosols, and (4) 2050 climate and emissions. Each case is integrated for 10 yr (driven by 1996– 2005 meteorological ﬁelds to represent year 2000 climate or by 2046–2055 meteorology to represent year 2050 climate) following 1 yr of model spin-up. All the results presented in this paper are 10 yr averages. Statistical analysis based on Student’s two-sample t test is applied to the meteorological ﬁelds and concentrations of the 10 yr simulations. For both radiative forcing in climate simulation and chem- ical reactions, present-day methane levels in the model are based on observations and set to 1750 ppb with a 5 % inter- hemispheric gradient (Wu et al., 2008). The future (2046– 2055) methane level in GEOS-Chem follows the IPCC A1B scenario and is set to 2400 ppb for simulations in which changes in anthropogenic emissions are considered (Pye et al., 2009). Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ 1 Introduction We present here a study to estimate the following: (1) the changes of aerosol levels in China over the years 2000–2050 as a result of projected changes in emissions and climate, (2) the changes of transboundary ﬂuxes of aerosols into or out of China over this time period. This study builds on two previous ones. Liao et al. (2007) used the Goddard Institute for Space Studies (GISS) general circulation model (GCM) 3 to drive GEOS-Chem to simulate climatological present- day aerosol levels in the United States, and Pye et al. (2009) investigated the effects of projected climate and emissions changes on 2000–2050 sulfate–nitrate–ammonium aerosols in the United States using the same GISS Model 3/GEOS- Chem combination. As a basis for the present study, the IPCC emission scenario A1B (Nakicenovic and Swart, 2000) is adopted; this scenario represents a future world with rapid economic growth and introduction of new and more energy- efﬁcient technologies. GISS Model 3 global meteorological ﬁelds are used to drive the atmospheric chemical transport model GEOS-Chem for both present day (1996–2005) and years 2046–2055. Effects of climate change alone, emission changes alone, and both climate and emissions changes to- gether on aerosol levels and transboundary ﬂuxes are simu- lated. The models in the present study have a relatively coarse spatial resolution of 4◦latitude by 5◦longitude, which are not expected to capture the characteristically high concentra- tions of aerosols in China’s major urban areas. Nevertheless, Another issue that is associated with the aerosol levels in China is the future transboundary aerosol transport. A num- ber of observational and modeling analyses have demon- strated the importance of present-day long-range transport of aerosols from East Asia. Surface observations at island sites (Huebert et al., 2001; Prospero et al., 2003) and air- craft observations in Asian outﬂow over the Northwest Pa- ciﬁc (Jordan et al., 2003; Maxwell-Meier et al., 2004) and the Northeast Paciﬁc (Clarke et al., 2001; Price et al., 2003) have documented the spring maximum in transpaciﬁc transport. By using the global atmospheric chemical transport model GEOS-Chem, Park et al. (2003) predicted that transpaciﬁc transport contributes about 10 % of the annual mean natural www.atmos-chem-phys.net/13/7937/2013/ www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ www.atmos-chem-phys.net/13/7937/2013/ H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions o ay and year 2050 (IPCC A1B scenario) anthropogenic emissions of aerosol precursors and aerosols. The N, 98◦–125◦E. Global Eastern China Species 2000 2050 Change, % 2000 2050 Change, % NOx (Tg N yr−1) Aircraft 0.5 0.5 0.0 0.02 0.02 0.0 Anthropogenic 23.7 47.9 +102.0 3.15 6.21 +97.1 Biomass burning 6.5 8.1 +24.6 0.30 0.34 +13.3 Biofuel 2.2 2.1 −4.5 0.53 0.36 −32.1 Fertilizer 0.5 0.9 +80.0 0.06 0.04 −33.3 Total 33.4 59.5 +78.1 4.06 6.97 +71.7 CO (Tg CO yr−1) Anthropogenic 420.1 413.6 −1.5 104.26 94.04 −9.8 Biomass burning 458.8 749.6 +63.4 21.69 34.46 +58.9 Biofuel 175.6 168.9 −3.8 46.34 31.24 −32.6 Total 1054.5 1332.1 +26.3 172.29 159.74 −7.3 NMVOCs (Tg C yr−1) Anthropogenic 24.5 42.2 +72.2 2.45 6.52 +166 Biomass burning 3.1 5.1 +64.5 0.14 0.23 +64.3 Biofuel 7.2 6.9 −4.2 1.65 1.12 −32.1 Total 34.8 54.2 +55.7 4.24 7.87 +85.6 SO2 (Tg S yr−1) Aircraft 0.1 0.1 0.0 < 0.01 < 0.01 0.0 Anthropogenic 66.1 88.7 +34.2 13.83 9.81 −29.1 Biomass burning 1.2 2.0 +66.7 0.06 0.09 +50.0 Biofuel 0.3 0.3 0.0 0.07 0.05 −28.6 Ship 4.2 5.4 +28.6 0.03 0.05 +66.7 Total 71.9 96.5 +34.2 13.99 10.00 −28.5 NH3 (Tg N yr−1) Anthropogenic 33.3 50.4 +51.4 6.68 5.90 −11.7 Biomass burning 5.9 6.1 +3.4 0.26 0.28 +7.7 Biofuel 1.6 1.7 +6.3 0.32 0.22 −31.3 Total 40.8 58.2 +42.6 7.26 6.40 −11.8 OC (Tg C yr−1) Anthropogenic 2.5 0.9 −64.0 0.58 0.16 −72.4 Biomass burning 21.6 21.8 +0.9 1.10 1.08 −1.8 Biofuel 6.4 2.7 −57.8 1.31 0.56 −57.3 Total 30.5 25.4 −16.7 2.99 1.80 −39.8 BC (Tg C yr−1) Anthropogenic 3.1 2.0 −35.5 0.84 0.27 −67.9 Biomass burning 2.6 2.7 +3.8 0.13 0.13 0.0 Biofuel 1.5 0.6 −60.0 0.36 0.15 −58.3 Total 7.2 5.3 −26.4 1.33 0.55 −58.6 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels . Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Projected changes are all statistically significant at the 95% level, as determined by the student’s two sample t-test. g. 1. Projected changes in surface air temperature (K) in China from the present day (1996–2005) to future (2046–2055) under the 1B scenario. Projected changes are all statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. 3 Projected climate change in China over 2000–2050 2000–2050 climate change. We have compared the Model 3 predictions of present-day (1996–2005) tem Table 2. Simulated changes in natural emissions due to predicted climate change (IPCC A1B scenario). The domain of eastern China is 20◦–55◦N, 98◦–125◦E. , Global Eastern China Species 2000 2050 Change, % 2000 2050 Change, % NOx (Tg N yr−1) Lightning 4.8 5.7 +18.8 0.28 0.37 +32.1 Soil 6.7 7.3 +9.0 0.36 0.40 +11.1 Total 11.5 13.0 +13.0 0.64 0.77 +20.3 Biogenic hydrocarbons (Tg C yr−1) Isoprene 432.4 533.2 +23.3 19.31 23.90 +23.8 Monoterpenes 120.9 144.4 +19.4 6.26 7.38 +17.9 Acetone 43.4 50.4 +16.1 1.28 1.54 +20.3 Propene 12.4 15.2 +22.6 0.55 0.68 +23.6 Methyl Butenol 5.4 6.8 +25.9 1.03 1.30 +26.2 Total 614.5 750.0 +22.1 28.43 34.80 +22.4 Fig. 1. Projected changes in surface air temperature (K) in China from the present day (1996-2005) to future (2046-2055) under the IPCC A1B scenario. Fig. 1. Projected changes in surface air temperature (K) in China from the present day (1996–2005) to future (2046–2055) under the IPCC A1B scenario. Projected changes are all statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. Fig. 1. Projected changes in surface air temperature (K) in China from the present day (1996-2005) to future (2046-2055) under the IPCC A1B scenario. Fig. 1. Projected changes in surface air temperature (K) in China from the present day (1996–2005) to future (2046–2055) under the IPCC A1B scenario. Projected changes are all statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. Projected changes are all statistic determined by the student’s two samp 2.3 Projected climate change in China over 2000–2050 t-test. 2000–2050 climate change. We have compared the GISS Model 3 predictions of present-day (1996–2005) tempera- ture, wind, and precipitation with assimilated meteorologi- cal parameters for 1996–2005 from the Goddard Earth Ob- serving System (GEOS)-4 of the NASA Global Modeling and Assimilation Ofﬁce (GMAO) (http://acmg.seas.harvard. edu/geos/geos sim.html). The GISS model captures fairly well the pattern and magnitude of surface air temperatures and zonal winds in China (see Supplement). Both the sim- ulated and assimilated surface air temperatures show higher Present-day meteorological conditions from the NASA GISS Model 3 were simulated with greenhouse gas levels cor- responding to years 1996–2005. Year 2046–2055 climate was obtained from a simulation in which CO2 and other greenhouse gases follow the IPCC A1B scenario. The GISS Model 3 was coupled with a “Q-ﬂux” ocean as described in Wu et al. (2008). Note that the direct and indirect ef- fects of aerosols were not considered in the simulation of Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7940 Table 1. Present-day and year 2050 (IPCC A1B scenario) anthropogenic emissions of aerosol precursors and aerosols. The domain of eastern China is 20◦–55◦N, 98◦–125◦E. Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 . Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7941 Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels ble 2. Simulated changes in natural emissions due to predicted climate change (IPCC A1B scenario). The domain of eastern C –55◦N, 98◦–125◦E. Global Eastern China Species 2000 2050 Change, % 2000 2050 Change, % NOx (Tg N yr−1) Lightning 4.8 5.7 +18.8 0.28 0.37 +32.1 Soil 6.7 7.3 +9.0 0.36 0.40 +11.1 Total 11.5 13.0 +13.0 0.64 0.77 +20.3 Biogenic hydrocarbons (Tg C yr−1) Isoprene 432.4 533.2 +23.3 19.31 23.90 +23.8 Monoterpenes 120.9 144.4 +19.4 6.26 7.38 +17.9 Acetone 43.4 50.4 +16.1 1.28 1.54 +20.3 Propene 12.4 15.2 +22.6 0.55 0.68 +23.6 Methyl Butenol 5.4 6.8 +25.9 1.03 1.30 +26.2 Total 614.5 750.0 +22.1 28.43 34.80 +22.4 Fig. 1. Projected changes in surface air temperature (K) in China from the present day (1996-2005) to future (2046-2055) under the IPCC A1B scenario. Projected changes are all statistically significant at the 95% level, as determined by the student’s two sample t-test. . 1. Projected changes in surface air temperature (K) in China from the present day (1996–2005) to future (2046–2055) under th B scenario. Projected changes are all statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. Projected climate change in China over 2000–2050 2000–2050 climate change. We have compared the Model 3 predictions of present-day (1996–2005) tem ture wind and precipitation with assimilated meteor . Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels able 2. Simulated changes in natural emissions due to predicted climate change (IPCC A1B scenario). The domain of eastern Ch ◦–55◦N, 98◦–125◦E. Global Eastern China Species 2000 2050 Change, % 2000 2050 Change, % NOx (Tg N yr−1) Lightning 4.8 5.7 +18.8 0.28 0.37 +32.1 Soil 6.7 7.3 +9.0 0.36 0.40 +11.1 Total 11.5 13.0 +13.0 0.64 0.77 +20.3 Biogenic hydrocarbons (Tg C yr−1) Isoprene 432.4 533.2 +23.3 19.31 23.90 +23.8 Monoterpenes 120.9 144.4 +19.4 6.26 7.38 +17.9 Acetone 43.4 50.4 +16.1 1.28 1.54 +20.3 Propene 12.4 15.2 +22.6 0.55 0.68 +23.6 Methyl Butenol 5.4 6.8 +25.9 1.03 1.30 +26.2 Total 614.5 750.0 +22.1 28.43 34.80 +22.4 Fig. 1. Projected changes in surface air temperature (K) in China from the present day (1996-2005) to future (2046-2055) under the IPCC A1B scenario. www.atmos-chem-phys.net/13/7937/2013/ H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7942 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Fig. 2. (a) Simulated precipitation (mm day-1) in China in present day; (b) Projected changes in precipitation (mm day-1) in China from the present day (1996 2005) t f t (2046 2055) d th IPCC A1B i ( ) Th Fig. 2. (a) Simulated precipitation (mm day−1) in China in present day; (b) projected changes in precipitation (mm day−1) in China from the present day (1996–2005) to future (2046–2055) under the IPCC A1B scenario; (c) the percentage changes in precipitation relative to present day. The dotted areas in (c) are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. Fig. 2. (a) Simulated precipitation (mm day-1) in China in present day; (b) Projected changes in precipitation (mm day-1) in China from the present day Fig. 2. (a) Simulated precipitation (mm day−1) in China in present day; (b) projected changes in precipitation (mm day−1) in China from the present day (1996–2005) to future (2046–2055) under the IPCC A1B scenario; (c) the percentage changes in precipitation relative to present day. The dotted areas in (c) are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. percentage changes in precipitation r in (c) are statistically significant at student’s two sample t-test. temperatures in southern China than in northern China and also higher temperatures in eastern China than in western China. The maximum temperatures in southeastern China are simulated to be 280–290 K in DJF, 290–300 K in MAM, 300–310 K in JJA, and 290–300 K in SON, which agree with the assimilated values in all seasons except that the as- similated maximum temperatures in southeastern China are lower than 305 K in JJA. Simulated zonal winds averaged over 100◦–120◦E longitudes show maximum wind speeds of the jet stream located at 200 hPa altitude of 60, 40, 20, and 30 m s−1 in DJF, MAM, JJA, and SON, respectively, which agree closely with the assimilated values. The present-day precipitation simulated by the GISS model shows larger val- ues in MAM and JJA than in DJF and SON, which agree ative to present day. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels ang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7943 g. 3. (a) Simulated cloud fraction (true fraction, 1.0= total overc hi i t d (b) P j t d h i l d f ti i Ch ig. 3. (a) Simulated cloud fraction (true fraction, 1.0 = total overcast cloud) in China in present day and (b) projec action in China from the present day (1996–2005) to future (2046–2055) under the IPCC A1B scenario. The do atistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. Fig. 3. (a) Simulated cloud fraction (true fraction, 1.0= total overcast cloud) in Chi i t d (b) P j t d h i l d f ti i Chi f th Fig. 3. (a) Simulated cloud fraction (true fraction, 1.0 = total overcast cloud) in China in present day and (b) projected changes in cloud fraction in China from the present day (1996–2005) to future (2046–2055) under the IPCC A1B scenario. The dotted areas in (b) are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. present day (1996-2005) to future (204 The dotted areas in (b) are statisti predicted to be more pronounced than under scenario B1 and less pronounced than under A2 (IPCC, 2007). -2055) under the IPCC A1B scenario. ally significant at the 95% level, as t-test. (MAM), year 2050 precipitation is estimated to increase by 20–40 % in southern China where the present-day sea- sonal precipitation is greatest. Precipitation is predicted to in- crease generally in eastern China in June-July-August (JJA) and September-October-November (SON). Note that the pro- jected patterns of precipitation changes (the increases in pre- cipitation in northern China in DJF and the increases in pre- cipitation in eastern China in JJA) from the GISS Model 3 e dotted a eas (b) a e stat st c determined by the student’s two sampl Projected changes in precipitation in China from present day to year 2050 are shown in Fig. 2. Relative to present day, year 2050 precipitation in DJF is estimated to increase by 50–80 % in northern China and decrease by about 20 % in southern China; note that the present-day precipitation in DJF is the smallest among all seasons. In March-April-May www.atmos-chem-phys.net/13/7937/2013/ www.atmos-chem-phys.net/13/7937/2013/ The dotted areas e 95% level, as determined by the with the assimilated precipitation, but the model overesti- mates precipitation in the middle and lower reaches of the Yangtze River in MAM whereas underestimates precipitation in that region in JJA. Figure 1 shows the projected changes in surface air tem- perature in China from present day to year 2050 by long- lived greenhouse gases under the IPCC A1B scenario. Over the period 2000–2050, surface air temperatures in December- January-February (DJF) are estimated to have large in- creases, with values of 1.5–3 K in most places of China and the strongest warming of 3–4 K over the Tibetan Plateau and northeastern China. In other seasons, surface air tempera- tures are estimated to increase by 1–3 K over a large frac- tion of China. Warming under scenario A1B is generally www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 3 Simulated present-day aerosols The projected changes in the planetary boundary layer (PBL) depth in China between present day and 2050 are shown in Fig. 4. In a warmer 2050, the PBL depths over the heavily populated eastern China are predicted to decrease generally in MAM and SON (see also Supplement). Reduc- tions in PBL depth are also predicted in northern China in www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol leve 7944 Fig. 4. (a) Simulated planetary boundary layer (PBL) depth (km) in China in present day; (b) Projected changes in PBL depth (km) in China from the Fig. 4. (a) Simulated planetary boundary layer (PBL) depth (km) in China in present day; (b) projected changes in PBL depth (km) in China from the present day (1996–2005) to future (2046–2055) under the IPCC A1B scenario; (c) the percentage changes in PBL depth relative to present day. The dotted areas in (c) are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. Fig. 4. (a) Simulated planetary boundary layer (PBL) depth (km) in China in present day; (b) Projected changes in PBL depth (km) in China from the Fig. 4. (a) Simulated planetary boundary layer (PBL) depth (km) in China in present day; (b) projected changes in PBL depth (km) in China from the present day (1996–2005) to future (2046–2055) under the IPCC A1B scenario; (c) the percentage changes in PBL depth relative to present day. The dotted areas in (c) are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. p y ( ) ( (c) The percentage changes in PBL d areas in (c) are statistically significant student’s two sample t-test. generally agree with those from the IPCC AR4 multi-model predictions for China under the A1B scenario (IPCC, 2007). Projected changes in cloud fraction in China from present day to year 2050 are shown in Fig. 3. Changes in cloud frac- tion correspond well with the changes in precipitation; de- creases (or increases) in clouds are associated with the de- creases (or increases) in precipitation (Fig. 2). ) ; pth relative to present day. The dotted t the 95% level, as determined by the DJF and JJA. Simulated changes in the seasonal mean PBL depth in China are in the range of ±10 %. The changes in PBL depth result from the simulated changes in atmospheric temperature (or atmospheric stability); a more unstable at- mosphere leads to higher PBL depths. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7945 ang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Fig 5 Simulated present day seasonal mean surface layer concentrations (g Fig. 5. Simulated present-day seasonal mean surface-layer concentrations (µg m−3) of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 in China. Fi Si l d d l f l i ( Fig. 5. Simulated present-day seasonal mean surface-layer concentrations (µg m−3) of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 in China. Fi Si l d d l f l Fig. 5. Simulated present-day seasonal mean surface-layer concentrations (µg m−3) of SO2− 4 , NO− 3 , NH+ 4 , B PM2.5 in China. to be in the range of 3–7 µg m−3 in eastern China in all sea- sons. Predicted BC and OC concentrations are high in DJF and SON and low in MAM and JJA, owing to the seasonal variation of precipitation (Fig. 2). Simulated PM2.5 concen- trations show the highest values of 18–32 µg m−3 in DJF and of 18–28 µg m−3 in JJA. In the surface layer, NO− 3 is pre- dicted to have been the most abundant aerosol species over eastern China in 1996–2005, followed by SO2− 4 , NH+ 4 , OC, and BC. m ) of SO4 , NO3, NH4, BC, OC, and maximum concentrations of 7–9 µg m−3 in JJA and of 5– 7 µg m−3 in DJF, MAM, and SON in eastern China. Although strong photochemistry facilitates maximum sulfate forma- tion in JJA, prevalent precipitation in southern China in JJA (Fig. 2) leads to enhanced wet removal of sulfate in that re- gion. Simulated concentrations of NO− 3 are generally higher than those of SO2− 4 in eastern China, which is likely caused by the overestimate of NH3 emissions (Wang et al., 2013b). The highest NO− 3 concentrations of 9–12 µg m−3 are simu- lated in DJF because of the relatively low temperatures and precipitation. In contrast, high temperatures and large rainfall in JJA lead to the lowest NO− 3 concentrations. Because of the excess amount of NH3 in eastern China (Wang et al., 2013b), ammonium aerosol exists predominantly as ammonium sul- fate or ammonium nitrate; its concentrations are simulated 3.2 Comparisons of simulated concentrations with measurements Three previous studies have compared the simulated aerosol concentrations in GEOS-Chem with measurements taken 3.1 Model results Figure 5 shows simulated 1996–2005 seasonal mean concen- trations of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 (sum of SO2− 4 , NO− 3 , NH+ 4 , BC, and OC) in China. Sulfate exhibits Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7946 g j g Fig. 6. Comparisons of simulated present-day concentrations of sulfate, nitrate, BC, and OC aerosols with measurements. Simulated values are seasonal 1996 2005 Al h i th 1 1 li (d h d) d li fit Fig. 6. Comparisons of simulated present-day concentrations of sulfate, nitrate, BC, and OC aerosols with measurements. Simulated values are seasonal averages over 1996–2005. Also shown is the 1 : 1 line (dashed) and linear ﬁt (solid line and equation). R is the correlation coefﬁcient between simulated and measured concentrations. Fig. 6. Comparisons of simulated present-day concentrations of sulfate, nitrate, BC, and OC aerosols with measurements. Simulated values are seasonal Fig. 6. Comparisons of simulated present-day concentrations of sulfate, nitrate, BC, and OC aerosols with measurements. Simulated values are seasonal averages over 1996–2005. Also shown is the 1 : 1 line (dashed) and linear ﬁt (solid line and equation). R is the correlation coefﬁcient between simulated and measured concentrations. (solid line and equation). R is the corre measured concentrations. during 2001–2009 in China (Zhang et al., 2010a; Wang et al., 2013b; Fu et al., 2012). At a horizontal resolution of 4◦latitude by 5◦longitude, Zhang et al. (2010a) found that GEOS-Chem tends to underestimate PM2.5 aerosol concen- trations in China, because measurements are usually taken in urban areas, whereas the simulated values represent grid cell averages. By using the one-way nested-grid capability of GEOS-Chem with a horizontal resolution of 0.5◦latitude by 0.6674◦longitude driven by the assimilated meteorological ﬁelds, Wang et al. (2013b) found that simulated concentra- tions of sulfate, nitrate, and ammonium at 22 sites in East Asia exhibited annual biases of −10 %, +31 %, and +35 %, respectively, and Fu et al. (2012) reported that the simulated annual mean concentrations of BC and OC averaged over ru- ral and background sites were underestimated by 56 % and 76 %, respectively. Underestimation of BC in China was also found in all AEROCOM models (Koch et al., 2009), suggest- ing that emissions of carbonaceous aerosols are currently un- derestimated in China. tion coefficient between simulated and at a few rural sites (such as Fenghuangshan, Gaolanshan, and Lin’an). See the Supplement for the locations of mea- surements. www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7947 Fig. 7. Predicted changes in surface-layer concentrations of aerosols (g m-3) due to changes in climate alone from the present day (1996–2005) to the f t (2046 2055) G h f ll th IPCC i A1B Fig. 7. Predicted changes in surface-layer concentrations of aerosols (µg m−3) due to changes in climate alone from the present day (199 2005) to the future (2046–2055). Greenhouse gases follow the IPCC scenario A1B. Anthropogenic emissions are held at present-day valu but natural emissions may change in response to climate. The dotted areas are statistically signiﬁcant at the 95 % level, as determined Student’s two-sample t test. Fig. 7. Predicted changes in surface-layer concentrations of aerosols (g m-3) due to changes in climate alone from the present day (1996–2005) to the f t (2046 2055) G h f ll th IPCC i A1B Fig. 7. Predicted changes in surface-layer concentrations of aerosols (µg m−3) due to changes in climate alone from the present day (1996– 2005) to the future (2046–2055). Greenhouse gases follow the IPCC scenario A1B. Anthropogenic emissions are held at present-day values, but natural emissions may change in response to climate. The dotted areas are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. Anthropogenic emissions are emissions may change in re statistically significant at the 95 4 Predicted 2000–2050 changes in surface-layer aerosols in China at present day values, but natural to climate. The dotted areas are , as determined by the student’s two the four seasons. Climate-induced changes in concentrations of SO2− 4 , BC, and OC correspond well with the changes in precipitation in all seasons (Fig. 2), with increases (or de- creases) in aerosol concentrations over places with decreases (or increases) in precipitation. Predicted reductions in nitrate and ammonium aerosol concentrations also result from the increases in temperature over 2000–2050, which lead to the shift of gas–aerosol equilibrium toward the gas phase. sample t-test. 4.1 Effect of changes in climate alone Figure 7 shows the predicted future changes in seasonal mean surface-layer aerosol concentrations as a result of the future changes in climate alone. Simulated changes in concentra- tions of the aerosol species are within the range of −1.5 to +0.8 µg m−3 and exhibit similar spatial patterns in each of As a result of the changes in individual aerosol species, PM2.5 concentrations in DJF are predicted to exhibit www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Scatterplots of simulated versus observed sea- sonal mean sulfate, nitrate, BC, and OC concentrations are displayed in Fig. 6. As anticipated, aerosol concentrations are generally underpredicted; the simulated concentrations of sulfate, nitrate, BC, and OC are about 38 %, 68 %, 29 %, and 19 % of the measured values, respectively. One notes the es- pecially low levels of BC and OC simulated, as compared with measurements, indicating the inadequacy of current emission inventories for these species. Comparisons of sim- ulated versus observed concentrations of all aerosol species show relatively high correlation coefﬁcients, with values of R2 ranging from 0.77 to 0.83. These relatively high R2 val- ues indicate that the model captures the spatial distributions and seasonal variations of each aerosol species despite the general low bias in simulated concentrations. Again, one should bear in mind that predicted concentrations in this study have been averaged over 4◦× 5◦grid cells. No publicly accessible in situ measurement network of aerosols in China exists (Chan and Yao, 2008). Therefore, we have compiled for model evaluation the monthly or sea- sonal mean measured concentrations of each aerosol species based on measurements reported in the literature. Most ob- servations were conducted between 2001 and 2009 at ur- ban sites (such as Beijing, Shanghai, and Guangzhou) and www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7949 Fig. 9. Predicted changes in surface-layer concentrations of aerosols (g m-3) due to changes in both climate and anthropogenic emissions from the present 9. Predicted changes in surface-layer concentrations of aerosols (µg m−3) due to changes in both climate and anthropogenic emissions m the present day (1996–2005) to the future (2046–2055). Almost all the changes over China are statistically signiﬁcant at the 95 % level etermined by Student’s two-sample t test. Fig. 9. Predicted changes in surface-layer concentrations of aerosols (g m-3) due to changes in both climate and anthropogenic emissions from the present Fig. 9. Predicted changes in surface-layer concentrations of aerosols (µg m−3) due to changes in both climate and anthropogenic emissions from the present day (1996–2005) to the future (2046–2055). Almost all the changes over China are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. day (1996–2005) to the future (2046–2 are statistically significant at the 95% sample t-test. 1.5 µg m−3 in large-scale simulations and up to 3.5 µg m−3 in ﬁne-scale simulations under the IPCC A1B scenario. ) g vel, as determined by the student’s two lie within the range of 0.5–3.5 µg m−3 throughout the year. Although the simulated present-day concentrations of BC and OC are lower than those of sulfate, the decreases in BC and OC are 0.5–2 µg m−3 and 0.5–3.5 µg m−3, respectively, in eastern China. 4.2 Effect of changes in anthropogenic emissions alone Unlike sulfate, BC, and OC, based on changes in emis- sions alone, nitrate concentrations are predicted to increase in the future due to changing anthropogenic emissions, with the largest increases of 1–2 µg m−3 in DJF, 3.5–4.5 µg m−3 in JJA, and 2–3.5 µg m−3 in MAM and SON in eastern China. These increases can be explained by the assumed 72 % increase in annual anthropogenic NOx emission over In the IPCC A1B scenario, year 2050 annual anthropogenic emissions of SO2, BC, and OC in eastern China are predicted to decrease by 29 %, 59 %, and 40 %, respectively, relative to the present-day values. Future concentrations of sulfate, BC, and OC in China are hence predicted to decrease (Fig. 8). The predicted reductions in sulfate aerosol in eastern China Atmos. Chem. Phys., 13, 7937–7960, 2013 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7948 Fig. 8. Predicted changes in surface-layer concentrations of aerosols (g m-3) due to changes in anthropogenic emissions alone from the present day (1996– g. 8. Predicted changes in surface-layer concentrations of aerosols (µg m−3) due to changes in anthropogenic emissions alone from the esent day (1996–2005) to the future (2046–2055). Almost all the changes over China are statistically signiﬁcant at the 95 % level, as termined by Student’s two-sample t test. Fig. 8. Predicted changes in surface-layer concentrations of aerosols (g m-3) due to changes in anthropogenic emissions alone from the present day (1996 Fig. 8. Predicted changes in surface-layer concentrations of aerosols (µg m−3) due to changes in anthropogenic emissions alone from the present day (1996–2005) to the future (2046–2055). Almost all the changes over China are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. ) ( ) statistically significant at the 95% lev sample t-test. increases of 1–2.5 µg m−3 along east and southeast coasts of China but decreases of 1.5–2.5 µg m−3 in central China. Increases in PM2.5 are predicted over a large fraction of east- ern China in MAM, with maximum increases in the range of 1–2.5 µg m−3. PM2.5 in eastern China shows general de- creases of 0.5–1.5 µg m−3 in JJA and of 1.5–2.5 µg m−3 in SON. These estimated climate-induced changes in PM2.5 concentrations over 2000–2050 represent about 10–20 % of the present-day values in those regions. g , as determined by the student’s two other studies for other regions. Based on the IPCC A1B sce- nario, Tagaris et al. (2007) found a 10 % decrease in PM2.5 throughout the United States and Pye et al. (2009) reported changes in annually averaged sulfate–nitrate–ammonium of up to 0.61 µg m−3 in the United States, as a result of 2000– 2050 climate change alone. Under the IPCC A2 scenario, climate change over 2000–2050 alone was found to reduce PM2.5 concentrations in the United States by −0.9 µg m−3 in the study of Avise et al. (2009). Juda-Rezler et al. (2012) reported that from 1991–2000 to 2091–2100 PM10 values over Central–Eastern Europe generally decrease, by up to Our simulated climate-induced changes in aerosol concen- trations are comparable in magnitude with those reported in Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ H. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels ang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels www.atmos-chem-phys.net/13/7937/2013/ www.atmos-chem-phys.net/13/7937/2013/ H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7950 2000–2050 (Table 1) as well as the fact that the future reduc- tions in SO2 favor the formation of ammonium nitrate. As a result, ammonium concentrations show increases of about 0.5 µg m−3 over those locations with large increases in ni- trate. Fig. 10. The locations of the 3 vertical planes through which fluxes of transboundary aerosols are calculated: the meridional plane along 135E from 20 to 55N to show the outflow from eastern China to the West Pacific, the meridional plane that along 75E from 35 to 55N to show the inflow from Europe and Central Asia to China, and the latitudinal plane along 21.7N from 90 to 125E to show the transport to or from South Asia and Southeast Asia. Fig. 10. The locations of the 3 vertical planes through which ﬂuxes of transboundary aerosols are calculated: the meridional plane along 135◦E from 20◦to 55◦N to show the outﬂow from eastern China to the West Paciﬁc; the meridional plane along 75◦E from 35◦to 55◦N to show the inﬂow from Europe and Central Asia to China; and the latitudinal plane along 21.7◦N from 90◦to 125◦E to show the transport to or from South Asia and Southeast Asia. The net effect of the changes in all aerosol species owing to changes in emissions alone is an overall decrease in PM2.5 concentrations in China except for the Tibet Plateau. Over eastern China, the projected largest decreases in PM2.5 as a result of the future changes in emissions are 7.5–8 µg m−3 (or 20–40 % relative to the present-day values) in DJF, 1– 5 µg m−3 (or 10–30 %) in MAM and SON, and 1–3 µg m−3 (or 10–40 %) in JJA. As noted, the increase in PM2.5 over or near the Tibet Plateau is caused by long-range transport from South Asia. Fig. 10. 4.3 Effect of changes in both climate and anthropogenic emissions Predicted future changes in aerosols as a consequence of fu- ture changes in both climate and anthropogenic emissions are summarized in Fig. 9. Concentrations of sulfate, BC, and OC in China are predicted to decrease in all seasons under future changes in both climate and anthropogenic emissions, although climate change can, to some extent, offset the effect of reductions in anthropogenic emissions on these species (Fig. 7). In eastern China, concentrations of sulfate are pre- dicted to decrease 0.5–3.5 µg m−3 in SON and 0.5–2 µg m−3 in other seasons, while those of BC and OC to decrease by 0.5–2 µg m−3 and 0.5–3.5 µg m−3, respectively, in all sea- sons. indicate that changes in concentrations of these aerosols are strongly driven by the changes in emissions. www.atmos-chem-phys.net/13/7937/2013/ The locations of the 3 vertical planes through which fluxes of transboundary aerosols are calculated: the meridional plane along 135E from 20 to 55N to show the outflow from eastern China to the West Pacific, the meridional plane that along 75E from 35 to 55N to show the inflow from Europe and Central Asia to China, and the latitudinal plane along 21.7N from 90 to 125E to show the transport to or from South Asia and Southeast Asia. Fig. 10. The locations of the 3 vertical planes through which ﬂuxes of transboundary aerosols are calculated: the meridional plane along 135◦E from 20◦to 55◦N to show the outﬂow from eastern China to the West Paciﬁc; the meridional plane along 75◦E from 35◦to 55◦N to show the inﬂow from Europe and Central Asia to China; and the latitudinal plane along 21.7◦N from 90◦to 125◦E to show the transport to or from South Asia and Southeast Asia. 5 Simulated 2000–2050 changes in transboundary transport of aerosols We next estimate 2000–2050 changes in aerosol ﬂuxes into and out of China. These ﬂuxes of aerosols are calculated through 3 vertical planes from the surface to 100 hPa altitude (Fig. 10): (1) the meridional plane along 135◦E from 20◦ to 55◦N that captures the outﬂow from eastern China to the western Paciﬁc, (2) the meridional plane along 75◦E from 35◦to 55◦N that captures the inﬂow from Europe and Cen- tral Asia to China, and (3) the latitudinal plane along 21.7◦N from 90◦to 125◦E to capture the transport to or from South Asia and Southeast Asia. The transboundary ﬂuxes are cal- culated within the model at every time step, and the seasonal and annual values are presented here. For nitrate aerosol in eastern China, while climate change exerts an effect opposite to that of changes in anthropogenic emissions in DJF, JJA, and SON, the effect of climate change enhances the changes due to emissions in MAM (Fig. 7). As a result of changes in both climate and anthropogenic emis- sions, concentrations of nitrate in eastern China are predicted to exhibit reductions of 0.5–1 µg m−3 in DJF and increases of 0.5–4.5 µg m−3 in other seasons. The simulated 2000–2050 changes in PM2.5 are dominated by future changes in emissions; concentrations of PM2.5 in eastern China are simulated to decrease by 3–7.5 µg m−3 (or 10–30 % relative to the present-day values) in DJF, 1– 5 µg m−3 (or 30–40 %) in JJA, and 3–7.5 µg m−3 (or 20– 40 %) in SON. In MAM, climate change has a dominant role in inﬂuencing the future changes in aerosols in the lower reaches of Yangtze River, where increases of up to 1 µg m−3 in PM2.5 are simulated. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7951 Fig. 11. (a) Simulated present-day mass fluxes of PM2.5 and zonal winds. Projected changes in mass fluxes of PM2.5 and zonal winds from the present day (1996-2005) to future (2046-2055) owing to (b) climate change alone, (c) changes in anthropogenic emissions alone and (d) changes in both climate Fig. 11. (a) Simulated present-day mass ﬂuxes of PM2.5 and zonal winds. Projected changes in mass ﬂuxes of PM2.5 and zonal winds the present day (1996–2005) to future (2046–2055) owing to (b) climate change alone, (c) changes in anthropogenic emissions alone (d) changes in both climate and anthropogenic emissions. Mass ﬂuxes of PM2.5 are shown by shades (units: Tg), and winds are repres by contours (units: m s−1). Both mass ﬂuxes and winds are those through the meridional plane along 135◦E from 20◦to 55◦N. The d areas are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. Fig. 11. (a) Simulated present-day mass fluxes of PM2.5 and zonal winds. Projected changes in mass fluxes of PM2.5 and zonal winds from the present day (1996-2005) to future (2046-2055) owing to (b) climate change alone, (c) Fig. 11. (a) Simulated present-day mass ﬂuxes of PM2.5 and zonal winds. Projected changes in mass ﬂuxes of PM2.5 and zonal winds from the present day (1996–2005) to future (2046–2055) owing to (b) climate change alone, (c) changes in anthropogenic emissions alone, and (d) changes in both climate and anthropogenic emissions. Mass ﬂuxes of PM2.5 are shown by shades (units: Tg), and winds are represented by contours (units: m s−1). Both mass ﬂuxes and winds are those through the meridional plane along 135◦E from 20◦to 55◦N. The dotted areas are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. uxes of PM2.5 are shown by shades by contours (Units: m s-1). Both mass meridional plane along 135E from 20 cally significant at the 95% level, as t-test. of these maximum ﬂuxes shift from 20◦–35◦N in DJF and MAM to 30◦–50◦N in JJA and SON, which are consistent with the changes in westerlies (Fig. 11). Our simulated ver- tical distributions of the strongest ﬂuxes of aerosols agree with those reported by Wang et al. (2009), who showed that the transport to North America occurs mainly in the mid- to upper troposphere. and anthropogenic emissions. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Mass (Units: Tg) and winds are represente fluxes and winds are those through th to 55N. The dotted areas are stat determined by the student’s two samp outﬂow of SO2− 4 is simulated to peak in DJF, which results from the simulated high concentrations of sulfate (Fig. 5) and the strong westerlies (Fig. 11) in that season. All other aerosol species exhibit maximum outﬂow in MAM, a re- sult that agrees with the conclusions from previous stud- ies (Holzer et al., 2005). Heald et al. (2006) and Chin et al. (2007) reported that the export from Asia is most efﬁ- cient in spring with nearly all East Asian air involved in transpaciﬁc transport. The outﬂow of aerosols is generally the weakest in JJA, as reported by Holzer et al. (2005) and Yu et al. (2008). 5.1.2 Effect of changes in climate alone Simulated future changes in seasonal and annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 through the merid- ional plane along 135◦E are also listed in Table 3. As a re- sult of the climate change alone, the annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 are simulated to change by The pressure–latitude cross sections of the simulated ﬂuxes of PM2.5 at 135◦E are shown in Fig. 11. The maxi- mum ﬂuxes of aerosols are found at 500–700 hPa in DJF and SON and at 400–600 hPa in MAM and JJA. The locations www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 5.1.1 Estimated present-day outﬂow Simulated present-day seasonal and annual total ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 across the meridional plane along 135◦E from 20◦to 55◦N are listed in Table 3. Among all aerosol species, the estimated present-day out- ﬂow of SO2− 4 of 5.2 Tg yr−1 across this plane is the largest, contributing 48 % of the annual outﬂow of PM2.5. The esti- mated ﬂuxes of NO− 3 , NH+ 4 , BC, and OC account for 19 %, 15 %, 5 %, and 13 % of annual outﬂow of PM2.5, respec- tively. Aerosol ﬂuxes show strong seasonal variations. The With reductions in SO2, BC, and OC in eastern China over 2000–2050 by 28.5 %, 58.6 %, and 39.8 % (Table 1), respec- tively, annual mean concentrations of sulfate, BC, and OC over eastern China are simulated to be reduced, respectively, by 19.2 %, 56.4 %, and 35.9 % with future changes in emis- sions alone whereas by 18.5 %, 56.6 %, and 36.5 % with fu- ture changes in both climate and emissions. These results Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ . Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7952 Table 3. Simulated outﬂow of aerosols from eastern China through the meridional plane along 135◦E from 20◦to 55◦N. Numbers in the parentheses are percentage changes relative to the present-day ﬂuxes. The units are Tg season−1 for seasonal ﬂuxes and Tg yr−1 for annual ﬂuxes of whole species (e.g., Tg(SO2− 4 ) season−1, Tg(SO2− 4 ) yr−1). Table 3. Simulated outﬂow of aerosols from eastern China through the meridional plane along 135◦E from 20◦to 55◦N. Numbers in the parentheses are percentage changes relative to the present-day ﬂuxes. The units are Tg season−1 for seasonal ﬂuxes and Tg yr−1 for annual ﬂuxes of whole species (e.g., Tg(SO2− 4 ) season−1, Tg(SO2− 4 ) yr−1). Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels a Seasonal and annual total ﬂuxes averaged over 1996–2005; b year 2050 ﬂuxes simulated with climate change alone; c year 2050 ﬂuxes simulated with changes in anthropogenic emissions alone; d year 2050 ﬂuxes simulated with changes in both climate and anthropogenic emissions; e NonL= (2050b−2000a)+(2050c−2000a) 2050d−2000a . −4.2 %, −15.6 %, −10.1 %, 0.0 %, −4.5 %, and −7.0 %, re- spectively, relative to the present-day values. Nitrate aerosol outﬂow is projected to exhibit the largest reduction, be- cause nitrate aerosol concentrations in China decrease signif- icantly in a warmer climate (Fig. 7). The ﬂuxes of all aerosol species show reductions in all seasons except a small increase (0.7 %) in ﬂux of SO2− 4 in MAM. and ammonium outﬂow ﬂuxes exhibit large increases owing to the projected increases in concentrations in China (Fig. 8), the ﬂuxes of SO2− 4 , BC, and OC decrease, corresponding to the future decreases in concentrations of these species (Fig. 8). Consequently, the annual outﬂow of PM2.5 is esti- mated to exhibit a modest change of −0.7 % over 2000–2050 as a result of changes in emissions alone. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Outﬂow from eastern China Species 2000a (Tg) 2050b (Tg) 2050c (Tg) 2050d (Tg) NonLe DJF 2.12 1.99 (−6.1 %) 2.07 (−2.4 %) 1.97 (−7.1 %) 1.20 MAM 1.50 1.51 (+0.7 %) 1.59 (+6.0 %) 1.58 (+5.3 %) 1.25 SO2− 4 JJA 0.45 0.43 (−4.4 %) 0.35 (−22.2 %) 0.34 (−24.4 %) 1.09 SON 1.12 1.04 (−7.1 %) 0.94 (−16.1 %) 0.87 (−22.3 %) 1.04 Annual 5.19 4.97 (−4.2 %) 4.95 (−4.6 %) 4.76 (−8.3 %) 1.07 DJF 0.52 0.45 (−13.5 %) 0.65 (+25.0 %) 0.50 (−3.8 %) −3.00 MAM 0.86 0.75 (−12.8 %) 0.89 (+3.5 %) 0.77 (−10.5 %) 0.89 NO− 3 JJA 0.41 0.34 (−17.1 %) 0.56 (+36.6 %) 0.46 (+12.2 %) 1.60 SON 0.32 0.24 (−25.0 %) 0.53 (+65.6 %) 0.40 (+25.0 %) 1.63 Annual 2.11 1.78 (−15.6 %) 2.63 (+24.6 %) 2.13 (+0.9 %) 9.50 DJF 0.45 0.41 (−8.9 %) 0.48 (+6.7 %) 0.44 (−2.2 %) 1.00 MAM 0.64 0.61 (−4.7 %) 0.70 (+9.4 %) 0.67 (+4.7 %) 1.00 NH+ 4 JJA 0.29 0.25 (−13.8 %) 0.29 (0.0 %) 0.26 (−10.3 %) 1.33 SON 0.30 0.24 (−20.0 %) 0.35 (+16.7 %) 0.30 (0.0 %) – Annual 1.68 1.51 (−10.1 %) 1.82 (+8.3 %) 1.67 (−0.6 %) 3.00 DJF 0.17 0.17 (0.0 %) 0.094 (−44.7 %) 0.090 (−47.1 %) 0.95 MAM 0.21 0.21 (0.0 %) 0.15 (−28.6 %) 0.15 (−28.6 %) 1.00 BC JJA 0.066 0.066 (0.0 %) 0.034 (−48.5 %) 0.033 (−50.0 %) 0.97 SON 0.085 0.080 (−5.9 %) 0.044 (−48.2 %) 0.041 (−51.8 %) 1.05 Annual 0.53 0.53 (0.0 %) 0.32 (−39.6 %) 0.31 (−41.5 %) 0.95 DJF 0.37 0.34 (−8.1 %) 0.24 (−35.1 %) 0.22 (−40.5 %) 1.07 MAM 0.64 0.62 (−3.1 %) 0.58 (−9.4 %) 0.57 (−10.9 %) 1.14 OC JJA 0.17 0.17 (0.0 %) 0.12 (−29.4 %) 0.12 (−29.4 %) 1.00 SON 0.16 0.15 (−6.3 %) 0.095 (−40.6 %) 0.086 (−46.3 %) 1.01 Annual 1.34 1.28 (−4.5 %) 1.04 (−22.4 %) 1.00 (−25.4 %) 1.06 DJF 3.63 3.36 (−7.4 %) 3.53 (−2.8 %) 3.22 (−11.3 %) 0.90 MAM 3.85 3.70 (−3.9 %) 3.92 (+1.8 %) 3.73 (−3.1 %) 0.67 PM2.5 JJA 1.38 1.26 (−8.7 %) 1.35 (−2.2 %) 1.22 (−11.6 %) 0.94 SON 1.98 1.76 (−11.1 %) 1.96 (−1.0 %) 1.69 (−14.6 %) 0.83 Annual 10.84 10.08 (−7.0 %) 10.76 (−0.7 %) 9.86 (−9.0 %) 0.86 a Seasonal and annual total ﬂuxes averaged over 1996–2005; b year 2050 ﬂuxes simulated with climate change alone; c year 2050 ﬂuxes simulated with changes in anthropogenic emissions alone; d year 2050 ﬂuxes simulated with changes in both climate and anthropogenic emissions; e NonL= (2050b−2000a)+(2050c−2000a) 2050d−2000a . www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol leve 5.1.4 Effect of changes in both climate and anthropogenic emissions Simulated year 2050 outﬂow ﬂuxes of aerosols through the meridional plane along 135◦E from 20◦to 55◦N with the future changes in both anthropogenic emissions and climate are listed in Table 3. The annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , Annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 are simulated to change by −4.6 %, +24.6 %, +8.3 %, −39.6 %, −22.4 %, and −0.7 %, respectively (Table 3), as a result of the changes in anthropogenic emissions alone. While nitrate Annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 are simulated to change by −4.6 %, +24.6 %, +8.3 %, −39.6 %, g y , , , , −22.4 %, and −0.7 %, respectively (Table 3), as a result of the changes in anthropogenic emissions alone. While nitrate Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ 7953 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 5.1 % in DJF, as a result of the future stronger westerlies in DJF (Fig. 12). In MAM, JJA, and SON, the westerlies show reductions in wind speed around 700 hPa (Fig. 12), leading to reductions in inﬂow of all aerosol species (Table 4). BC, OC, and PM2.5 are simulated to change by −8.3 %, +0.9 %, −0.6 %, −41.5 %, −25.4 %, and −9.0 %, respec- tively. Climate change mitigates the effects of increases in NOx emissions on annual ﬂuxes of NO− 3 and NH+ 4 , but slightly enhances the effects of reductions in emissions of SO2, BC, and OC on outﬂow of SO2− 4 , BC, and OC. 5.2.3 Effect of changes in anthropogenic emissions alone 4 With all aerosol species accounted for, the 2000–2050 changes in seasonal and annual outﬂow ﬂuxes of PM2.5 re- sulting from future climate alone always exceed those caused by future changes in emissions alone (Table 3), indicating that future climate change can be inﬂuential in estimates of future intercontinental transport of aerosols. The annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 are simulated to change by +1.7 %, +91.5 %, +39.3 %, −28.6 %, −23.3 %, and +19.5 %, respectively (Table 4), as a result of the future changes in anthropogenic emissions alone. The magnitudes of changes in ﬂuxes of NO− 3 , NH+ 4 , BC, OC, and PM2.5 by anthropogenic emissions alone are generally larger than those of changes by climate change alone on either a seasonal or annual mean basis. As a re- sult of future changes in emissions, the seasonal ﬂuxes of NO− 3 and NH+ 4 show large increases of 68–146 % and 25– 50 %, respectively, while those of BC and OC show large decreases of 18–30 % and 12–34 %, respectively, and the in- ﬂow of PM2.5 is predicted to increase by about 20 % in all seasons (Table 4). It is of interest to examine the rate of nonlinearity of changes in aerosol ﬂuxes with respect to the changes in cli- mate and emissions. The last column of Table 3 shows the rate of nonlinearity deﬁned as NonL = (Change in ﬂux by cli- mate change alone + Change in ﬂux by changes in emissions alone)/Change in ﬂux by changes in both climate and emis- sions. Values of NonL of seasonal and annual outﬂow aerosol ﬂuxes mostly deviate from 1.0 (perfect linearity), with abso- lute values ranging from 0.67 to 9.5. 5.3.1 Present-day transport The simulated present-day seasonal and annual ﬂuxes of aerosols through the latitudinal plane along 21.7◦N from 90◦–125◦E are listed in Table 5. The annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 are −0.22, −0.15, −0.09, +0.08, +0.63, and +0.26 Tg, respectively, with the posi- tive (or negative) values indicating northward (or southward) transport of aerosols. The signs of the aerosol ﬂuxes are de- termined by the directions of winds and the simulated distri- butions of aerosols. In DJF and SON, while southward ﬂuxes of all aerosol species are found in the lower troposphere over 100◦–125◦E because of the prevailing northerlies over east- ern China, northward ﬂuxes of aerosols are found through the depth of the troposphere over 90◦–100◦E and in the 5.2.1 Present-day inﬂow Year 2050 inﬂow ﬂuxes of aerosols through the meridional plane along 75◦E calculated with the future changes in both anthropogenic emissions and climate are listed in Table 4. The annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, and OC are sim- ulated to change by +0.9 %, +49.2 %, +27.9 %, −29.3 %, and −30.0 %, respectively. Climate change mitigates the ef- fects of increases in NOx emissions on annual ﬂuxes of NO− 3 and NH+ 4 , but enhances the effects of changes in emissions of BC and OC. As a net result of the changes in inﬂow of all aerosol species, the annual inﬂow of PM2.5 to western China is projected to increase by 10.5 % over 2000–2050 with fu- ture changes in both emissions and climate. Simulated present-day seasonal and annual ﬂuxes of aerosols through the meridional plane along 75◦E from 35◦to 55◦N are listed in Table 4. On an annual basis, the estimated ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, and OC are 2.3, 0.6, 0.6, 0.1, and 0.3 Tg, respectively. The annual inﬂow of PM2.5 is 3.9 Tg, which is about 35 % of the annual outﬂow of PM2.5 from eastern China. The inﬂow of SO2− 4 peaks in DJF, when sim- ulated SO2− 4 concentrations are high in Europe, and in MAM, when the westerlies are strong (Fig. 12). The ﬂuxes of NH+ 4 , BC, OC, and PM2.5 all peak in MAM because of the domi- nant effect of strong westerlies in this season. The inﬂow of NO− 3 is largest in JJA, when simulated NO− 3 concentrations are highest in Europe (not shown). 5.3 Transport of aerosols to/from southern China The pressure–latitude cross sections of the simulated ﬂuxes of PM2.5 at 75◦E are shown in Fig. 12. The ﬂuxes of aerosols are the largest north of 40◦N, which is the principal predicted transport path from Europe. The strongest inﬂow of all aerosol species is located at about 700 hPa altitude. 5.2.2 Effect of changes in climate alone Relative to the present-day values, the annual ﬂuxes of SO2− 4 , NO− 3 , NH+ 4 , BC, OC, and PM2.5 through the merid- ional plane along 75◦E from 35◦to 55◦N are simulated to change by −0.9 %, −25.4 %, −11.5 %, −7.1 %, −6.7 %, and −5.9 %, respectively, as a result of the climate change alone. As expected, the warmer future temperatures contribute to large reductions of 20–29 % in estimated seasonal mean in- ﬂow of nitrate. The ﬂux of SO2− 4 is simulated to increase by Atmos. Chem. Phys., 13, 7937–7960, 2013 www.atmos-chem-phys.net/13/7937/2013/ www.atmos-chem-phys.net/13/7937/2013/ H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7954 Table 4. Simulated inﬂow of aerosols to western China through the meridional plane along 75◦E from 35◦to 55◦N. Numbers in the parentheses are percentage changes relative to the present-day ﬂuxes. The units are Tg season−1 for seasonal ﬂuxes and Tg yr−1 for annual ﬂuxes of whole species (e.g., Tg(SO2− 4 ) season−1, Tg(SO2− 4 ) yr−1). Table 4. Simulated inﬂow of aerosols to western China through the meridional plane along 75◦E from 35◦to 55◦N. Numbers in the parentheses are percentage changes relative to the present-day ﬂuxes. The units are Tg season−1 for seasonal ﬂuxes and Tg yr−1 for annual ﬂuxes of whole species (e.g., Tg(SO2− 4 ) season−1, Tg(SO2− 4 ) yr−1). www.atmos-chem-phys.net/13/7937/2013/ a Seasonal and annual total ﬂuxes averaged over 1996–2005; b year 2050 ﬂuxes simulated with climate change alone; c year 2050 ﬂuxes simulated with changes in anthropogenic emissions alone; d year 2050 ﬂuxes simulated with changes in both climate and anthropogenic emissions; e NonL = (2050b−2000a)+(2050c−2000a) 2050d−2000a . upper troposphere over 100◦–125◦E (Fig. 13). In MAM and JJA, the southerlies associated with the summer monsoon generally favor northward transport (Fig. 13). The ﬂuxes of aerosols across the southern boundary of China are gener- ally much smaller than those through the eastern and western boundaries. One exception is that the ﬂuxes of BC and OC to China in MAM are ampliﬁed as a result of the predominant biomass burning in South Asia in this season (Zhang et al., 2010b). upper troposphere over 100◦–125◦E (Fig. 13). In MAM and JJA, the southerlies associated with the summer monsoon generally favor northward transport (Fig. 13). The ﬂuxes of aerosols across the southern boundary of China are gener- ally much smaller than those through the eastern and western boundaries. One exception is that the ﬂuxes of BC and OC to China in MAM are ampliﬁed as a result of the predominant biomass burning in South Asia in this season (Zhang et al., 2010b). increase by 26.7 % relative to the present-day value. The per- centage changes in seasonal ﬂuxes are usually much larger. In DJF, the northerlies in the lower troposphere east of 100◦E are predicted to be stronger and the southerlies west of 100◦E become weaker (Fig. 13), which lead to increases in transport of SO2− 4 , NO− 3 , and PM2.5 from China to South Asia by 33 %, 11 %, and 67 %, respectively. In JJA, the southerlies associated with the summer monsoon are sim- ulated to strengthen in the future climate and hence favor the northward transport of aerosols. The ﬂuxes of all aerosol species to South Asia show reductions in SON, which can be explained by the weaker northerlies in the future atmosphere (Fig. 13). www.atmos-chem-phys.net/13/7937/2013/ Inﬂow to western China Species 2000a (Tg) 2050b (Tg) 2050c (Tg) 2050d (Tg) NonLe DJF 0.78 0.82 (+5.1 %) 0.84 (+7.7 %) 0.84 (+7.7 %) 1.67 MAM 0.71 0.67 (−5.6 %) 0.73 (+2.8 %) 0.70 (−1.4 %) 2.00 SO2− 4 JJA 0.27 0.26 (−3.7 %) 0.23 (−14.8 %) 0.24 (−11.1 %) 1.67 SON 0.53 0.52 (−1.1 %) 0.53 (+0.8 %) 0.53 (+0.8 %) −0.50 Annual 2.29 2.27 (−0.9 %) 2.32 (+1.7 %) 2.31 (+0.9 %) 1.00 DJF 0.075 0.053 (−29.3 %) 0.15 (+100.0 %) 0.10 (+33.3 %) 2.12 MAM 0.15 0.11 (−26.7 %) 0.29 (+93.3 %) 0.22 (+46.7 %) 1.43 NO− 3 JJA 0.25 0.19 (−24.0 %) 0.42 (+68.0 %) 0.34 (+36.0 %) 1.22 SON 0.11 0.088 (−20.0 %) 0.27 (+145.5 %) 0.22 (+100.0 %) 1.25 Annual 0.59 0.44 (−25.4 %) 1.13 (+91.5 %) 0.88 (+49.2 %) 1.34 DJF 0.12 0.11 (−8.3 %) 0.18 (+50.0 %) 0.17 (+41.7 %) 1.00 MAM 0.18 0.16 (−11.1 %) 0.25 (+38.9 %) 0.23 (+27.8 %) 1.00 NH+ 4 JJA 0.16 0.14 (−12.5 %) 0.20 (+25.0 %) 0.18 (+12.5 %) 1.00 SON 0.15 0.13 (−13.3 %) 0.22 (+46.7 %) 0.20 (+33.3 %) 1.00 Annual 0.61 0.54 (−11.5 %) 0.85 (+39.3 %) 0.79 (+27.9 %) 1.00 DJF 0.038 0.038 (0.0 %) 0.030 (−21.1 %) 0.030 (−21.1 %) 1.00 MAM 0.045 0.043 (−4.4 %) 0.037 (−17.8 %) 0.034 (−24.4 %) 0.91 BC JJA 0.023 0.022 (−4.3 %) 0.016 (−30.4 %) 0.015 (−34.8 %) 1.00 SON 0.030 0.029 (−3.3 %) 0.021 (−30.0 %) 0.020 (−33.3 %) 1.00 Annual 0.14 0.13 (−7.1 %) 0.10 (−28.6 %) 0.099 (−29.3 %) 1.22 DJF 0.077 0.077 (0.0 %) 0.054 (−29.9 %) 0.054 (−29.9 %) 1.00 MAM 0.11 0.10 (−9.1 %) 0.097 (−11.8 %) 0.090 (−18.2 %) 1.15 OC JJA 0.053 0.046 (−13.2 %) 0.042 (−20.8 %) 0.036 (−32.1 %) 1.06 SON 0.056 0.052 (−7.1 %) 0.037 (−33.9 %) 0.033 (−41.1 %) 1.00 Annual 0.30 0.28 (−6.7 %) 0.23 (−23.3 %) 0.21 (−30.0 %) 1.00 DJF 1.08 1.09 (+0.9 %) 1.25 (+15.7 %) 1.20 (+11.1 %) 1.50 MAM 1.19 1.09 (−8.4 %) 1.41 (+18.5 %) 1.28 (+7.6 %) 1.33 PM2.5 JJA 0.75 0.66 (−12.0 %) 0.91 (+21.3 %) 0.81 (+8.0 %) 1.17 SON 0.87 0.82 (−5.7 %) 1.08 (+24.1 %) 1.01 (+16.1 %) 1.14 Annual 3.89 3.66 (−5.9 %) 4.65 (+19.5 %) 4.30 (+10.5 %) 1.29 a Seasonal and annual total ﬂuxes averaged over 1996–2005; b year 2050 ﬂuxes simulated with climate change alone; c year 2050 ﬂuxes simulated with changes in anthropogenic emissions alone; d year 2050 ﬂuxes simulated with changes in both climate and anthropogenic emissions; e NonL = (2050b−2000a)+(2050c−2000a) 2050d−2000a . 5.3.2 Effect of changes in climate alone As a result of the future climate change alone, the changes in annual ﬂuxes of aerosol species are generally small ex- cept that the annual southward ﬂux of NO− 3 is simulated to uxes and winds are those through th 55N. 5.3.4 Effect of changes in both climate and anthropogenic emissions The annual ﬂuxes of SO2− 4 and NH+ 4 are simulated to change from a net southward outﬂow to a net northward inﬂow, as a result of the changes in anthropogenic emissions over 2000– 2050 (Table 5). The annual southward ﬂux of NO− 3 increases by 60 %, and the annual northward ﬂuxes of BC, OC, and PM2.5 increase by 48 %, 14 %, and 281 %, respectively. The annual ﬂuxes of SO2− 4 and NH+ 4 exhibit large increases in northward transport because of the large increases in aerosol concentrations in South Asia and Southeast Asia (Fig. 8), and the annual inﬂow of BC and OC also increases because the reductions in carbonaceous aerosol concentrations in eastern China are projected to be larger than those in South Asia and Southeast Asia (Fig. 8). In contrast, the annual ﬂux of NO− 3 exhibits southward outﬂow because of the large increases in NO− 3 levels in eastern China (Fig. 8). Simulated ﬂuxes of aerosols through the latitudinal plane along 21.7◦N from 90◦to 125◦E with the future changes in both anthropogenic emissions and climate are listed in Ta- ble 5. The year 2050 annual ﬂuxes of SO2− 4 , NH+ 4 , BC, OC, and PM2.5 are simulated as northward into China, and the annual ﬂux of NO− 3 is southward out of China. The 2000– 2050 changes in ﬂuxes of all aerosol species are dominated by the contributions from changes in anthropogenic emis- sions. However, the role of future climate change can exceed that of future changes in emissions for the ﬂuxes of NO− 3 in DJF, BC in JJA, as well as OC in DJF (Table 5). Atmos. Chem. Phys., 13, 7937–7960, 2013 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7955 Fig. 12. Same as Fig. 11 but mass ﬂuxes and winds are those through the meridional plane along 75◦E from 35◦to 55◦N. Fig. 12. Same as Fig. 11 but mass ﬂuxes and winds are those through the meridional plane along 75◦E from 35◦to 55◦N. Fig. 12. Same as Fig. 11 but mass ﬂuxes and winds are those through the meridional plane along 75◦E fr www.atmos-chem-phys.net/13/7937/2013/ www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 . Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels www.atmos-chem-phys.net/13/7937/2013/ H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol leve H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7956 g j g Fig. 13. (a) Simulated present-day mass fluxes of PM2.5 and meridional winds. Projected changes in mass fluxes of PM2.5 and meridional winds from the present day (1996-2005) to future (2046-2055) owing to (b) climate change alone, (c) changes in anthropogenic emissions alone, and (d) changes in both climate and anthropogenic emissions Mass fluxes of PM are shown by Fig. 13. (a) Simulated present-day mass ﬂuxes of PM2.5 and meridional winds. Projected changes in mass ﬂuxes of PM2.5 and meridi winds from the present day (1996–2005) to future (2046–2055) owing to (b) climate change alone, (c) changes in anthropogenic emiss alone, and (d) changes in both climate and anthropogenic emissions. Mass ﬂuxes of PM2.5 are shown by shades (units: Tg), and w are represented by contours (units: m s−1). Both mass ﬂuxes and winds are those through latitudinal plane along 21.7◦N from 90 125◦E. Positive (negative) ﬂuxes indicate northward (southward) transport. The dotted areas are statistically signiﬁcant at the 95 % leve determined by Student’s two-sample t test. Fig. 13. (a) Simulated present-day mass fluxes of PM2.5 and meridional winds. Projected changes in mass fluxes of PM2.5 and meridional winds from the present day (1996-2005) to future (2046-2055) owing to (b) climate change alone, (c) changes in anthropogenic emissions alone, and (d) changes in both Fig. 13. (a) Simulated present-day mass ﬂuxes of PM2.5 and meridional winds. Projected changes in mass ﬂuxes of PM2.5 and meridional winds from the present day (1996–2005) to future (2046–2055) owing to (b) climate change alone, (c) changes in anthropogenic emissions alone, and (d) changes in both climate and anthropogenic emissions. Mass ﬂuxes of PM2.5 are shown by shades (units: Tg), and winds are represented by contours (units: m s−1). Both mass ﬂuxes and winds are those through latitudinal plane along 21.7◦N from 90◦to 125◦E. Positive (negative) ﬂuxes indicate northward (southward) transport. The dotted areas are statistically signiﬁcant at the 95 % level, as determined by Student’s two-sample t test. H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7957 Table 5. Simulated transport of aerosols to or from southern China through the latitudinal plane along 21.7◦N from 90◦to 125◦E. Positive (negative) values indicate northward (southward) transport. Percentage change is listed in the parentheses as the 2050 ﬂux has the same sign as 2000 ﬂux. The units are Tg season−1 for seasonal ﬂuxes and Tg yr−1 for annual ﬂuxes of whole species (e.g., Tg(SO2− 4 ) season−1, Tg(SO2− 4 ) yr−1). Table 5. Simulated transport of aerosols to or from southern China through the latitudinal plane along 21.7◦N from 90◦to 125◦E. Positive (negative) values indicate northward (southward) transport. Percentage change is listed in the parentheses as the 2050 ﬂux has the same sign as 2000 ﬂux. The units are Tg season−1 for seasonal ﬂuxes and Tg yr−1 for annual ﬂuxes of whole species (e.g., Tg(SO2− 4 ) season−1, Tg(SO2− 4 ) yr−1). H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 2050d−2000a NonL = 2050d−2000a eastern China to the western Paciﬁc (through the meridional plane along 135◦E from 20◦to 55◦N) is simulated to be 10.8 Tg yr−1, and the annual outﬂow of PM2.5 is estimated to change by −7.0 %, −0.7 %, and −9.0 %, respectively, over 2000–2050 owing to future climate change alone, future changes in emissions alone, and future changes in both cli- mate emissions. Climate change is predicted to have a larger impact on future ﬂuxes of aerosols than changes in emis- sions. While annual ﬂuxes of all aerosol species show reduc- tions by climate change alone, future increases in outﬂow of nitrate and ammonium offset to a large extent the future de- creases in outﬂow of sulfate, BC, and OC, leading to a small nitrate concentrations are predicted to increase in eastern China over 2000–2050, with the largest increases of 1– 2 µg m−3 in DJF, 3.5–4.5 µg m−3 in JJA, and 2–3.5 µg m−3 in MAM and SON. The projected changes in PM2.5 owing to the changes in emissions alone show decreases in PM2.5 con- centrations of 1–8 µg m−3 in eastern China over 2000–2050 at a 4◦× 5◦resolution. Under the IPCC A1B scenario, future changes in anthropogenic emissions exert a larger effect on year 2050 PM2.5 concentrations in eastern China than does projected future climate change. The estimated year 2050 transboundary ﬂuxes of aerosol species to and from China are sensitive to future changes in climate and emissions. The present-day ﬂux of PM2.5 from Atmos. Chem. Phys., 13, 7937–7960, 2013 shade mass 6 Conclusions ented by contours (Units: m s 1). Both gh latitudinal plane along 21.7N from xes indicate northward (southward) cally significant at the 95% level, as t-test. changes in individual aerosol species, future PM2.5 concen- trations in China are simulated to decrease generally, except that increases are predicted along east and southeast coasts of China in DJF and over a large fraction of eastern China in MAM. The simulated climate-induced changes in PM2.5 concentrations in China are within ±2.5 µg m−3, which rep- resent about 10–20 % of the present-day concentrations in those regions. 90 to 125E. Positive (negative) f transport. The dotted areas are statis determined by the student’s two samp We estimate changes in aerosol levels in China and the trans- boundary transport of aerosols in and out of China over the period 2000–2050 by using the global chemical transport model GEOS-Chem with meteorological input from the gen- eral circulation model GISS GCM 3 (at 4◦× 5◦resolution) and changes in emissions based on the IPCC A1B scenario. The projected 2000–2050 change in climate alone is esti- mated to lead to changes in seasonal mean concentrations of individual aerosol species at 4◦× 5◦resolution over China ranging from −1.5 to +0.8 µg m−3. As a result of these 90 to 125E. Positive (negative) f transport. The dotted areas are statis determined by the student’s two samp We estimate changes in aerosol levels in China and the trans- boundary transport of aerosols in and out of China over the period 2000–2050 by using the global chemical transport model GEOS-Chem with meteorological input from the gen- eral circulation model GISS GCM 3 (at 4◦× 5◦resolution) and changes in emissions based on the IPCC A1B scenario. As a result of the future changes in anthropogenic emis- sions alone, concentrations of sulfate, BC, and OC are simu- lated to decrease by 0.5–3.5 µg m−3, 0.5–2 µg m−3, and 0.5– 3.5 µg m−3, respectively, in eastern China. On the contrary, The projected 2000–2050 change in climate alone is esti- mated to lead to changes in seasonal mean concentrations of individual aerosol species at 4◦× 5◦resolution over China ranging from −1.5 to +0.8 µg m−3. As a result of these www.atmos-chem-phys.net/13/7937/2013/ Atmos. Chem. Phys., 13, 7937–7960, 2013 H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels Transport to or from southern China Species 2000a (Tg) 2050b (Tg) 2050c (Tg) 2050d (Tg) NonLe DJF −0.11 −0.15 (+33.4 %) −0.0094 (−91.5 %) −0.047 (−57.3 %) 0.96 MAM 0.088 0.11 (+25.0 %) 0.41 (+365.9 %) 0.43 (+388.6 %) 1.01 SO2− 4 JJA −0.011 −0.0051 (−53.6 %) 0.023 (–) 0.047 (–) 0.69 SON −0.19 −0.16 (−15.8 %) −0.13 (−31.6 %) −0.11( −42.1 %) 1.13 Annual −0.22 −0.21 (−4.5 %) 0.29 (–) 0.32 (–) 0.96 DJF −0.047 −0.052 (+10.6 %) −0.050 (+6.4 %) −0.059 (+25.5 %) 0.67 MAM 0.10 0.045 (−55.0 %) 0.19 (+90.0 %) 0.045 (−55.0 %) −0.64 NO− 3 JJA −0.12 −0.11 (−8.3 %) −0.31 (+158.3 %) −0.29 (+141.7 %) 1.06 SON −0.086 −0.069 (−19.8 %) −0.069 (−19.8 %) −0.050 (−41.9 %) 0.94 Annual −0.15 −0.19 (+26.7 %) −0.24 (+60.0 %) −0.35 (+133.3 %) 0.65 DJF −0.043 −0.054 (+25.6 %) −0.0035 (−91.9 %) −0.017 (−60.5 %) 1.10 MAM 0.066 0.055 (−16.7 %) 0.22 (+233.3 %) 0.18 (+172.7 %) 1.25 NH+ 4 JJA −0.035 −0.029 (−17.1 %) −0.085 (+142.9 %) −0.070 (+100.0 %) 1.26 SON −0.073 −0.058 (−20.5 %) −0.047 (−35.6 %) −0.039 (−46.6 %) 1.21 Annual −0.085 −0.086 (+1.2 %) 0.085 (–) 0.054 (–) 1.22 DJF −0.00088 −0.0054 (+513.6 %) 0.013 (–) 0.0097 (–) 0.88 MAM 0.095 0.097 (+2.1 %) 0.11 (+15.8 %) 0.11 (+15.8 %) 1.13 BC JJA 0.0029 0.0043 (+48.3 %) 0.0039 (+34.5 %) 0.0050 (+72.4 %) 1.14 SON −0.016 −0.012 (−25.0 %) −0.0033 (−79.4 %) −0.0017 (−89.4 %) 1.17 Annual 0.081 0.084 (+3.7 %) 0.12 (+48.1 %) 0.12 (+48.1 %) 1.08 DJF 0.076 0.057 (−25.0 %) 0.090 (+18.4 %) 0.074 (−2.6 %) 2.50 MAM 0.56 0.56 (0.0 %) 0.63 (+12.5 %) 0.63 (+12.5 %) 1.00 OC JJA 0.017 0.022 (+29.4 %) 0.0083 (−51.2 %) 0.011 (−35.3 %) 0.62 SON −0.020 −0.012 (−40.0 %) −0.0075 (−62.5 %) −0.0046 (−77.0 %) 1.33 Annual 0.63 0.63 (0.0 %) 0.72 (+14.3 %) 0.71 (+12.7 %) 1.13 DJF −0.12 −0.20 (+66.7 %) 0.040 (–) −0.040 (−66.7 %) 1.00 MAM 0.91 0.87 (−4.4 %) 1.57 (+72.5 %) 1.40 (+53.8 %) 1.27 PM2.5 JJA −0.14 −0.12 (−14.3 %) −0.36 (+157.1 %) −0.30 (+114.3 %) 1.25 SON −0.39 −0.31 (−20.5 %) −0.26 (−33.0 %) −0.21 (−46.2 %) 1.17 Annual 0.26 0.24 (−7.7 %) 0.99 (+280.8 %) 0.85 (+226.9 %) 1.20 a Seasonal and annual total ﬂuxes averaged over 1996–2005; b year 2050 ﬂuxes simulated with climate change alone; c year 2050 ﬂuxes simulated with changes in anthropogenic emissions alone; d year 2050 ﬂuxes simulated with changes in both climate and anthropogenic emissions; e NonL = (2050b−2000a)+(2050c−2000a) 2050d−2000a . Atmos. Chem. Phys., 13, 7937–7960, 2013 ng et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels H. Jiang et al.: Projected effect of 2000–2050 changes in climate and emissions on aerosol levels 7958 negative change in annual outﬂow of PM2.5 as a result of changes in emissions alone. Avise, J., Chen, J., Lamb, B., Wiedinmyer, C., Guenther, A., Salath´e, E., and Mass, C.: Attribution of projected changes in summertime US ozone and PM2.5 concentrations to global changes, Atmos. Chem. Phys., 9, 1111–1124, doi:10.5194/acp- 9-1111-2009, 2009. The present-day inﬂow of PM2.5 aerosols from Europe and Central Asia to western China (the ﬂuxes through the merid- ional plane along 75◦E from 35◦to 55◦N) is calculated to be 3.9 Tg yr−1. 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https://openalex.org/W3186610887	https://www.scielo.br/j/rae/a/yN9ZkXFfcNwyxg8T9mQxGbL/?lang=pt&format=pdf	Portuguese	null	GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO	RAE	2,021	cc-by	6,967	GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Uberização, gig economy, on-demand economy, economia de plataforma e crowdsourcing são exemplos de termos usados em diversos trabalhos acadêmicos para se referir ao grande número de transformações promovidas pelas tecnologias de comunicação e informação (doravante abreviado como “TCI”) nos universos das organizações e do trabalho, sem que haja consenso sobre seus significados (Filgueiras & Antunes, 2020, p. 30). O termo “uber ização” (Slee, 2017) remete diretamente à Uber, porém não se restringe a ela, sendo usado em sentido amplo como uma tendência global que atinge ocupações com diferentes qualificações e rendimentos, materializando processos e subjetividades associados à nova razão neoliberal do mundo (Dardot e Laval, 2016). A socióloga Ana Claudia Moreira cunhou a expressão “uberismo” para se referir a um modelo de organização e gestão do tra balho, tal qual taylorismo, fordismo e toyotismo, que surgiu no lastro das TCI e das plataformas (Santos, 2020). Além da Uber, são exemplos de “empresas-aplicativo” (Abílio, 2019) Loggi, iFood, Rappi e Lyft. Inicialmente as transformações promovidas pelas TCI ofereciam inúmeras oportunidades aos trabalhado res, por exemplo, a ausência de rigidez dos empregos tradicionais, permitindo que as pessoas acumulassem atividades e gerassem renda extra. A geração de renda aconteceria supostamente de modo divertido no tempo livre do prestador do serviço (Stefano, 2017), ou quando aquele bem entendesse e desejasse (Valenduc, 2019), por meio da sua atuação ou como criador, ou como parceiro da empresa-aplicativo. Outra vantagem do modelo de plataforma é a redução da distância espacial entre as oportunidades de renda e os trabalhadores, que podem exercer as atividades independentemente da sua localização geográfica (Kittur et al., 2013), como no caso da plataforma de microtrabalho Amazon Mechanical Turk (Moreschi, Pereira, & Cozman, 2020). A distância tornou -se menor também entre provedores de serviços e consumidores graças às plataformas digitais e aos aplicativos, reduzindo preços e agilizando a prestação de uma gama enorme de serviços (Manyika et al., 2016) de transporte, estética, limpeza e serviços gerais, entregas, entre outros. Mais vantagens figuram nesse rol, como a democratiza ção dos meios de produção e o fomento ao neoempreendedorismo (com oportunidades para pequenos negócios, como citam Filgueiras e Antunes, 2020). PENSATA Submetido 29.07.2020. Aprovado 05.01.2021 Avaliado pelo sistema double blind review. Editor Científico convidado: Alcides Barrichello Versão original \| DOI: http://dx.doi.org/10.1590/S0034-759020210407 Submetido 29.07.2020. Aprovado 05.01.2021 Renata Couto de Oliveira1 \| renatacouto@yahoo.com \| 0000-0001-5839-8814 ¹Universidade do Grande Rio, Escola De Ciências Sociais Aplicadas, Rio de Janeiro, RJ, Brasil RAE-Revista de Administração de Empresas \| FGV EAESP RAE-Revista de Administração de Empresas \| FGV EAESP eISSN 2178-938X PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Renata Couto de Oliveira Renata Couto de Oliveira estruturando-se a partir de um modelo capitalista extrativista (Morrell, Espelt, & Cano, 2020). Contrastam com as oportunidades citadas no parágrafo anterior os baixos salários e falta de autodeterminação (Schor & Eddy, 2020), além de longas jornadas de trabalho, da transferência dos custos e riscos associados às atividades para os trabalhadores, que ainda precisam ser geridos, controlados e subordinados (Abílio, 2019) após aderirem aos termos e condições de uso das plataformas. Em um contexto como esse, no qual fontes e estruturas de signifi cados (como práticas, valores, rituais e hierarquias, por exemplo) são influenciados, criados ou expressos pelas TCI (Kozinets, 2019, p. 621), a gamificação emerge como uma das ferramentas que as plataformas digitais utili zam para controlar, gerenciar e vigiar seus “parceiros”. Nos últimos oito anos, a “gamificação” tomou as organizações, a mídia e as pesquisas acadêmicas de assalto (e.g. Dymek & Zackariasson, 2016a). Uma busca no Google Scholar aponta cerca de 51 mil resultados sobre o tema entre 2002 e 2020. Neste ano (2020), destacamos o trabalho de Doorn e Chen (no prelo), que versa sobre a gamificação no contexto de duas plataformas de entrega de comida, uma chinesa e outra norte-ameri cana; e o de Abílio, que define gamificação como a “transferência de riscos e em estímulos à produtividade, que também são novas formas de controle sobre o trabalho” (Abílio, 2020, p. 20), uma forma-desafio lançada a quem se arrisca trabalhando, sem nenhuma garantia, uma vez que cabe à empresa-aplicativo definir as regras do jogo e, possivelmente, o vencedor (o que sugere que, em vez da aleatoriedade algorítmica, há uma distribuição pro gramada de bonificações, por exemplo). O objetivo mais imediato da gamificação é promover o engajamento de trabalhadores e consumidores por meio de desafios e competições baseados em desempenho e incentivos e, em decorrência disso, obter melhores resultados (Dymek & Zackariasson, 2016b). A definição seminal de Deter ding, Khaled, Nacke e Dixon (2011) inspirou autores como Woodcock e Johnson (2018) a apreenderem gamificação como a aplicação de sistemas de jogos (competição, prêmios, quantificação do comportamento do usuário) em domínios que não têm relação com aqueles, como no trabalho. Nesta pensata, analisamos a relação entre gamificação e trabalho uberizado no bojo das empresas-aplicativo, plataformas digitais (Srnicek, 2017) que representam um novo modelo de negócio com infraestrutura vinculada às TCI e aos algoritmos. PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Partimos de uma posição crítica sobre esses temas, uma vez que a gamificação é comumente abordada pela literatura gerencialista como uma estratégia capaz de promover engajamento e aumentar o desem penho dos funcionários, promovendo satisfação em uma espécie de win-win situation para organizações e seus colaboradores. Como a gamificação também apresenta grande potencial de solucionar problemas que podem ser resolvidos influenciando a motivação e o comportamento humano (Zichermann & Cunningham, 2011), nos per guntamos quais seriam os desdobramentos da união entre essa estratégia e o uberismo em um contexto no qual a precariedade do trabalho se associa ao capitalismo de vigilância (Zuboff, 2015) e à governança algorítmica (Castro, 2018). Assim, visamos discutir a gamificação como uma ferramenta de gestão, controle e subordinação do traba lho, associada também à opacidade algorítmica (Bridle, 2019), à manutenção da multidão de trabalhadores e à figura do trabalhador just in time (Abílio, 2019). Por fim, destacaremos sucintamente algumas opções à economia de plataforma tradicional que podem minimizar o uso da gamificação, como a possibilidade de economia de pla taforma sustentável e o cooperativismo de plataforma (Morrell et al., 2020; Scholz, 2016; Schor & Eddy, 2020). GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Apesar do entusiasmo inicial associado à autonomia, às oportunidades econômicas e à equidade (Schor & Eddy, 2020) promovidas pelas plataformas digitais, o contexto atual, principalmente o da pandemia de Covid- 19, apontou para a dependência de alguns serviços, como o de entregas, além de mostrar que, apesar de alguns resultados positivos, muitos trabalhadores que atuam nessas condições sofrem as consequências negativas da economia de plataforma. Essa nova economia é capitaneada por startups que se definem como empresas de tec nologia e intermediárias entre consumidores e produtores/prestadores de serviços (Filgueiras & Antunes, 2020), 1 © RAE \| São Paulo \| V. 61 \| n. 4 \| jul-ago 2021 \| 1-10 \| e2020-0762 eISSN 2178-938X PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Renata Couto de Oliveira são exemplos de gamificação pré-digital (Vesa & Harviainen, 2019). Por um lado, essa estratégia é exaltada como uma solução divertida para engajar e motivar consumidores e trabalhadores (McGonigal, 2011). Assim, as estru turas, organizações ou sistemas tornam-se mais intrinsecamente motivadores, apoiando a criação geral de valor (Vesa, Hamari, Harviainen, & Warmelinl, 2016). Por outro lado, surgiram críticas e dúvidas sobre a gamificação, questionando sua eficácia sem motivação preexistente (e.g. Hamari, 2013) e a duração de seu efeito em longo prazo (e.g. Koivisto & Hamari, 2014). são exemplos de gamificação pré-digital (Vesa & Harviainen, 2019). Por um lado, essa estratégia é exaltada como uma solução divertida para engajar e motivar consumidores e trabalhadores (McGonigal, 2011). Assim, as estru turas, organizações ou sistemas tornam-se mais intrinsecamente motivadores, apoiando a criação geral de valor (Vesa, Hamari, Harviainen, & Warmelinl, 2016). Por outro lado, surgiram críticas e dúvidas sobre a gamificação, questionando sua eficácia sem motivação preexistente (e.g. Hamari, 2013) e a duração de seu efeito em longo prazo (e.g. Koivisto & Hamari, 2014). A diferença proposta por Caillois (2001/1958) entre ludus (formas mais explícitas de jogos orientados por regras, sem conotação negativa) e paida (jogo livre, improvisação, espontaneidade e impulsividade) orientou algumas dessas críticas. Por exemplo, Bogost (2011) cunhou a expressão exploitationware como sinônimo de gamificação, enquanto Escribano (2013) afirma que a gamificação não passa de uma “ditadura lúdica”. Wood cock e Johnson (2018) destacam dois tipos distintos de gamificação: a vinda de cima (gamification-from-above), associada ao conceito de ludus, enfatizando formas de interação e feedbacks desenhados a partir de jogos, mas separados de seus contextos lúdicos originais, e a vinda de baixo (gamification-from-below), identificada com contextos lúdicos associados à paida e que teriam por escopo tornar a realização do trabalho mais fácil e, em certos momentos, servir como prática de resistência. A gamificação do trabalho parece orientá-lo através de um viés comportamental, virtual e instrumental, tornando-o um jogo competitivo e (em tese) divertido, retribuindo trabalhadores com recompensas de prestígio imaterial (Vesa et al., 2017), como distintivos virtuais (badges) de excelência de serviço e de motorista divertido, como no caso da Uber (Scheiber, 2017). GAMIFICAÇÃO E PLATAFORMAS Em 2002, o termo “gamificação” surge como o processo por meio do qual métodos, metáforas, valores e atribu tos de jogos permeiam nossa sociedade (Fuchs, 2014). Placas de funcionário do mês e programas de fidelidade 2 © RAE \| São Paulo \| V. 61 \| n. 4 \| 1-10 \| e2020-0762 eISSN 2178-938X VIGILÂNCIA E GOVERNANÇA ALGORÍTMICA DO TRABALHADOR JUST IN TIME A coleta e a análise de dados devem ser entendidas como “um processo dinâmico no qual sistemas de vigilância e criação de perfis e informações pessoais informam-se continuamente a cada nova interação entre os sistemas e consumidores” (Pridmore & Zwick, 2011, p. 272). Como resultado, as empresas-aplicativo customizam e estru turam o mundo de maneira pouco transparente (Morozov, 2018, p. 53), por meio da “mineração da realidade” (reality mining, Zuboff [2015]). Extrapola-se o mero saber sobre o que as pessoas fazem todo o tempo, criando-se a possibilidade de intervenção e alteração do comportamento de modo que “o que é real e o que é resultado da manipulação tecno-cibernética em tempo real se torne indistinguível” (Charitsis, Zwick, & Bradshaw, 2018, p. 822). Isso se aplica também aos “parceiros” das empresas-aplicativo, que são, ao mesmo tempo, consumi dores das TCI empregadas por elas e trabalhadores que aderem (grifo nosso) a ela (uma vez que não é firmado um contrato entre as partes). A operação das plataformas digitais foi viabilizada, entre outros fatores, pelo cenário tecnocultural e ope rações associados ao capitalismo de vigilância (Zuboff, 2015), nos remetendo, assim, ao conceito de sociedade de controle e à “implantação progressiva e dispersa de um novo regime de dominação” (Deleuze, 1992, p. 225). O capitalismo de vigilância instaura um regime institucional ubíquo, um novo tipo de mão invisível, denominado Big Other, que aniquila as liberdades conquistadas por meio da instituição do Estado de direito, configurando -se como um “regime de fatos independentes e independentemente controlados que suplanta a necessidade de contratos, de governança e o dinamismo de uma democracia de mercado” (Zuboff, 2015, p. 81). O futuro adap tado à normalização do caos e do terror é o que emerge em substituição à “comunidade de iguais vinculados pelas leis na inevitável e, em última análise, frutífera luta humana contra a incerteza” (Zuboff, 2015, p. 81). A pre visão de Zuboff contempla também a instauração de um sistema de conformidade baseado em recompensas e punições que contribui para a acumulação “não apenas de capital e ativos de vigilância, mas também de direi tos” (Zuboff, 2015, p. 83), na medida que os direitos individuais seriam tomados dos indivíduos pelo Big Other e, posteriormente, redistribuídos unilateralmente por ele. Apesar do que afirma Zuboff (2015) sobre ausência de governança, Castro (2018) aponta “uma nova forma de governança exercida pelas organizações através de algoritmos” (Castro, 2018, p. PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira 4 \| 1-10 \| e2020-0762 eISSN 2178-938X PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Renata Couto de Oliveira regulatórias (Gillespie, 2010, p. 349). Esse enquadramento é estratégico, pois posiciona as empresas-aplicativo na busca de lucros atuais e futuros em um ponto regulatório entre as proteções legislativas que as beneficiam e as obrigações que não as beneficiam, apresentando um imaginário cultural no qual seus serviços fazem sentido aos seus usuários e à sociedade como um todo (Gillespie, 2010, p. 348). Assim, as plataformas estabelecem “os próprios critérios pelos quais essas tecnologias serão julgadas” (Gillespie, 2010, p. 359), assumindo a forma de “um arranjo progressivo e igualitário, [que promete] apoiar aqueles que a sustentam” (Gillespie, 2010, p. 350). PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Nosso posicionamento é que a gamificação associada ao trabalho uberizado das empresas-aplicativo resulta na intensificação e na exploração do trabalho, evidenciando a nova forma que as práticas de gestão, controle e vigilância assumem no contexto tecnocultural. Nesse sentido, a promessa utópica da sociedade da informação de inserção social emancipada no e pelo trabalho desmorona (Braga, 2009, p. 65). A gamificação se cristaliza por meio da uberização do trabalho, sendo difícil identificá-la, pois ela não se materializa em um espaço dis ciplinar (como uma fábrica). Tudo ocorre por meio dos aplicativos, com ínfimo contato humano e de maneira automatizada. A vigilância ostensiva torna-se um elemento fundamental para a eficácia da gamificação e é via bilizada pelas plataformas digitais, que atuam como intermediários entre a oferta de trabalho e a demanda de serviço, exibindo tendências de monopólio decorrentes dos efeitos de rede (quanto maior o número de usuários, mais valiosa a plataforma e melhores seus algoritmos) (Srnicek, 2017). Assim, ao pensarmos sobre gamificação, devemos atentar ao escopo dessa estratégia vis-à-vis o processo do trabalho nas empresas-aplicativo. Sabemos que o capitalismo se reestrutura de tempos em tempos e que, desde 2008, sua última versão está associada a uma narrativa focada na mudança e na ascensão da tecnologia, e que, desde então, vivemos numa economia informacional que o autor denomina “economia de plataforma”, caracterizada pela extração e uso de uma nova forma de matéria-prima: dados (Srnicek, 2017). Dizer que os dados são o petróleo do século XXI pode ser um clichê, mas, levando em consideração a escala das transformações digitais em andamento, não podemos subestimá-lo (Morozov, 2018, p. 8). De modo similar ao petróleo, os dados devem ser extraídos e refinados para então serem usados. Esse processo envolve sua captação, seu registro em algum tipo de mídia material e sua manutenção em sistemas de armazenamento maciço (Srnicek, 2017). As plataformas são projetadas para gover nar as possibilidades de interação, sendo as regras de desenvolvimento de seus produtos e serviços definidas pelo seu proprietário, o que indica que ela não é um espaço vazio nem apolítico (Srnicek, 2017). O próprio termo “plataforma” institui um modo de ser que sanciona um estado particular das coisas, enqua drando discursivamente os serviços e tecnologias de empresas no contexto de demandas financeiras, culturais e 3 © RAE \| São Paulo \| V. 61 \| n. PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira tamos, ainda, que, associada a essa instituição, está a opacidade das TCI e seus algoritmos, esses entendidos como um conjunto de instruções para realizar uma tarefa e cada vez mais incumbidos de decisões, avaliações e análises que impactam diretamente nossas vidas (Doneda & Almeida, 2018, p. 141). O trabalho algorítmico é tão complexo que a ele é atribuída a opacidade tecnológica, uma vez que o algo ritmo em si é dotado de aprendizagem automática, sendo capaz de tomar sozinho a decisão “de reorganizar seu funcionamento interno com base nos dados que está analisando” (Doneda & Almeida, 2018, p. 142). Nesse sentido, fala-se em “opacidade dos sistemas tecnológicos” (Bridle, 2019, p. 132), ou seja, a atuação contempo rânea da tecnologia produz efeitos no mundo real que são quase imperceptíveis a priori e, portanto, mais difíceis de serem dimensionados por nós (Bridle, 2019, p. 137). No contexto do uberismo, é comum que trabalhadores não apreendam o funcionamento de diversas atividades que fazem parte de suas rotinas, como o preço dinâ mico, os desligamentos promovidos pelas plataformas, a distribuição de corridas e tarefas, entre outras (Abílio, 2019, p. 3). A presença de algoritmos nas empresas-aplicativo é explicitamente associada aos sistemas opacos e imprevisíveis de gerenciamento e de tomada de decisão automática, substituindo supervisores e gerentes de turno (Mason, 2019). A lógica do algoritmo é desconhecida pelos “parceiros” das plataformas, a quem só resta especular em fóruns virtuais e em mídias sociais sobre seu funcionamento, confabulando sobre táticas para “enganá-lo”, como por meio da interrupção do trabalho em massa visando “obrigar” o algoritmo a gerar tarifas mais altas. Contudo, e dentro do contexto aqui observado, a tradicional técnica de interrupção do trabalho perde a conotação de luta contra a exploração, tornando-se uma mera tentativa de gaming the game (ludibriar o jogo algorítmico), o que dá aos trabalhadores um sentimento de controle sobre o processo de trabalho ao mesmo tempo que não demonstra um posicionamento contra a lógica capitalista, apenas contra obstáculos dentro do próprio jogo (Mason, 2019). É interessante pensar sobre essa colocação à luz das reivindicações dos motofretistas responsáveis pelas paralisações realizadas no mês de julho de 2020, em meio à pandemia de Covid-19, quando os serviços de entrega se tornaram essenciais. VIGILÂNCIA E GOVERNANÇA ALGORÍTMICA DO TRABALHADOR JUST IN TIME 169, itálico no original) que mineram (data mining) grandes volumes de dados e metadados (daí o termo big data) capturados por meio das atividades mediadas pelas empresas-aplicativo. Trata-se da governança algorítmica que reflete uma generali zação dos instrumentos de gestão, criando uma espécie de governo sem governo e distanciando-se da ideia de governo como administração estatal, privilegiando, assim, estilos de comando horizontais ou em rede (Castro, 2018, p. 169). Destacamos, ainda, que a governança algorítmica encontra esteio no ponto de vista tecnocrático, segundo o qual “as decisões políticas baseiam-se em fatos neutros ou argumentos racionais” (Lemke, 2007, p. 54). Tal constatação nos remete aos trabalhos de Gillespie (2010) e Srnicek (2017) sobre como as plataformas se instituem, inclusive discursivamente, como empresas de tecnologia mediadoras entre demanda e oferta. Ressal 4 © RAE \| São Paulo \| V. 61 \| n. 4 \| 1-10 \| e2020-0762 eISSN 2178-938X eISSN 2178-938X UBERISMO E EXPERIMENTOS GAMIFICADOS A gamificação é uma ferramenta que consegue esconder o trabalho embaixo de camadas de desafios e brincadeiras, nos remetendo ao conceito de playbor, neologismo que sinaliza a sobreposição entre trabalho e lazer, produção e con sumo, capaz de tornar o trabalho tão viciante quanto jogos de videogame (Scholz, 2017). O caráter experimental das estratégias gamificadas das empresa-aplicativos é enfatizado por autores e comentaristas: Scheiber (2017) escreve sobre a Uber, enquanto Mason (2019) fala sobre sua experiência trabalhando para a Lyft. Scheiber (2017) é explícito ao afirmar que a Uber está furtivamente envolvida em experimentos comportamentais para manipular seus motoris tas em prol do crescimento corporativo. Por um lado, a empresa insiste em dizer que seus motoristas estão vinculados por meio de contratos, sendo parceiros ou empreendedores que gozam de flexibilidade e que, por meio da plataforma, se conectam aos consumidores que precisam de seus serviços (mediante o pagamento de uma comissão de entre 20% e 30%, segundo Rosenblat e Stark [2016]). Por outro lado, a empresa-aplicativo precisa competir com outras, o que significa atender seus consumidores no menor prazo e com o menor custo possíveis. E, como elas não dispõem dos meios de coerção das organizações tradicionais, a gamificação é usada para incentivar comportamentos lucrati vos dos seus “colaboradores”, ainda que aquelas possam ser também prejudiciais aos trabalhadores. Motoristas recebem “mensagens de texto, emails, pop-ups e gráficos cuidadosamente projetados para mantê-los atrás do volante e direcioná-los, ostensivamente, para áreas de maior demanda” (Süsser, Roessler, & Nissenbaum, 2019, p. 8). Dessa maneira, como em jogos de videogame, os trabalhadores das plataformas são impulsionados discretamente, resultando em mais engajamento emocional e mais investimento na finalização de tarefas. Lembramos, ainda, que o contato com a empresa é feito majoritariamente por meio do aplicativo, sem interação humana, viabilizando que todas as atividades sejam quantificadas e transformadas em dados que são coletados e analisados, sugerindo, assim, a possibilidade de implementação ilimitada das estratégias gamifica das. Elencamos abaixo exemplos dessas estratégias, lembrando que não se trata de um rol exaustivo. PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Renata Couto de Oliveira sas on-demand. A formação de uma multidão de trabalhadores representa um novo estágio da terceirização do trabalho, já que não existem mais subcontratados, mas uma multidão de “parceiros” que constantemente se gerenciam e buscam se diferenciar individualmente por meio de jornadas de trabalho mais longas e assun ção de custos maiores. Esses são os empreendedores de si, neossujeitos modulados pela “cultura de empresa” (Dardot e Laval, 2016, p. 328) e que gerenciam o próprio trabalho de maneira subordinada (Abílio, 2019, p. 5). A governança ou gerenciamento algorítmico torna possível uma previsibilidade maior do comportamento da multidão de trabalhadores, e é justamente essa previsibilidade que torna possível governar a massa, tradicional mente associada à desordem (Castro, 2018). Das numerosas movimentações monitoradas pelos aplicativos e seus algoritmos, “tiram-se pressuposições sobre padrões futuros, que se projetam sobre o presente, como seu itinerário e guia, provocando a preempção da incerteza pela via da mensurabilidade, que por seu turno pavimenta o caminho para a gestão do risco” (Castro, 2018, p. 175). Nesse sentido, “o capitalismo da informação visa predizer e modi ficar o comportamento humano como um meio [de] produzir receita e controle de mercado” (Zuboff, 2015, p. 75). PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Segundo publicação no perfil do Instagram “Treta no Trampo” (@tretanotrampo), do dia 21 de julho de 2020, “a luta é pra ganhar mais, não pra virar CLT ou placa vermelha e acabar ganhando menos. Ninguém quer cumprir hora e ter chefe.” O objetivo dos motofretistas é autonomia “pra valer”, o que possibilita ria a negociação do preço do trabalho, afinal não é possível negociar com o aplicativo nem com o algoritmo. Isso se evidencia por meio das punições em caso de recusa de tarefas. Os entregadores e motoristas dos aplicativos reclamam que eles param de tocar, que perdem pontuação e eventualmente são bloqueados. Por um lado, ainda que o desafio imposto aos trabalhadores pelo gerenciamento algorítmico seja evidente, nenhum deles deseja ver seu trabalho regulamentado pela CLT. Por outro lado, a Reforma Trabalhista (Lei n. 13.467/2017) legalizou e passou a fomentar o uberismo (entendido também como informalidade), por meio do reconhecimento do trabalho intermitente (Krein, Abílio, Freitas, Borsari, & Cruz, 2018), por exemplo. Nesse sentido, além da eliminação das garantias conquistadas por meio do Estado de direito pelo Big Other infotecnológico (Zuboff, 2015), agora o pró prio Estado colabora para que essas garantias (entre elas, as trabalhistas) sejam apagadas da legislação vigente. O gerenciamento algorítmico desponta como uma nova faceta do mundo do trabalho, tendo como estraté gia a gamificação e possibilitando, assim, que o trabalho esteja cada vez mais disperso e a sua gestão e controle, cada vez mais concentrados (“organização através da dispersão”, Harvey [1992]). A dispersão é representada pela multidão de trabalhadores, disponíveis e engajados, que aderem à empresa-aplicativo, arcam com riscos e custos da sua atividade e não possuem nenhuma garantia, direito ou segurança (Abílio, 2019). Eles também são denominados “trabalhadores just in time“ (Abílio, 2019, p. 4), uma vez que as empresas-aplicativo são empre 5 © RAE \| São Paulo \| V. 61 \| n. 4 \| 1-10 \| e2020-0762 eISSN 2178-938X eISSN 2178-938X PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Renata Couto de Oliveira dos uma técnica de gamificação (Süsser et al., 2019). Os motoristas são atraídos pela possibilidade de corridas mais lucrativas (“preço dinâmico”), mas as chances de conseguir uma são ínfimas e extremamente imprevisí veis, porque as áreas em destaque aparecem e desaparecem em questão de minutos (Mason, 2019). Ao que tudo indica, a tática mantém os motoristas dirigindo para atender a demanda (Calo & Rosenblat [2017, p. 1662] chamam essa técnica de “mecanismo isca e chibata”). A imprevisibilidade é viciante e muito explorada também em jogos de azar para fazer com que os jogadores façam só mais uma aposta (Mason, 2019). A manutenção do trabalhador just in time ou on-demand é realizada por meio de técnicas como notifica ções que são recebidas quando, por exemplo, estão prontos para se desconectar do aplicativo: “Tem certeza de que deseja ficar offline? A demanda é muito alta na sua área. Ganhe mais dinheiro, não pare agora!" (Rosenblat & Stark, 2016, p. 3768). Outra notificação comum é a que avisa sobre a proximidade de atingir uma meta de ganho arbitrária: “Você está a US$ 10 de ganhar US$ 330!”, acompanhada por comandos logo abaixo da mensagem (e em destaque), que oferecem duas opções ao motorista – se desconectar ou seguir dirigindo (Scheiber, 2017), como uma tela de um jogo de videogame. Essas são “tentativas de explorar uma conhecida vulnerabilidade na tomada de decisão – a preocupação das pessoas com metas” (Süsser et al., 2019, p. 8). Efeitos semelhantes são obtidos por meio do recurso interativo dos aplicativos que mostram aos motoristas “o status do trabalho (horas, ganhos, viagens, etc.) em formatos semelhantes a jogos, conhecidos por seu poder de manter jogado res ao console e, presumivelmente, motoristas ao volante” (Süsser et al., 2019, p. 8). Uma técnica adotada pela Uber e que é considerada viciante (Mason, 2019; Scheiber, 2017; Süsser et al., 2019) é o despacho de corridas em fila (recurso automático e padrão, que a Uber garante que pode ser desati vado pelo motorista, porém é automaticamente reativado após cada pausa, segundo Scheiber [2017]), ou seja, antes de terminar uma corrida, os motoristas são informados de outras solicitações, o que as torna praticamente irresistíveis. Essa técnica é comparável àquela utilizada por serviços de streaming, como Netflix e Amazon Prime, que promove uma nova forma de consumir denominada “binge watching” (e.g. Pittman & Sheehan, 2015, o “maratonar séries” em português). eISSN 2178-938X © RAE \| São Paulo \| V. 61 \| n. 4 \| 1-10 \| e2020-0762 UBERISMO E EXPERIMENTOS GAMIFICADOS Além disso, chamamos atenção para a obsolescência que ronda os estudos sobre novas mídias e as TCI (Crary, 2013), o que sinaliza a ampliação do rol de técnicas gamificadas ao longo do tempo, tornando fundamental uma investiga ção aprofundada para entender como o universo do trabalho está sendo reconfigurado por meio da aceleração promovida pelas TCI e as novas formas de gerenciamento e controle do trabalho que emergem nesse contexto. Além das recompensas imateriais, como os distintivos de motorista divertido (Scheiber, 2017), gráficos elaborados, que mostram aos motoristas áreas nas quais há probabilidade de tarifas mais altas, são considera 6 © RAE \| São Paulo \| V. 61 \| n. 4 \| 1-10 \| e2020-0762 eISSN 2178-938X PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Renata Couto de Oliveira sentam queda na média (Mason, 2019). Como as avaliações são calculadas com base nas últimas 100 corridas, os motoristas sentem-se obrigados a dirigir ainda mais em troca de avaliações melhores (Mason, 2019). Abílio (2019, p. 4) aponta que as avaliações atuam como uma certificação informal que afeta a identidade profissional do trabalhador, isto é, seu reconhecimento profissional está intimamente ligado ao seu ranqueamento ao longo do tempo. Logo, até mesmo a possibilidade de acessar trabalhos futuros está em jogo. Os experimentos conduzidos pelas empresas-aplicativo com seus “parceiros” revelam a “vulnerabilidade humana a formas de pensamento irracional, tendencioso e limitado, bem como tendências a rotinas compulsi vas e até viciantes” (Süsser et al., 2019, p. 9). Mason (2019) vai mais longe, sustentando que essas empresas on-demand adotam os mesmos elementos de empresas de apostas com o escopo de manter seus trabalhado res conectados e disponíveis por períodos maiores, checando o aplicativo frequentemente. Em tese, a vigilância contribui ao triangular os dados produzidos a cada interação com outros dados demográficos, formando perfis que permitem que motoristas recebam comunicação personalizada. Contudo, existem autores (e.g. Schüll, 2014) que defendem que o laço lúdico (ludic loop), um sentimento de imprevisibilidade que é viciante, é responsá vel pela manutenção dos trabalhadores à disposição. Afinal, eles nunca sabem quando surgirão áreas de preço dinâmico nem quando chegará a próxima tarefa que permitirá a conquista do bônus. Assim, os trabalhadores são condicionados a fazer só mais uma corrida, como o jogador que precisa jogar sempre uma última partida. Existe, ao que parece, uma dimensão temporal envolvendo a gestão e controle do trabalho e que se combina com a vigilância e o funcionamento das plataformas que merece ser investigada. Teme-se, assim, um retorno a uma era anterior ao New Deal (Scheiber, 2017), quando vigorava uma assustadora assimetria de poder entre organizações e trabalhadores, representada pela ausência de garantias e seguranças, viabilizando a explora ção daqueles. Estamos diante de uma possível servidão digital, que se assemelha muito com as condições de trabalho do século XIX (Ordaz, 2019). À guisa de conclusão, sinalizamos que o uberismo se afirma como tendência global no mundo do tra balho (Abílio, 2019), aparecendo em outros contextos associados à gig economy, como o trabalho doméstico, o microtrabalho e o online freelancing (Woodcock & Graham, 2020). Em todos esses cenários, a gamificação opera, modificando a forma da gestão e controle dos trabalhadores. PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira No âmbito do trabalho uberizado, certamente a técnica promove uma nova (e viciante) forma de trabalhar, o que diminui o tempo de espera para os consumidores enquanto passa por cima do autocontrole dos motoristas (Scheiber, 2017). Como último exemplo, apontamos o próprio sistema de avaliações que os aplicativos usam para classi ficar seus motoristas, associado à vigilância e ao gerenciamento do trabalhador. Além de terceirizar a função de gerente para os consumidores (Rosenblat & Stark, 2016), criando uma economia da reputação (Slee, 2017), as avaliações evidenciam o sistema de monitoramento constante do trabalho dos motoristas com o intuito de identificar e criar fluxos de trabalho eficientes, tal qual na tradição taylorista (Rosenblat & Stark, 2016, p. 3772). No bojo da tecnocultura, a vigilância é a via de coleta dos dados produzidos por trabalhadores e usuários dos aplicativos. Esses dados viabilizam que outras técnicas gamificadas sejam implementadas, evitando medidas disciplinares diretas por meio do uso métricas semanais de desempenho, por exemplo (Rosenblat & Stark, 2016, p. 3772). A estabilidade de emprego é ameaçada. Os trabalhadores limitam-se a especular em comunidades nas redes sociais como essas avaliações são feitas. Algumas empresas distribuem as tarefas em faixas de horário de alta demanda de acordo com as avaliações acumuladas. Quando a pontuação cai, as chamadas tornam-se escassas ou os trabalhadores são desligados pela empresa-aplicativo (Ordaz, 2019). Tentando manter médias altas, os trabalhadores recorrem ao que é descrito como trabalho emocional, a combinação de tentativas amigáveis de conversação com contato visual, visando adivinhar as preferências dos passageiros em troca de boas avaliações em vez de gorjetas (Rosenblat & Stark, 2016, p. 3775). Além disso, o empenho em agradar os usuários torna-se uma obsessão conforme os relatórios de avaliação chegam e apre 7 © RAE \| São Paulo \| V. 61 \| n. 4 \| 1-10 \| e2020-0762 eISSN 2178-938X eISSN 2178-938X REFERÊNCIAS Abílio, L. C. (2019). Uberização: Do empreendedorismo para o autogerenciamento subordinado. Psicoperspectivas, 18(3), 1-11. doi: 10.5027/psicoperspectivas-vol18-issue3-fulltext-1674 Review. 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Recuperado de https://columbialawreview.org/ content/the-taking-economy-uber-information-and-power/ Filgueiras, V., & Antunes, R. (2020). Plataformas digitais, uberização do trabalho e regulação no capitalismo contemporâneo. Contracampo, 39(1), 27-43. doi: 10.22409/ contracampo.v39i1.38901 Castro, J. C. L. (2018). Redes sociais como modelo de governança algorítmica. MATRIZes, 12(2), 165-191. doi: 10.11606/ issn.1982-8160.v12i2p165-191 Fuchs, M. (2014). Predigital precursors of gamification. In M. Fuchs, S. Fizek, P. Ruffino, & N. Schrape (Eds.), Rethinking gamification. Hybrid Publishing Lab, Centre for Digital Cultures, United Kingdom: Lightning Source UK Ltd, 119-140. Charitsis, V., Zwick, D., & Bradshaw, A. (2018) Creating worlds that create audiences: Theorising personal data markets in the age of communicative capitalism. Triple C, 16(2), 820- 834. doi: 10.31269/triplec.v16i2.1041 Gillespie, T. (2010). The politics of ‘platforms’. New Media and Society, 12(3), 347-364. doi 10.1177/1461444809342738 Hamari, J. (2013). Transforming Homo economicus into Homo ludens: A field experiment on gamification in a utilitarian peer- to-peer trading service. Electronic Commerce Research and Applications, 12, 236-245. doi: 10.1016/j.elerap.2013.01.004 Crary, J. (2013). 24/7: Late capitalism and the ends of sleep. London, UK: Verso. Dardot, P., & Laval, C. (2016). PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Renata Couto de Oliveira existe e conta com o trabalho voluntário de profissionais de diversas áreas (advogados, programadores, econo mistas, entre outros), apesar do alto custo inicial do desenvolvimento de um aplicativo simplificado (cerca de 500 mil reais, segundo site da revista Época Negócios [2020]). PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Contudo, estudos recentes aventam a possibilidade de uma economia de plataforma sustentável, bem como do cooperativismo de plataforma (Morrell et al., 2020; Scholz, 2016; Schor & Eddy, 2020), contextos nos quais o uso de técnicas gamificadas é bastante reduzido: quatro das 20 plataformas que fizeram parte do estudo de Morrell et al. (2020) usam gamificação. Três dessas quatro plataformas são unicórnios (ou seja, startups avaliadas em 1 bilhão de dóla res antes de abrirem seus capitais em bolsas de valores), e uma é uma plataforma com design alternativo. Além disso, duas em 20 afirmaram que seus empregados podem rejeitar tanto o gerenciamento algorítmico quanto as técnicas gamificadas quando esses aspectos estiverem presentes (Morrell et al., 2020). Ainda, essas duas plataformas são do tipo alternativo (ou seja, é impossível rejeitar tanto o gerenciamento algorítmico quanto a gamificação quando se trabalha para plataformas digitais unicórnios, segundo Morrell et al. [2020]). Por seu turno, o cooperativismo de plataforma é pensado a partir dos valores da Economia Social e Solidá ria e dos princípios do cooperativismo com o intuito de ser uma alternativa verdadeiramente colaborativa (Morrell et al., 2020). Schor e Eddy (2020) investigaram plataformas sem fins lucrativos que visam promover benefícios sociais e que, apesar de usarem a mesma tecnologia, em versões de baixo custo, de outras plataformas, ou não tiveram sucesso, ou tiveram um crescimento bastante lento. De toda forma, a expectativa é que nesses modelos a gamificação seja reduzida ou até mesmo opcional. A tentativa de criar cooperativas de entregadores no Brasil 8 © RAE \| São Paulo \| V. 61 \| n. 4 \| 1-10 \| e2020-0762 eISSN 2178-938X PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Krein, J. D., Abílio, L., Freitas, P., Borsari, P., & Cruz, R. (2018). Flexibilização das relações de trabalho: Insegurança para os trabalhadores. Revista do Tribunal Regional do Trabalho da 15ª Região, (52), 41-66. Recuperado de https://juslaboris.tst. jus.br/handle/20.500.12178/141969 Scheiber, N. (2017). How Uber uses psychological tricks to push its drivers’ buttons. N.Y. Times. Recuperado de https://www. nytimes.com/interactive/2017/04/02/technology/uber- drivers-psychological-tricks.html Scholz, T. (2016). Cooperativismo de plataforma: Contestando a economia do compartilhamento corporativa. São Paulo, SP: Autonomia Literária. Lemke, T. (2007). An indigestible meal? Foucault, governmentality and state theory. 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Reality is broken: Why games make us better and how they can change the world. New York, USA: Penguin Books. Slee, T. (2017). Uberização: A nova onda do trabalho precarizado. São Paulo, SP: Editora Elefante. Moreschi, B., Pereira, G., & Cozman, F. G. (2020). The Brazilian workers in Amazon Mechanical Turk: Dreams and realities of ghost workers. Contracampo, 39(1), 46-64. doi: 10.22409/ contracampo.v39i1.38252 Srnicek, N. (2017). Platform capitalism [ePub]. Cambridge, UK: Polity Press. Stefano, V. De. (2017). Labour is not a technology: Reasserting the declaration of Philadelphia in times of platform-work and gig-economy. IUSLabor, 2, 1-16. Recuperado de https://core. ac.uk/download/ pdf/155003521.pdf Morozov, E. (2018). Big Tech: A ascensão dos dados e a morte da política. São Paulo, SP: Ubu Editora Morrell, M. F., Espelt, R., & Cano, M. R. (2020). Sustainable platform economy: Connections with the sustainable development goals. Sustainability, 12(18), 7640. doi: 10.3390/su12187640 Süsser, D., Roessler, B., & Nissenbaum, H. (2019). Online manipulation: Hidden influences in a digital world. Georgetown Law Techonology Review, 4(1), 1-45. Recuperado de https:// georgetownlawtechreview.org/online-manipulation-hidden- influences-in-a-digital-world/GLTR-01-2020/ Ordaz, P. (2019). Os entregadores de comida são reféns do seu ‘like’. Recuperado de https://brasil.elpais.com/ brasil/2019/01/23/economia/1548260634_440077.html Valenduc, G. (2019). New form of work and employment in the digital economy. In A. Serrano-Pascual, & M. REFERÊNCIAS A nova razão do mundo: Ensaio sobre a sociedade neoliberal. São Paulo, SP: Boitempo. Harvey, D. (1992). A condição pós-moderna: Uma pesquisa sobre as origens da mudança cultural. São Paulo, SP: Loyola. Deleuze, G. (1992). Conversações, 1972-1990. São Paulo, SP: Editora 34. Kittur, A., Nickerson, J. V., Bernstein, M., Gerber, E., Shaw, A., Zimmerman, J., Lease, M., & Horton, J. 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Journal of Information Technology, 30(1), 75-89. doi: 10.1057/jit.2015.5 PENSATA \| GAMIFICAÇÃO E TRABALHO UBERIZADO NAS EMPRESAS-APLICATIVO Renata Couto de Oliveira Renata Couto de Azevedo de Oliveira trabalhou na pesquisa, na redação e na revisão final do manuscrito deste ensaio teórico. CONTRIBUIÇÃO DA AUTORA Renata Couto de Azevedo de Oliveira trabalhou na pesquisa, na redação e na revisão final do manuscrito deste ensaio teórico. Renata Couto de Azevedo de Oliveira trabalhou na pesquisa, na redação e na revisão final do manuscrito deste ensaio teórico. © RAE \| São Paulo \| V. 61 \| n. 4 \| 1-10 \| e2020-0762 eISSN 2178-938X 10
https://openalex.org/W2089355869	https://europepmc.org/articles/pmc4014551?pdf=render	English	null	Quality of Obstetric Referral Services in India's JSY Cash Transfer Programme for Institutional Births: A Study from Madhya Pradesh Province	PloS one	2,014	cc-by	10,106	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. pyright: 2014 Chaturvedi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by EU FP 7 grant to project MATIND. Support was also received from Swedish Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sarikabharat2005@gmail.com Quality of Obstetric Referral Services in India’s JSY Cash Transfer Programme for Institutional Births: A Study from Madhya Pradesh Province Quality of Obstetric Referral Services in India’s JSY Cash Transfer Programme for Institutional Births: A Study from Madhya Pradesh Province Sarika Chaturvedi1,2, Bharat Randive1,3, Vishal Diwan1,2,4, Ayesha De Costa2 Sarika Chaturvedi , Bharat Randive , Vishal Diwan , Ayesha De Costa 1 Department of Public Health and Environment, R D Gardi Medical College, Ujjain, India, 2 Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden, 3 Department of Public Health and Clinical Medicine, Umea University, Umea, Sweden, 4 International Centre for Health Research, R D Gardi Medical College, Ujjain, India and Environment, R D Gardi Medical College, Ujjain, India, 2 Department of Public Health Sciences, Karolinska Institutet, Stockholm Health and Clinical Medicine, Umea University, Umea, Sweden, 4 International Centre for Health Research, R D Gardi Medical College Abstract Background: India launched JSY cash transfer programme to increase access to emergency obstetric and neonatal care (EmONC) by incentivising in-facility births. This increased in-facility births from 30%in 2005 to 73% in 2012 however, decline in maternal mortality follows a secular trend. Dysfunctional referral services can contribute to poor programme impact on outcomes. We hence describe inter- facility referrals and study quality of referral services in JSY. Methods and Results: Women accessing intra natal care (n = 1182) at facilities (reporting .10 deliveries/month, n = 96) were interviewed in a 5 day cross sectional survey in 3 districts of Madhya Pradesh province. A nested matched case control study (n = 68 pairs) was performed to study association between maternal referral and adverse birth outcomes. There were 111 (9.4%) in referrals and 69 (5.8%) out referrals. Secondary level facilities sent most referrals and 40% were for conditions expected to be treated at this level. There were 36 adverse birth outcomes (intra partum and in-facility deaths). After matching for type of complication and place of delivery, conditional logistic regression model showed maternal referral at term delivery was associated with higher odds of adverse birth outcomes (OR- 2.6, 95% CI: 1.0–6.6 p = 0.04). Maternal death record review (April 10–March 12) was conducted at the CEmOC facility in one district. Spatial analysis of transfer time from sending to the receiving CEmOC facility among in-facility maternal deaths was conducted in ArcGIS10 applying two hours (equated to 100 Km) as desired transfer time. There were 124 maternal deaths, 55 of which were among mothers referred in. Buffer analysis revealed 98% mothers were referred from ,2 hours. Median time between arrival and death was 6.75 hours. Conclusions: High odds of adverse birth outcomes associated with maternal referral and high maternal deaths despite spatial access to referral care indicate poor quality of referral services. Editor: Hamid Reza Baradaran, Iran University of Medical Sciences, Iran (Islamic Republic Of) Received October 9, 2013; Accepted April 11, 2014; Published May 8, 2014 turvedi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Chaturvedi et al. Study site and context The study was conducted in Madhya Pradesh (MP) province in central India. MP has a population of 72 million, 70% of which is rural [16]. It has relatively poor health indicators compared to the rest of the country (Table 1). The public sector, through which the JSY is implemented, is the dominant provider of obstetric services in the province. The private sector is relatively small and concentrated in urban areas. Despite the criticality of obstetric referral services for effective EmONC and reductions in maternal and neonatal mortality, no studies have examined the functioning of these, especially in the context of the JSY program in India. Questions that remain unanswered are about the processes and characteristics of referral like proportion of mothers participating in the program that are referred, indications for which they are referred, facility levels from (and to) which referrals are made, transport used for referral, outcomes of delivery in referred mothers and their newborns. Importantly the quality of referral services in the program also needs study. Thus the objectives of this study were (1) to describe the inter-facility obstetric referrals in the JSY cash transfer programme in terms of (a) the proportion of mothers referred (b) referral indications (c) referral patterns and (d) delivery outcomes, and (2) to study the quality of referral services in the JSY by examining (a) association between maternal referral and birth outcomes and (b) spatial access to CEmOC among mothers who were referred to and subsequently died at a CEmOC facility. The province is divided into fifty independent administrative districts. Each district has approximately a million people and a separate district health department that oversees health facilities, health workers and program implementation within its boundar- ies. Three districts were purposively selected for this study to reflect different levels of maternal mortality, institutional delivery proportions, and heterogeneity in socio demographic profiles and variations in geographical parts of the province. (Table 1). The JSY: In MP, the JSY provides a cash transfer to all mothers (,31 USD if rural, 22 USD if urban) conditional on delivering in public health institutions. Delivery care in public institutions is formally free of charge. All public health facilities, regardless of the level of functionality are eligible for the JSY i.e. they provide the cash incentive to mothers delivering there. Introduction neonatal mortality by promoting in-facility delivery. The criteria for women to qualify as beneficiaries, as well as the size of the cash transfer, vary between provinces of the Indian Union [5]. The program has thus far had 54 million beneficiaries. National surveys [6] [7] have documented a steep increase in institutional delivery proportions since the JSY began- from 30% in 2005 to 73% in 2012 [8].Studies published to date have failed to detect an effect of the JSY on reduction in maternal [9] [10] and neonatal mortality [11]. Maternal mortality is considered to be the greatest health inequity of the 21stcentury [1]. It continues to be a problem largely for poor women in low and middle income countries (LMICs).Five obstetric complications cause over two thirds of maternal deaths [2].While most fatal complications cannot be prevented or predicted, they can be effectively treated if women have access to good quality emergency obstetric care (EmOC). Given that most maternal deaths occur during labour, delivery and the first 24 hours post partum, an effective intra partum care strategy including EmOC services has been identified as a priority to reduce maternal deaths[3] [4]. Effective referral services are central to a program like the JSY, which aims to provide EmOC care to save lives. It is known that reductions in maternal mortality and morbidity are not possible without an effective referral system for obstetric complications [12].Obstetric complications are unpredictable and offer a short time window to start medical care, failing which they can be fatal. The capacity of different tiers of public sector health facilities (that are all also JSY institutions) to function as EmONC facilities is India, which contributes to a fifth of all maternal deaths, adopted an intra-partum care strategy, under its Janani Suraskha Yojana (JSY) program. The JSY, launched in 2005, is a cash transfer paid to women when they give birth in a health facility. The objective of the JSY has been to reduce maternal and May 2014 \| Volume 9 \| Issue 5 \| e96773 May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org 1 Quality of Referral Services in JSY, India sending and receiving facilities in the JSY program is shown in Figure 1. sending and receiving facilities in the JSY program is shown in Figure 1. Introduction varied, with some being competent Comprehensive emergency obstetric and newborn care (CEmONC)facilities, while others function at less than Basic EmONC (BEmONC) levels. Given this variation, it is all the more important that an effective referral system is in place to facilitate effective first line management at the first facility a mother attends, and efficient transfer to more comprehensive care facilities when complications may necessitate this. A dysfunctional referral system can contribute to a poor program impact on maternal and neonatal mortality outcomes. Study site and context The program has raised institutional birth proportion in MP from 30% in 2005 to 81% in 2010[17].Thus far, 7 million women have been beneficiaries of the program across the province [18].Apart from the cash incentive to mothers, under the JSY programme; the government also provides a cash incentive to rural female health volunteers called ASHAs (Accredited Social Health Activists) to escort women to institutions for delivery. Large investments are underway in a number of LMICs to strengthen their capacities to provide EmOC and achieve MDGs 4 and 5, however research into the functioning of the referral services for EmOC in these areas is limited [13]. This study of referral services for EmOC in the JSY in India has implications for the JSY program as well as for other low middle income settings working to strengthen EmOC to achieve their MDGs. y The public health system and referrals: The public healthcare delivery system is organised in three tiers comprising Primary Health Centres (PHCs), Community Health Centres (CHCs) at secondary level and tertiary District Hospitals (DHs). The PHCs are expected to provide normal delivery care and referrals when there are complications; the CHCs are expected to provide specialist services, and some of these are designated as First Referral Units (FRUs) providing Caesarean section while the district hospitals are expected to be tertiary care facilities handling complicated cases. Although varying by district size, on average each district has about 40 PHCs, 6–9 CHCs and one DH. While this hierarchy of healthcare facilities exists, patients can choose to access any level of care directly, without moving through the hierarchy. There is no formal preferential treatment for referred cases. Ideally when patients are referred from one facility to Study Framework The term referral is often used to indicate an advice given by a health worker to seek care at higher level facility, whether followed or not [14]. In this study, within the context of the JSY program, we used the term referral to mean movement of women seeking intra-natal care from one health facility to another, because of an inability to receive the necessary obstetric care at the first facility attended. We used the referral chain model proposed by Jahn et al[15]. The model conceptualises referrals as composed of three main components- sender, transport and receiver. Although the importance of the family or village level in the referral chain in Jahn’s model cannot be denied, we limited this study to referrals between facilities, as this is relevant in the context of the JSY. An adaptation of Jahn’s model of the referral chain that focuses on the Figure 1. Model of referral chain, adapted from Jahn A and De Brouwer V 2001. doi:10.1371/journal.pone.0096773.g001 Figure 1. Model of referral chain, adapted from Jahn A and De Brouwer V 2001. doi:10.1371/journal.pone.0096773.g001 May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org 2 Quality of Referral Services in JSY, India Table 1. Profile of study districts.* Health/Development indicators District 1 District 2 District 3 Madhya Pradesh India Maternal mortality ratio* 415 386 206 277 212 Neonatal mortality rate 46 65 32 43 33 Literacy (%) 69 67 74 74 74 Institutional delivery (%) 58 72 81 76 73 Human development Index 0.5 0.4 0.6 - Population(million) 1 1 1.9 72 1000 MMR estimates are regional; * Most recent figures available at time of district selection. Source-Annual Health Survey and Census of India 2011, Registrar General of India, Government of India;,Government of India; Human Development Indices of districts of MP 2001. doi:10.1371/journal.pone.0096773.t001 e at time of district selection. nd Census of India 2011, Registrar General of India, Government of India;,Government of India; Human Development Indices of district complications including Caesarean section and blood transfusion. Maternal death records are required to be maintained at all health facilities in a prescribed format by the government. These records (Apr 2010–Mar2012) were studied at the CEmOC facility to extract data regarding deaths among those mothers referred into this hospital for obstetric care and subsequently died there. In particular data on sending facility locations, delivery details, cause of death, and times of arrival and death were extracted. The two main variables of interest were (a) spatial access measured by travel time between the sending facility and the receiving CEmOC facility and (b) time between the mother’s arrival at the CEmOC facility and death. All sending and the receiving facility were geo- positioned onto a digitised map of the district. The distance between the sending and receiving facilities was converted into travel time assuming the average speed of a van in the study area is 50 km/hour. This average for van speed was based on informa- tion provided by key informants including nursing staff and ambulance drivers in the study district. Travel time between sending and the receiving facility (the district hospital) was categorised into ,1 hour, between 1and 2 hours and .2 hours. Time between arrival and death was derived from the medical record entries. another, a referral slip is to be sent with the patient and a duplicate record is maintained at the sending facilities, although this is not always done in practice. There is no reply form or information sent back from the receiving facility to the sending facility on the case referred. The MP provincial government operates a transport system called Janani Express (JE)[19] (started 2006) to transport pregnant mothers to/from or between health facilities. The JE service is formally free of charge. Data collection Cross sectional survey. A list of delivery facilities along with the average number of deliveries each facility performed was obtained from the district health office in the three districts. The facilities in three study districts- both public and private that performed 10 or more deliveries a month were invited to participate in a cross sectional survey. At the time of the survey, researchers visiting each facility also enquired with facility personnel about availability of other delivery centres in the vicinity. This way snowballing was used to complete the list of delivery facilities. Trained women research assistants visited each study facility for five consecutive days. All women admitted or referred into a facility for intra natal or early post natal care were interviewed to gather data on socio demographic profile, delivery and referral details. During the same five day period details of obstetric referrals sent out from study facilities were obtained by meeting the nursing staff soon after the referral. The survey was done during February 2012 through to April 2013. Data on reasons for referral were sought from the referral slips and by meting nursing staff at facilities while data on other variables were obtained from mothers interviewed. The description of inter- facility referrals and the nested case control study draw on this survey data. Ethics statement. All the study participants in the survey provided written informed consent to participate. Consent was administered in the local language. The study was approved by the Institutional Ethics Committee of the R D Gardi Medical College, MP, India. Study design The objectives of this paper were addressed through different studies. The first objective to describe the inter facility referrals was studied using a cross sectional survey of mothers delivering under the JSY program. The quality of referral services is studied in two ways (a) by using a nested matched case control design to examine association between maternal referral and birth outcomes, and (b) a retrospective maternal death record review to examine spatial access to CEmOC. Analysis To describe inter facility referrals, we determined the propor- tion of mothers referred for intra partum care, among those who accessed the study facilities for delivery care during the 5 study days. Referral pathways of mothers who were referred were traced and analysed by facility levels to produce referral patterns. Descriptive statistics were used to analyse data on time spent at facilities in the referral chain, referral indications, distance travelled, transport used and delivery outcomes. In the matched case control design, cases were women who delivered at term but had adverse birth outcome defined as intra partum foetal death or in-facility neonatal death within 48 hours post partum. Controls were women who delivered at term and had live neonates at 48 hours post partum matched individually to cases for the type of obstetric complication and place of delivery. The exposure of interest was maternal referral. Types of complications matched included (1) haemorrhage (2) pre eclamp- sia or eclampsia (3) obstructed labour (4) infection and (5) indirect Record review. Data for our spatial analysis was sourced from maternal death records maintained at the only Comprehen- sive Emergency Obstetric Care (CEmOC) facility in district 1. This facility is the only one that provides services for obstetric May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org May 2014 \| Volume 9 \| Issue 5 \| e96773 3 Quality of Referral Services in JSY, India Table 2. Study facilities by level and distribution of mothers who accessed intra-partum care at these levels. Facility levels/type No of facilities (%) No of mothers accessed care (%) Primary 47 (49) 203 (17) Secondary 24 (25) 493 (42) Tertiary 3 (3) 362 (31) Private 22 (23) 124 (10) Total 96 (100) 1182 (100) doi:10.1371/journal.pone.0096773.t002 In keeping with our objective of studying referrals, we included 180 mothers (111 mothers who were referred into the study facilities and the 69 who were referred out from the study facilities) in this part of study. Of the referred in mothers, 60% belonged to families living below the poverty line; 35% had a caesarean section delivery. Only 4% were grand mutiparas. causes (anaemia). The second matching criterion was place of delivery. This was chosen to limit differences in case management and quality of care received. Each case was matched to two controls. Analysis Odds ratio and 95% confidence intervals were calculated using conditional logistic regression to study association of referral with birth outcomes controlling for other potential confounders. Referral indications. For the 180 referred mothers, pro- longed labour (obstruction/malposition) was the most common reason (39%) for referral followed by leaking (13%), haemorrhage (8%) and pre/eclampsia (7%). Importantly, facility dysfunction- ality that included non availability of staff, or power and water shortages at sending facilities was a reason for referral in 7% cases; all such sending facilities were primary or secondary level ones. Referral indications by level of sending facilities are presented in Table 3. Of the referrals from the CHC level over one third (40%) were for prolonged labour and a fifth were for leaking membranes, both are conditions ideally expected to be managed at the CHC level. Referral indications. For the 180 referred mothers, pro- longed labour (obstruction/malposition) was the most common reason (39%) for referral followed by leaking (13%), haemorrhage (8%) and pre/eclampsia (7%). Importantly, facility dysfunction- ality that included non availability of staff, or power and water shortages at sending facilities was a reason for referral in 7% cases; all such sending facilities were primary or secondary level ones. A spatial analysis of transfer time from sending to the receiving CEmOC facility among in-facility maternal deaths was conducted. The CEmOC facility and all sending facilities from where the deceased mothers were referred to the CEmOC facility in District 1 were plotted on the map. Buffers of a radius of 50 km (equated to one hour travel time) and 100 km (equated to two hours travel time) were constructed around the CEmOC facility. We used the two hour travel time for categorisation since a travel time of two hours to a CEmOC facility is considered a standard for adequate spatial access to EmOC[20] [21]. Hence deaths among mothers who were referred to CEmOC facility from a facility located within the 2 hour buffer indicated poor quality referral services, either at the sender or receiver levels or at both these levels in the referral chain. Time spent at the CEmOC facility before death was used to assess care at the receiver level facility. Characteristics of referrals. Of all the referrals, 97% were made prior to delivery. Analysis There were 2% referrals that were made directly from the facility entrance without any examination or treatment, 38% after examination, and 60% were admitted into the sending facility before being referred (median duration of admission = 4.5 hours). A referral slip was given to 72% referred mothers. The referral slip mainly contained patient name, date and the facility to which she was referred. Two thirds (63%) of inter facility transfers used the JE transport. The average inter facility travel time was 1.25 hours. y Quantitative data were analysed in STATA. Spatial analysis was conducted using ArcGIS 10. Results Objective 1: To describe inter facility referrals in the JSY Study facilities and mothers. The facility survey yielded a 97% response rate resulting in 96 facilities included in the study. The study facilities by levels of care are presented in Table 2. All the private facilities surveyed provided Caesarean section and have been categorised separately as they are not JSY program facilities. All the three district hospitals provided Caesarean section, while none of the CHCs (secondary level) except one in District 3 did so. For all primary and secondary level facilities, the respective District Hospitals were the referral centres of first choice equipped with blood and obstetric surgery facilities. The average distance to these higher centres was 47 km (IQR 25–63 km). All facilities had 24 hour seven days a week access to free Janani Express van for referral transportation. The distribution of mothers who accessed intra-partum care at these facilities during the 5 day study period is presented in Table 2. Referral pattern. The referral patterns for referred in and referred out mothers are presented separately below. Of the total 111 referred in mothers, 98 were referred once, 11 were referred twice and two were referred thrice resulting in a total of 126 inter facility referrals among the referred in mothers. As seen in Figure 3, in the first referral, sending facilities for mothers referred in were secondary level CHCs in 62% of cases and primary levels in 26% of cases. Of mothers referred from both these levels, 70% went to tertiary level receiving facilities in the public sector. This pattern depicted in Figure 3 below shows that the tertiary level facilities receive most referrals, while the secondary level facilties receive very few referrals at all. In all 13 (12%) mothers who had been referred once required a further referral, half of whom went to a private facility, indicating greater involvement of the private sector in higher order referrals. As seen in Figure 3, in the first referral, sending facilities for mothers referred in were secondary level CHCs in 62% of cases and primary levels in 26% of cases. Of mothers referred from both these levels, 70% went to tertiary level receiving facilities in the public sector. This pattern depicted in Figure 3 below shows that the tertiary level facilities receive most referrals, while the secondary level facilties receive very few referrals at all. Discussion In the matched design when examined separately, odds for intra partum foetal deaths were higher (OR = 3.1, 95% CI: 1.3–8.7) than those for early neonatal death (OR = 1.3, 95% CI: 0.3– 4.9).When association of referral with intra partum foetal deaths was examined in the conditional logistic regression model, maternal referral had four times higher odds of intra partum foetal deaths (OR = 4, 95% CI 1.2–13.8) and the type of provider showed a protective effect – such deaths were less likely in deliveries conducted by doctors than those conducted by nursing personnel or other unqualified staff (OR = 0.15). Results In all 13 (12%) mothers who had been referred once required a further referral, half of whom went to a private facility, indicating greater involvement of the private sector in higher order referrals. A total of 1182 mothers arrived at the 96 study facilities for delivery care during the 5 day survey period. Of these, 1071 came directly from home while 111(9.4%) came on referral from another facility (Figure 2). Of the 1182 mothers who arrived at the study facilities, 69 (5.8%) mothers were referred out for higher care while 1113 were admitted and received delivery care in study facilities. All the 111 mothers who arrived on referral into study facilities completed their care there, none were referred further on. Referral patterns for the 69 mothers who were referred out reveal the same pattern as above –secondary level facilities (CHCs) were the common senders and tertiary level facilities (DHs) were the common intended receivers of referrals. About half the referrals from primary level were sent directly to tertiary level. Referrals were rarely sent from public to private facilities, while few referrals were sent to the medical college. The referral patterns of these 69 inter facility referrals are shown in Figure 4. May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org 4 Quality of Referral Services in JSY, India Figure 2. Mothers who reached study facilities for intra-partum care by referral status. doi:10.1371/journal.pone.0096773.g002 Figure 2. Mothers who reached study facilities for intra-partum care by referral status. doi:10.1371/journal.pone.0096773.g002 Table 6 presents the age, parity, and causes of deaths for the 55 referred mothers. The buffer analysis of travel time between sending facilities and the CEmOC facility (receiver) is shown in Figure 5. It reveals that for 54 maternal deaths (98%) the sending facilties were located within a two hour buffer from CEmOC facility indicating good geographic access. The median time between arrival of mothers at the CEmOC facility and death was 6.75 hours (IQR-2.1–32 hours). Delivery outcomes. Outcomes of delivery among mothers referred out during the study period are not known, we present here outcomes for all mothers admitted at study facilities (n = 1113). There were no maternal deaths while 36 adverse birth outcomes (intra partum foetal deaths and early in-facility neonatal deaths) occurred including two in preterm deliveries. Discussion Association between maternal referral and birth outcomes. The median age of cases (n = 34) was 22 years and that of controls (n = 68) was 23 years. When compared to controls, more cases belonged to families living below the poverty line (65% vs 41%) and received a referral (50% vs 25%p,0.05) An equal proportion(32%) in the two groups were illiterate and 50% of the cases and 44% of controls were primiparous.(Table 4). Global maternal and neonatal health outcomes could be significantly improved if ways of overcoming barriers to wide use of effective interventions including EmONC are in place, especially in low income settings. Conditional cash transfers are an increasingly popular mechanism to address the demand side barriers to utilisation of services. The JSY cash transfer program in India has sharply increased the utilisation of institutional care for delivery and thus is assumed to have increased access to EmONC. In order to ensure access to EmONC, a well functioning referral system is a critical more so when demand has been inflated through financial incentives and different levels of facilities in the program are known to have different functional capacities with regard to EmONC services. By systematically studying the referral services under the JSY, we identify the current gaps that need to be addressed to ensure access to effective EmONC under the JSY. This also provides lessons for policy makers interested in adopting CCTs, especially for attaining MDGs 4 and 5. In the matched design, odds of adverse birth outcomes were higher in mothers referred for obstetric care (OR = 2.3 (95% CI: 1.1–5) than those not referred. When potential confounding from age, education, parity, poverty, antenatal care, and provider at delivery was controlled in the conditional logistic regression model, the odds of adverse birth outcomes among women who were referred remained significantly higher(OR = 2.6, 95% CI: 1.0–6.6 p = 0.04)(Table 5). Results The mothers whose infants had adverse birth outcomes had spent a median time of 2 hours at the sending facility. Deaths despite access to referral facilities indicate poor quality referral services Our findings are in concordance with a recent study at a tertiary care hospital in urban India that reports lack of care before referral is a predictor of neonatal mortality [22]. A recent multi-country cohort study which included India reports most still borns were fresh (not macerated), born at term deliveries and had normal birth weight (.2500 gm), indicating compromised intra partum care and high proportion of potentially preventable still births in LMICs[23]. Also our finding of a protective effect of deliveries conducted by doctors compared to other staff suggests need for further exploration into competence of other staff at providing intra-partum care, and could be a possible reason for the bypassing of lower and mid level facilities. Most maternal deaths among referred mothers who died at CEmOC facility in our study were due to haemorrhage. Mothers with haemorrhage face a risk of death within 2 hours after the onset of the complication in the absence of medical treatment [24][25].Since these mothers were referred from a facility that should be able to provide first line management and died at a facility equipped to treat haemorrhage, it is possible that these mothers did not receive basic essential care like intra venous oxytocin and intra venous fluids at the sending facility prior to referral. Besides lack of care at the sending facilities, there are possibly problems in appropriate management of haemorrhage at the CEmOC facility, given that mothers spent a median of 6.75 hrs there before death. This long interval between arrival of referred mothers and their death at the CEmOC facility is Although we found obstructed labour to be the commonest indication for referral, haemorrhage and eclampsia were the leading causes of deaths among referred mothers indicating that inefficiencies in management are more likely in these cases. Increased risk of in-facility mortality in women with haemorrhage and prolonged labour, and if they are referred from another facility has been reported from a cluster randomised trial in Mali [26]. It is important to note that time to death after onset of complication in absence of appropriate medical care is relatively shorter in haemorrhage - 2 hours than in obstructed labour (12 hours) [20]. Our findings hence indicate inability at providing a rapid response in life threatening conditions, especially haemorrhage, and compromise in the quality of care at the receiving CEmOC facility. Deaths despite access to referral facilities indicate poor quality referral services A recent multi-country cohort study which included India reports most still borns were fresh (not macerated), born at term deliveries and had normal birth weight (.2500 gm), indicating compromised intra partum care and high proportion of potentially preventable still births in LMICs[23]. Also our finding of a protective effect of deliveries conducted by doctors compared to other staff suggests need for further exploration into competence of other staff at providing intra-partum care, and could be a possible reason for the bypassing of lower and mid level facilities. Most maternal deaths among referred mothers who died at CEmOC facility in our study were due to haemorrhage. Mothers with haemorrhage face a risk of death within 2 hours after the onset of the complication in the absence of medical treatment [24][25].Since these mothers were referred from a facility that should be able to provide first line management and died at a facility equipped to treat haemorrhage, it is possible that these mothers did not receive basic essential care like intra venous oxytocin and intra venous fluids at the sending facility prior to referral. Besides lack of care at the sending facilities, there are possibly problems in appropriate management of haemorrhage at the CEmOC facility, given that mothers spent a median of 6.75 hrs there before death. This long interval between arrival of referred mothers and their death at the CEmOC facility is indicative of critical delays in providing adequate emergency care, leading to deaths. the study area. In a separate study (unpublished), we found that nurse birth attendants at facilities included in the present study had poor competence at diagnosing and initiating first line management of obstetric complications. Such compromise in role of sending facilities in the referral system could lead to deterioration of the condition and negatively influence outcomes. While the purpose of referrals is to increase likelihood of better birth outcomes, the result from our matched case control study show referral was associated with worse birth outcomes even after controlling for complications and other confounders. Although we could not account for the severity of the complication and are limited by of sample size and diagnostic accuracy, our findings indicate that use of referral services did not ensure access to appropriate life saving care. Deaths despite access to referral facilities indicate poor quality referral services The high numbers of maternal deaths inspite of access to a functioning CEmOC facility under the JSY scheme indicate ineffective referral services at one or more of the levels in the referral chain. This could result from multiple causes as shown in Jahn et al’s model for referral services in Figure 1, however issues at receiving facility (and possibly) the sending facilities in our study appear more important than those related to transport as most mothers had arrived from facilities within the optimal two hour travel time. Availability of free transport services and the reasonably good condition of major roads in the study district (as found by our research team that travelled on these roads for the survey of health facilities) make problems with transport less likely in this context. Delay in timely recognition of the condition at the sending facility could be an important reason contributing to ineffective referral services and ultimately morbidity/mortality in Spatial access to CEmOC among mothers who were referred to and subsequently died at CEmOC facility. The single CEmOC facility in District 1 conducted 10474 deliveries during the two study years (April 2010 to March 2012). There were 124 maternal deaths in the facility during this period. Of the 124 maternal deaths, 55 (44%) were among mothers who were referred to the CEmOC facility from another sending facility. These 55 deaths and were included in this part of the study, excluding the rest. Most referrals were from CHCs (Figure 5). Referrals were also received from other tertiary level facilities in neighbouring districts. Spatial access to CEmOC among mothers who were referred to and subsequently died at CEmOC facility. The single CEmOC facility in District 1 conducted 10474 deliveries during the two study years (April 2010 to March 2012). There were 124 maternal deaths in the facility during this period. Of the 124 maternal deaths, 55 (44%) were among mothers who were referred to the CEmOC facility from another sending facility. These 55 deaths and were included in this part of the study, excluding the rest. Most referrals were from CHCs (Figure 5). Referrals were also received from other tertiary level facilities in neighbouring districts. May 2014 \| Volume 9 \| Issue 5 \| e96773 May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org 5 Quality of Referral Services in JSY, India Table 3. Reasons for referral by sending facility levels. Deaths despite access to referral facilities indicate poor quality referral services Reason for referral PHC CHC DH Private Total % Prolonged labour 23 42 3 2 70 38.9 Leaking 2 20 0 2 24 13.3 Haemorrhage 6 7 1 0 14 7.8 Pre Eclampsia/Eclampsia 2 7 2 2 13 7.2 Bad Obstetric History 6 3 1 0 10 5.5 Primiparity 4 1 0 1 6 3.3 Anaemia 1 4 0 0 5 2.8 Foetal distress 1 4 0 0 5 2.8 Twins 1 3 0 0 4 2.2 Retained placenta 0 1 0 0 1 0.5 Other reasons(medical) 2 8 1 0 11 6.1 Facility dysfunctionality 8 5 0 0 13 7.2 Not mentioned 2 1 0 1 4 2.2 Total 58 106 8 8 180 100 doi:10.1371/journal.pone.0096773.t003 Reason for referral PHC CHC DH Private Total % Prolonged labour 23 42 3 2 70 38.9 Leaking 2 20 0 2 24 13.3 Haemorrhage 6 7 1 0 14 7.8 Pre Eclampsia/Eclampsia 2 7 2 2 13 7.2 Bad Obstetric History 6 3 1 0 10 5.5 Primiparity 4 1 0 1 6 3.3 Anaemia 1 4 0 0 5 2.8 Foetal distress 1 4 0 0 5 2.8 Twins 1 3 0 0 4 2.2 Retained placenta 0 1 0 0 1 0.5 Other reasons(medical) 2 8 1 0 11 6.1 Facility dysfunctionality 8 5 0 0 13 7.2 Not mentioned 2 1 0 1 4 2.2 Total 58 106 8 8 180 100 doi:10.1371/journal.pone.0096773.t003 doi:10.1371/journal.pone.0096773.t003 the study area. In a separate study (unpublished), we found that nurse birth attendants at facilities included in the present study had poor competence at diagnosing and initiating first line management of obstetric complications. Such compromise in role of sending facilities in the referral system could lead to deterioration of the condition and negatively influence outcomes. While the purpose of referrals is to increase likelihood of better birth outcomes, the result from our matched case control study show referral was associated with worse birth outcomes even after controlling for complications and other confounders. Although we could not account for the severity of the complication and are limited by of sample size and diagnostic accuracy, our findings indicate that use of referral services did not ensure access to appropriate life saving care. Our findings are in concordance with a recent study at a tertiary care hospital in urban India that reports lack of care before referral is a predictor of neonatal mortality [22]. Deaths despite access to referral facilities indicate poor quality referral services Our finding is in agreement with conclusions of a systematic review of ‘third delay’ to explore reasons for maternal deaths despite institutional care by Knight et al that highlights how a focus on patient side delays can conceal that many health facilities in low income settings are yet unable to effectively treat serious obstetric complications [27]. Findings similar to ours are reported from other high maternal mortality regions- a maternal death inquiry in African region[28] and a study in Ghana[29] reported that a large part of avoidable maternal deaths were due to problems in managing emergencies at hospitals. Dogba et al in a study of maternal referral system in Kayes, Mali show that that maternal-newborn survival in the referral system is influenced by combined effects of point of care, the skill configuration of CHC personnel and distance travelled [30]. Improvement in performance at referral facilities under the JSY is essential for access to EmONC to be effective. Besides death reviews that are recently being emphasised under the JSY in India, routine measures like number of deliveries with adverse outcomes, number of such events per delivery and severity of outcomes could be useful [31] as also near miss audits and hospital standardised mortality rates[32]. May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Quality of Referral Services in JSY, India Figure 3. Referrals by facility levels among referred in mothers. (Arrows point to receiving facilities, encircled numbers on arrows indicate numbers of mothers who went to the receiving facility.) doi:10.1371/journal.pone.0096773.g003 Figure 3. Referrals by facility levels among referred in mothers. (Arrows point to receiving facilities, encircled numbers on arrows indicate numbers of mothers who went to the receiving facility.) doi:10.1371/journal.pone.0096773.g003 Figure 3. Referrals by facility levels among referred in mothers. (Arrows point to receiving facilities, encircled numbers on arrows indicate numbers of mothers who went to the receiving facility.) doi:10.1371/journal.pone.0096773.g003 Dysfunctional secondary level facilities and overcrowding at tertiary level facilities facilities [35]. Another noteworthy example in this regard comes from the UK where hospitals had ‘functional splits’- complicated cases were treated in separate wards by specially trained senior providers while others cared for non-complicated births [36], as also in a hospital in Nepal[37]. The referral patterns and indications in our study show secondary level facilities in the JSY scheme merely serve as sending facilities. They received very few referrals and were unable to handle obstetric complications- 40% referrals from CHCs were for obstructed labour. The practice of direct referrals sent from primary to tertiary referral level facilities and not to mid level facilities as found by us, was also reported over decade ago by a study from India [33], and more recently from Tanzania [34]. Perceived poor quality of care at bypassed facilities was an important reason for the bypassing behaviour. In the present study context, where most mid (secondary) level facilities lack the ability to provide even basic EmOC, patient interests are best served by direct referrals made to tertiary level facilities. This puts an additional burden on the higher level facilities compromising their quality of care. Preferential treatment to referred cases is mentioned as one of the influencing factors at receiving level in Jahn’s model. A system of triage at receiving facilties to identify mothers requiring emergency care and those requiring additional monitoring would be beneficial. A study from Rajasthan, India reports use of colour coded cards sent with patients referred for EmOC helped in seizing immediate attention of staff at referral Provider practices regarding referral Although we found referral slips were provided to most referred mothers, there was no mention of adequate details of treatment provided and progress of the labour. Our findings indicate lack of adequate communication between sending and receiving facilities. Authors of a recent maternal death enquiry in a district of Madhya Pradesh report that staff refrain from intervening in complicated cases and attempt to evade responsibility by referring out mothers with complications. It is likely that staff fear being blamed by patients’ families and questioned by higher authorities in the event of adverse outcomes at their facility premises. [38]. Such practices place women at further risk of death in multiple transits as has been reported by George et al from a south Indian province [39]. Although we found referral slips were provided to most referred mothers, there was no mention of adequate details of treatment provided and progress of the labour. Our findings indicate lack of adequate communication between sending and receiving facilities. Authors of a recent maternal death enquiry in a district of Madhya Pradesh report that staff refrain from intervening in complicated cases and attempt to evade responsibility by referring out mothers with complications. It is likely that staff fear being blamed by patients’ families and questioned by higher authorities in the event of adverse outcomes at their facility premises. [38]. Such practices place women at further risk of death in multiple transits as has been reported by George et al from a south Indian province [39]. The referrals sent from district hospitals in our study were for conditions that ordinarily ought to be managed there. Though referrals sent out from district hospitals were few in number, this is PLOS ONE \| www.plosone.org May 2014 \| Volume 9 \| Issue 5 \| e96773 7 Figure 4. Referrals by facility levels among referred out mothers. doi:10.1371/journal.pone.0096773.g004 Quality of Referral Services in JSY, India Quality of Referral Services in JSY, India Figure 4. Referrals by facility levels among referred out mothers. doi:10.1371/journal.pone.0096773.g004 Weak monitoring and accountability measures concerning as it indicates failure to provide required care at the highest centres for the JSY program in the district. Such patients are usually sent to medical colleges for critical care; which then does not necessarily remain free of cost to users as intended by the JSY program. Provider practices regarding referral In our setting, referrals from district hospitals possibly happen when either an obstetrician or an anaesthetist is not available because precarious human resources do not allow adequate cover when obstetricians are deputed to conduct sterilisations in various facilities or are on leave. Our findings indicate that there are inefficiencies in the referral services studied and that these are complex, multi factorial and demand a systemic approach. The inefficiencies this study finds could possibly stem from a varying mix of incompetence of providers at sending facilities at recognition and stabilisation of obstetric emergencies, lack of motivation of providers to perform to the best of their abilities contributing to phase III delays (delays in receiving appropriate care on reaching a health facility [40]), a lack of monitoring of referral services and poor accountability of the system to the users. Although all these elements have not been Table 4. Descriptive data for cases and controls. Characteristic Cases (n = = 34) Controls (n = 68) Age in years, median (range) 22 (18–35) 23 (18–40) Below poverty line* 65% 41% Illiterate 32% 32% Referred* 50% 25% Primiparous 50% 44% Caesarean section delivery 15% 28% ANC visits ,2 38% 25% Delivery care by doctor 21% 30% *Difference between cases and controls significant P,0.05. doi:10.1371/journal.pone.0096773.t004 PLOS ONE \| www plosone org 8 May 2014 \| Volume 9 \| Issue 5 \| e96773 Table 4. Descriptive data for cases and controls. May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org Quality of Referral Services in JSY, India Table 5. Output of conditional logistic regression for association between maternal referral and adverse birth outcomes. Variable Odds ratio (95% CI) P value Maternal referral 2.61 (1.08; 6.6) 0.04 Age 1.83 (0.60, 5.51) 0.28 Education 1.2 (0.43–3.43) 0.70 Below poverty line 2.06 (0.80–5.31) 0.13 Parity 0.63 (0.19–2.02) 0.43 ANC visit 1.19 (0.57, 2.48) 0.63 Provider 0.30 (0.07, 1.25) 0.10 doi:10.1371/journal.pone.0096773.t005 Table 5. Output of conditional logistic regression for association between maternal referral and adverse birth outcomes. Table 5. Output of conditional logistic regression for association between maternal referral and adverse birth outcomes. doi:10.1371/journal.pone.0096773.t005 documented [13].Suggested methods of monitoring referrals include use of specific comparable indicators for referral service, conducting audits [42][43] and GIS modelling combined with population data[44]. Conclusions To the best of our knowledge, this is the first study of referral services in the context of the JSY in India. We report on important aspects that have thus far remained under researched. This paper demonstrates how a simple GIS method - buffer analysis of transfer time among deaths in obstetric referrals can be used as a tool to assess quality of referral services. Acknowledgments We acknowledge Dr J P Muliyil for advice with statistical analysis. We thank to our colleagues Sayyed Ali, Dr Manish Singh, Zia Ur Rehman for research assistance, Dr Yogesh Sabde for data base management and the MATIND study team in Ujjain for their cooperation. We thank all study participants for their interest and participation. We thank Raven Joanna for critical comments on an earlier version of this manuscript. We thank the three anonymous peer reviewers for their comments and suggestions. Acknowledgements are due to National Rural Health Mission, Govern- ment of Madhya Pradesh. Birth weight and congenital anomalies could be possible confounders not considered in our case control study; however we were limited by inability to obtain reliable data on these variables and suggest interpretation of our findings in this consideration. Also our study has wide CIs because of small numbers and our focus being inter facility referrals, we have not studied the family/community level in the referral chain. Our sample had 34 cases and 68 controls. Given that referrals were 50% in cases and 25% in controls, our study had a power of 47% at alpha = 0.05. Therefore our results should be interpreted with this caution. Methodological discussion and limitations First, the delivery outcomes of mothers referred out from study facilities are not known. However the pattern is unlikely to be very different from those of mothers referred into study facilities and included in our study. Secondly, it could be argued that findings could vary at different times and in different settings. Although generalizability of our findings is limited, we believe that the key action areas we draw up are likely to be similar in other low income settings. For the spatial analysis, our assumption of van speed of 50 km/hour and hence 100 km distance being the equivalent to 2 hours of travel time, was arrived at from the triangulation of information from van drivers, nurses and our own experience of using these roads. Even if one was to assume this speed to be less or more by 10 km/hour, our results remain unchanged. In this part of our study on in -facility maternal deaths among users of referral services; it is not known if mothers arrived early on onset of complication or not and what modes of transport were used. We would argue though that once the mothers reach a health facility, the responsibility for care rests with the services including referral services and not with the users. This is in line with Knight et al who caution against shifting the blame to users in situations when facilities are dysfunctional. Fourth, the staff at participating facilities could have reported reasons for referral differently leaving a potential for misclassification of referral indications in our descriptive results, although this non-differential does not affect results from our matched case control design. q y The JSY cash transfer intervention is currently implemented through facilities that are not backed by good quality referral services. Hence these facilities fail to provide the life saving care essential for achievement of the program goals to reduce maternal and neonatal mortality. Our findings call for attention of policy makers and managers of the JSY program to monitoring effectiveness of referral services. An urgent improvement in referral system by ensuring first line care before referral, care during transfer, appropriate and preferential treatment to referred cases at referral facilities besides strengthening sub optimally performing facilities to function at desired levels is recommended Countries interested in raising demand for services by way of conditional cash transfer schemes should necessarily ensure a strong supply side prior to adopting JSY like schemes. EmONC in the context of a CCT programme for institutional delivery care. EmONC in the context of a CCT programme for institutional delivery care. Provider practices regarding referral Our findings highlight the need to institute effective data recording on referrals and appropriate monitoring mechanisms to support improvement in the referral process while reemphasising the criticality of effective referral services for explored in our study, overall it shows weaknesses in the referral system in the JSY and calls for measures to improve the functioning of these critical elements. It is important to emphasise what Murray et al. [41] include in their requisites for successful referral system- supervision and accountability of providers performance and the capacity to monitor effectiveness. The need to improve monitoring of referral services in developing contexts is Figure 5. Buffer analysis of distance between CEmOC facility and facilities that referred the 55 mothers who died at CEmOC facility. (Circles in the figure are buffers with radius of 50 Km and 100 Km around the CEmOC facility.) doi:10.1371/journal.pone.0096773.g005 Figure 5. Buffer analysis of distance between CEmOC facility and facilities that referred the 55 mothers who died at CEmOC facility. (Circles in the figure are buffers with radius of 50 Km and 100 Km around the CEmOC facility.) doi:10.1371/journal.pone.0096773.g005 May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9 9 Quality of Referral Services in JSY, India Table 6. Details of in-facility maternal deaths who were referred to CEmOC facility (n = 55). Details of mothers who were referred to CEmOC facility and died (n = 55) Median age 25years (Range 19–35) Primiparous 26 (47%) Death Before delivery 16 (29%) Post abortion 2 (4%) After Vaginal delivery 27 (49%) After C section 10 (18%) Cause of death Haemorrhage/Anaemia 25 (45%) Hypertensive disorders 15 (27%) Sepsis 6 (11%) Obstructed labour 6 (11%) Indirect causes 3 (4%) Mothers who were referred from facilities ,2 hr distance from CEmOC facility 54 (98%) Mothers who were referred from facilities ,1 hr distance from CEmOC facility 45(82%) Median time between arrival and death at CEmOC facility 6.75 Hours (IQR-2.1–32) doi:10.1371/journal.pone.0096773.t006 doi:10.1371/journal.pone.0096773.t006 EmONC in the context of a CCT programme for institutional delivery care. References 1. World Health Organisation (2012) WHO, UNICEF, UNFPA, World Bank estimates-Trends in Maternal Mortality:1990 to 2010, World Health Organi- zation, Geneva. 23. McClure EM, Pasha O, Goudar SS, Chomba E, Graces A, et al. (2011) Epidemiology of still birth in low-middle income countries: A global network study. Acta Obstet Gynecol Scand 90(12): 1379–1385. y y ( ) 24. AbouZahr C (1998) Antepartum and postpartum haemorrhage. Chapter 4.Murray C, Lopez A, editors. Health dimensions of sex and reproduction.Bos- ton: Harvard School of Public. 2. Khan KS, Wojdyla D, Say L, Gu¨lmezoglu AM, Van Look PF (2006) WHO analysis of causes of maternal death: a systematic review. Lancet 367: 1066– 1074. 25. Khan RU, El-Rafaey H (2006) Pathophysiology of post partum haemorrhage and third stage of labour Chapter 8. B-Lynch C, Keith LG, Lalonde AB, Karoshi M, A textbook of post partum haemorrhage. London: Sapiens publishing.pp 62–69. 3. Campbell O, Graham WJ (2006) Lancet Maternal Survival Series Steering Group. Strategies for reducing maternal mortality: Getting on with what works. Lancet, 368:1284–99. 4. Paxton A, Maine D, Freedman L, Fry D, Lobis S (2005) The evidence for emergency obstetric care. Int J Gynaecol Obstet 88: 181–193. 26. Ndour C, Dossou Gbe´te´ S, Bru N, Abrahamowicz M, Fauconnier A, et al. (2013) Predicting in-hospital maternal mortality in senegal and mali. PLoS One 8: e64157. g y y 5. Ministry of Health and Family Welfare (2006) Janani Suraksha Yojana: Revised Guidelines for implementation. Government of India, New Delhi. 5. Ministry of Health and Family Welfare (2006) Janani Suraksha Yo Guidelines for implementation. Government of India, New Delhi 27. Knight HE, Self A, Kennedy SH (2013) Why are women dying when they reach hospital on time? A systematic review of the ‘third delay’. PLoS One 8: e63846. 6. United Nations Population Fund–India (2009) Concurrent assessment of Janani Suraksha Yojana (JSY) in selected states: Bihar, Madhya Pradesh,Orissa, Rajasthan, Uttar Pradesh. Uniited Nations Population Fund–India, New Delhi. 28. National committee on confidential enquiries into maternal deaths (2000) A review of maternal deaths in South Africa during 1998. South Afr Med J 90(4): 367–73. j p 7. National Health Systems Resource Centre (2011) Programme evaluation of the Janani Suraksha Yojana, Ministry of Health and Family welfare, Government of India, New Delhi. 29. Ansong-Tornui J, Amar-Klemensu M, Arhinful D, Penfold S, Hussein J (2007) Hospital based maternity care in Ghana: Findings of a confidential enquiry into maternal deaths. References Ghana Med J 41(3): 125–32. 8. Registrar General of India (2013) Special bulletin of maternal mortality in India 2010–12. New Delhi: Government of India. 30. Dogba M, Fournier P, Dumont A, Zunzunegui MV, Tourigny C, et al. (2011) Mother and newborn survival according to point of entry and type of human resources in a maternal referral system in Kayes (Mali). Reprod Health 8: 13. 9. Lim SS, Dandona L, Hoisington JA, James SL, Hogan MC, et al. (2010) India’s Janani Suraksha Yojana, conditional cash transfer programme to increase births in health facilities: an impact evaluation. Lancet 375: 2009–2023. 31. Mann S, Pratt S, Gluck P, Nielssen P, Reisser D (2006) Assessing quality in obstetrical care: Development of standardized measures. Journal on Quality and Patient Safety 32(9): 497–505. 10. Randive B, Diwan V, De Costa A (2013) India’s Conditional Cash Transfer Programme (the JSY) to Promote Institutional Birth: Is There an Association between Institutional Birth Proportion and Maternal Mortality? PLoS One 8: e67452. 32. Ploemacher J, Israe¨ls AZ, van der Laan DJ, de Bruin A (2013) [Standardised in- hospital mortality decreasing over time]. Ned Tijdschr Geneeskd 157: A5267. 11. Powell-Jackson T (2011) Financial Incentives in Health: New Evidence from India’s Janani Suraksha Yojana. Available at http://dx.doi.org/10.2139/ssrn. 1935442 Accessed Oct 8, 2013. 33. Gupta J, Gupta H (2000) Perceptions of and constraints upon pregnancy-related referrals in rural Rajasthan, India. Health Serv Manage Res 13: 6–15. referrals in rural Rajasthan, India. Health Serv Manage Res 13: 12. Murray SF, Davies S, Phiri RK, Ahmed Y (2001) Tools for monitoring the effectiveness of district maternity referral systems. Health Policy Plan 16: 353– 361. 34. Kruk ME, Mbaruku G, McCord CW, Moran M, Rockers PC, et al. (2009) Bypassing primary care facilities for childbirth: A population based study in rural Tanzania. Health Policy and Planning 24:279–288. 13. Hussein J, Kanguru L, Astin M, Munjanja S (2012) The effectiveness of emergency obstetric referral interventions in developing country settings: a systematic review. PLoS Med 9: e1001264. 35. Jaimala H (1996) Perceptions and constraints of pregnancy related referrals in rural Rajasthan. The Journal of Family welfare 42(1): 24–29. 36. Hundley VA, Cruickshank FM, Lang GD, Glazener CM, Milne JM, et al. (1994) Midwife managed delivery unit: a randomised controlled comparison with consultant led care. BMJ 309: 1400–1404. y 14. World Health Organisation (1994) Mother baby package: Implementing safe motherhood in countries. References Practical Guide: Maternal Health and Safe Motherhood Programme. Division of Family Health. Geneva. J 37. Rana T, Rajopadhyaya R, Bajracharya B, Karacharya M, Osrin D (2003) Comparison of midwife-led and consultant-led maternity care for low risk deliveries in Nepal. Health Policy and Planning 18(3): 330–37. 15. Jahn A, de Brouwere V (2001) Referral in pregnancy and childbirth: concepts and strategies. In. Safe motherhood strategies: a review of the evidence. Edited by Brouwere V, van Leberghe W. Antwerp: ITG press. 38. Sri B, Naidu S, Khanna R (2012) An investigation of maternal deaths following public protests in a tribal district of Madhya Pradesh, Central India. Reproductive Health Matters 20(39):11–20. , g p p 16. Registrar General of India (2012) Census of India 2011. Government of India, New Delhi. 17. United Nations Children Fund (2011) Coverage evaluation survey report 2009. Accessed online from http://www.unicef.org/india/MP_Fact_Sheet.pdf Oct8, 2013. 39. George A (2007) Persistence of high maternal mortality in Koppal district, Karnataka, India: observed service delivery constraints. Reprod Health Matters 15: 91–102. 18. National Rural Health Mission (2013) Ministry of Health and Family welfare Government of India, New Delhi. Available online at http://nrhm.gov.in/ images/pdf/mis-report/March-2013/2High_Focus_NonNE_States_as_on_31. 03.2013.pdf Accessed Oct 8,2013. 40. Thhadeus S, Maine D (1994) Too far to walk: maternal mortality in context. Soc Sc Med 38(8):1091–1110. ( ) 41. Murray SF, Pearson SC (2006) Maternity referral systems in developing countries: current knowledge and future research needs. Soc Sci Med 62: 2205– 2215. 19. National Rural Health Mission (2006) Government of Madhya Pradesh. http:// www.indiagovernance.gov.in/files/gkc_oneworld_janani_express_yojana.pdf Accessed online Oct 8, 2013. 42. Strand RT, de Campos PA, Paulsson G, de Oliveria J, Bergstorm S (2009) Audit of referrals of obstetric emergencies in Angola: A tool for assessing quality of care. Afr J Reprod Health: 13(2):75–85. 20. World Health Organisation (2009) Monitoring emergency obstetric care: A handbook. Geneva. p ( ) 43. Kongnyuy EJ, Mlava G, van den Broek N (2008) Criteria-based audit to improve a district referral system in Malawi: a pilot study. BMC Health Serv Res 8: 190. 21. National Rural Health Mission (2010) Operational guidelines on maternal and newborn health. Ministry of Health and Family Welfare. New Delhi. newborn health. Ministry of Health and Family Welfare. New D 44. Bailey PE, Keyes EB, Parker C, Abdullah M, Kebede H, et al. (2011) Using a GIS to model interventions to strengthen the emergency referral system for maternal and newborn health in Ethiopia. Author Contributions Conceived and designed the experiments: SC BR. Performed the experiments: SC. Analyzed the data: SC BR. Contributed reagents/ materials/analysis tools: VD ADC. Wrote the paper: SC ADC. May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org 10 Quality of Referral Services in JSY, India Quality of Referral Services in JSY, India References Int J Gynaecol Obstet 115: 300–309. 22. Narang M, Kaushik JS, Sharma AK, Faridi MM (2013) Predictors of mortality among the neonates transported to referral centre in Delhi, India. Indian J Public Health 57: 100–104. May 2014 \| Volume 9 \| Issue 5 \| e96773 PLOS ONE \| www.plosone.org 11
https://openalex.org/W2998781646	http://www.scielo.br/pdf/sdeb/v43nspe3/en_0103-1104-sdeb-43-spe03-0165.pdf	English	null	Panorama da pesquisa sobre tratamento e reúso de efluentes da indústria de antibióticos	Saúde em Debate	2,019	cc-by	8,613	1 Fundação Oswaldo Cruz (Fiocruz), Escola Nacional de Saúde Pública Sergio Arouca (Ensp) – Rio de Janeiro (RJ), Brasil. ac.limarocha@gmail.com 165 165 REVIEW \| REVISÃO This article is published in Open Access under the Creative Commons Attribution license, which allows use, distribution, and reproduction in any medium, without restrictions, as long as the original work is correctly cited. Panorama da pesquisa sobre tratamento e reúso de efluentes da indústria de antibióticos Antônio Carlos de Lima Rocha1, Débora Cynamon Kligerman1, Jaime Lopes da Mota Oliveira1 Antônio Carlos de Lima Rocha1, Débora Cynamon Kligerman1, Jaime Lopes da Mota Oliveira1 DOI: 10.1590/0103-11042019S312 ABSTRACT This work carried out an integrative review of scientific articles indexed between 2007 and 2017 in different databases on treatment and reuse of effluents from the antibiotic industry. Thirty-one articles were found and only four addressed effluent reuse, and one used a full-scale treatment system. Most of these studies were conducted in Asia, with emphasis on China. In Brazil, which is one of the largest producers and consumers of drugs in the world, this type of research is still incipient. The most commonly found processes were oxidative advanced processes that showed greater efficiency in removing antibiotics, but may generate by-products, which might pose an even greater risk depending on the substance formed. Biological processes must first be acclimated to antibiotics in order not to be impacted, however, the release of these resistant microorganisms into the water bodies also presents an environmental risk. Biological integrated membrane systems were also very efficient, but attention should be given to the risk in the final destination of these membranes that were able to retain those compounds. Overall, further studies on this approach are needed to reduce the risks of developing multi-resistant microorganisms in the environment. KEYWORDS Drug industry. Anti-bacterial agents. Industrial effluent treatment. Drug resistance, bacterial. Wastewater use. KEYWORDS Drug industry. Anti-bacterial agents. Industrial effluent treatment. Drug resistance, bacterial. Wastewater use. RESUMO Este trabalho realizou uma revisão integrativa de artigos científicos indexados entre 2007 e 2017 em diferentes bases de dados sobre o tratamento e o reúso de efluentes provenien- tes da indústria de antibióticos. Foram encontrados 31 artigos, sendo que somente 4 abordaram o reúso de efluente, e 1 utilizou um sistema de tratamento em escala real. A maior parte desses estudos foi realizado na Ásia, com destaque para a China. Observa-se que, no Brasil, que é um dos grandes produtores e consumidores de fármacos do mundo, esse tipo de pesquisa ainda é in- cipiente. Os processos mais encontrados foram os oxidativos avançados que mostraram maior eficiência na remoção de antibióticos, mas podem gerar subprodutos, o que pode representar um risco ainda maior dependendo da substância formada. Os processos biológicos devem ser primei- ramente aclimatados aos antibióticos para não serem impactados, entretanto, a liberação desses SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 Rocha ACL, Kligerman DC, Oliveira JLM 166 micro-organismos resistentes no corpo receptor também apresenta um risco ambiental. Os siste- mas integrados de membranas ao biológico também foram bem eficientes, mas atenta-se ao risco na destinação final dessas membranas que foram capazes de reter esses compostos. No geral, são necessários mais estudos sobre essa abordagem para reduzir os riscos no desenvolvimento de micro-organismos multirresistentes no meio ambiente. micro-organismos resistentes no corpo receptor também apresenta um risco ambiental. Os siste- mas integrados de membranas ao biológico também foram bem eficientes, mas atenta-se ao risco na destinação final dessas membranas que foram capazes de reter esses compostos. No geral, são necessários mais estudos sobre essa abordagem para reduzir os riscos no desenvolvimento de micro-organismos multirresistentes no meio ambiente. PALAVRAS-CHAVE Indústria farmacêutica. Antibacterianos. Tratamento de efluentes indus- triais. Farmacorresistência bacteriana. Uso de águas residuárias. PALAVRAS-CHAVE Indústria farmacêutica. Antibacterianos. Tratamento de efluentes indus- triais. Farmacorresistência bacteriana. Uso de águas residuárias. Introduction and treatment lies with the generator. This effluent may be discharged into the public collection system or into receiving bodies as defined during the environmental licensing of the company3. It is noteworthy that effluents from the pharmaceutical industry may pose risks to human health4-6. The increased con- sumption of pharmaceutical products and the incentive to expand the pharmaceutical park lead to a greater generation of effluents with drug waste7-9. In the modern concept, sanitation includes water supply, waste management (solid, liquid and/or gaseous), urban drainage, rational use of the soil and control of communicable diseases. Such actions are essential for the physical, mental and social well-being of the population1. Thus, the non-accomplishment of any of these actions may result in damage to human and environmental health. An impor- tant component with direct effects on human health is the collection and treatment of efflu- ents. Brazil has alarming values in relation to this component: in 2017, the Country collected 74% of the sewage generated and treated only 46%, which totals 4.2 billion m³ of untreated domestic sewage being discarded in the dif- ferent water sources2. The Brazilian pharmaceutical industry presents financial profits superior to the other producing activities, having only the auto- mobile industry ahead. The pharmaceutical sector obtained, in 2018, a 6.1% production growth compared to 201710. Such a produc- tive result is due to the fact that Brazil has recorded an accumulated increase of 10.8% in the consumption of pharmaceutical prod- ucts between 2014 e 2018, with an estimated growth of 15% to 18% until 2023. This brings the Country from the seventh placing on the world drug market in 2018 to the fifth in the list of the 20 largest drug users in the world as shown in chart 111. The collection and treatment of domestic effluents is the responsibility of the public authorities, and may be granted to a private company, but supervised by the control agen- cies. A very critical type of effluent and that deserves attention is the industrial one, in which the responsibility for its collection SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 167 Overview of research on the treatment and reuse of effluents from the antibiotics industry rt 1. Introduction 165-180, DEZ 2019 Rocha ACL, Kligerman DC, Oliveira JLM 168 seas and coastal areas that may have been developed by the presence of antibiotics from effluent disposal18-20. of modern humanity. It is noteworthy that, among human activities, the industrial sector is considered the third largest con- sumer of water resources in the world30. The effluent of the pharmaceutical industry is normally composed of wastewater from the production lines, waste from production and leftovers of substances removed from machin- ery and equipment. It has specific character- istics that can vary depending on the products that are manufactured by the industrial unit. The effluent generated by the production of antibiotics may carry residues of the active substance. In this way, this residue must first go through a process of inactivation or elimina- tion of this active principle to prevent it from being carried into the environment. In the pharmaceutical industry, water must have purity levels appropriate to its multiple uses. Water used in production, for example, must be treated at the level of purified water; while other support sectors, such as utility units, control labo- ratories, and research and development sectors, require different levels of water quality30,31. Therefore, the importance of water resources management in the phar- maceutical industry is highlighted. Effective integrated water management (water consumed and effluent generated) in the industry can reduce the risk of en- vironmental pollution and contribute to lower water consumption. One way to reduce water consumption in the industrial sector is to reuse treated effluents. While the petrochemical and food sectors are already doing a lot of work on this topic, little is known about this practice in the pharmaceutical industry30. On the other hand, the pharmaceutical industry must be aware of the risk of this reuse to public health and productive activity. Different processes may be used for the treatment of pharmaceutical industrial ef- fluents. Among the most widespread and least expensive technologies, biological processes stand out22, but the efficiency of these pro- cesses in degrading these compounds may be questionable. In some cases, it is necessary to use other processes combined with biologi- cal processes, such as physical and chemical processes. Chemical and physicochemical processes are more efficient than biological processes in the removal of refractory sub- stances such as drugs23. Introduction Ranking of the 20 countries with higher spending on medicines in relation to the United States of America showing il’s growth estimate from 2013 to 2023 Chart 1. Ranking of the 20 countries with higher spending on medicines in relation to the United States of America showing Brazil’s growth estimate from 2013 to 2023 2013 2018 2023 Ranking Country Ranking Country Ranking Country 1º USA 1º USA 1º USA 2º China 2º China 2º China 3º Japan 3º Japan 3º Japan 4º Germany 4º Germany 4º Germany 5º France 5º France 5º Brazil 6º Italy 6º Italy 6º Italy 7º United Kingdom 7º Brazil 7º France 8º Brazil 8º United Kingdom 8º United Kingdom 9º Spain 9º Spain 9º India 10º Canada 10º Canada 10º Spain 11º India 11º India 11º Canada 12º South Korea 12º South Korea 12º Russia 13º Australia 13º Russia 13º South Korea 14º Russia 14º Australia 14º Turkey 15º Mexico 15º Mexico 15º Argentina 16º Saudi Arabia 16º Poland 16º Australia 17º Poland 17º Turkey 17º Mexico 18º Belgium 18º Saudi Arabia 18º Poland 19º Netherlands 19º Argentina 19º Saudi Arabia 20º Switzerland 20º Belgium 20º Vietnam Source: IQVIA, 201911. Source: IQVIA, 201911. Among the most commonly used drugs, antibiotics stand out. According to the World Health Organization (WHO) report, in 2016 alone the Brazilian population consumed 22.75 daily doses of antibiotics per thousand inhabitants12. The WHO also points out that among the 65 countries in the world, Brazil is ranked 19th in the antibiotic consumption ranking and the highest among the countries of the Americas12. developing some resistance to antibiotics, requiring the development of new sub- stances or their combined use13-15. There is also a warning about the development of multi-resistant microorganisms that has become a concern today. As a result, WHO has been searching for new antibiotics to prevent populations from dying again from previously controlled infections16. If nothing is done, it is estimated that 10 million people could die from a disease caused by multi-resistant microorganisms by 205017. Environmental studies have been raising evidence of increased antimicrobial resis- tance by microorganisms found in rivers, The high consumption of antibiotics is due to the fact that this medicine has given the population a new hope against various diseases. However, microorganisms can adapt to environmental conditions, SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. Introduction Activated charcoal adsorption, acid or alkaline hydrolysis and ozone oxidation are examples of promising processes in the removal and/or degradation of drug wastes24. Reuse modalities may be adopted accord- ing to the need of the industry. Indirect reuse is the most common, in which treated ef- fluents are thrown into rivers and lakes and, subsequently, captured, treated and used. Direct reuse is related to the use of effluents from one particular activity in another with lower requirements. In general, it is very common the reuse of industrial effluents in towers of cooling, gardening, washing of patios and sidewalks, water for toilets and in the activities of civil construction. For more specific uses of the industry, as in the manufacturing process itself, such ef- fluents shall undergo treatment appropriate to the quality required for their application, observing the risks that this practice may have32-34. Therefore, effluent treatment The level of treatment to be adopted by the industry is associated with the legal release requirements and the desired quality standard of the treated effluent24. In rela- tion to the Brazilian regulation regarding the launch of drugs in the environment, there is a need for its suitability, mainly due to new evidences of effects on wild biota and men25,26. However, the countries of the European Union already have standardiza- tion for the subject27-29. Another important component in sanita- tion actions is water supply. Scarcity and low water availability represent the challenges SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 169 Overview of research on the treatment and reuse of effluents from the antibiotics industry the term ‘reuse’ was excluded as it did not aggregate any results. efficiency should be evaluated for different types of reuse, especially those containing drug wastes35. After the recovery of the publications, a screening process was performed from the reading of the title and the summary, adopting as inclusion criterion the presence of data on ‘treatment of industrial effluents from the production of antibiotics’. Despite the use of the filters offered by the data- bases, books, opinion articles and patent were observed, which were also removed after detailed reading of the title and the summary. A second evaluation was per- formed with the help of the Zotero Program Standalone® for the exclusion of duplicate references. Introduction At the end of this screening, the articles were fully read, being excluded, restricted or paid access articles or, still, that presented divergent information to the object of this study. In view of this, this article brings an in- tegrative literature review on the studies performed in the treatment of effluents in the pharmaceutical industry of antibiotics and on the possibility of reuse of this treated effluent. Methodology This article was elaborated through an in- tegrative literature review36-38. Inclusion criteria for this review were indexed articles written in Portuguese, English or Spanish, from 2007 to 2017, retrieved from the search for descriptors and keywords in the Science Direct, Scopus, Web of Science, PubMed and Virtual Health Library (VHL) data- bases through the website of the Journal of the Coordination for the Improvement of Higher Education Personnel (Capes). Results From the search criteria and the filters used in the databases, 400 references were cataloged as shown in figure 1. By reading the title and the summary of these articles, 341 articles that had divergent themes from the one proposed in this paper were excluded. Of the remaining 59, 15 were in duplicate and were excluded (figure 1B), thus, leaving 44 articles. Finally, with the full reading, only 31 articles were selected (figure 1C). From the selection of descriptors and keywords, the retrieval of references in the indexed databases was performed by two forms of search. The first was composed of the terms ‘antibiotic’ AND ‘wastewater treatment’ OR ‘industrial effluents treatment’ OR ‘industrial waste’ AND ‘recycling’ OR ‘reuse’ which are vocabularies controlled by DeCS and MeSH. These indices were used in all search bases except PubMed, where SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 170 Rocha ACL, Kligerman DC, Oliveira JLM Figure 1. Path used for integrative review. (A) shows how was the recovery of articles based on descriptors using the VHL (Virtual Health Library) bases, Science Direct, Scopus, Web of Science and PubMed; (B) screening from the reading of the title and the abstract of the articles obtained as well as the removal of duplicate articles; (C) shows the exclusion from the full reading of the articles selected ((“antibiotic”) AND (wastewater treatment” OR “industrial efuent treatment” OR “industrial waste”) AND (“recycling” OR “reuse”)) ((“antibiotic”) AND (wastewater treatment” OR “industrial efuent treatment” OR “industrial waste”) AND (“recycling”)) BVS 26 Science Direct 33 Scopus Recovery of references 134 Where: Title, abstract and Subject Period: 2007 to 2017 Language: English Type of Document: Original articles or of Review Where: Title, abstract and Keywords Period: 2007 to 2017 Language: English Type of Document: Original articles or of Review Where: Title, abstract and Keywords Period: 2007 to 2017 Language: English Type of Document: Original articles or of Review Web of Science 29 Where: Topic Period: 2007 to 2017 Language: Portuguese, English and Spanish Type of Document: Original articles or of Review Pub Med 178 Where: All Fields When: 2007 to 2017 Language: Portuguese, English and spanish Type of Document: Original articles or of Review A Screening: Reading of Titles and Abstracts Full Reading of Articles 44 Articles Duplicate removal 07 19 12 21 03 26 12 166 25 109 BVS Science Direct Scopus 44 Articles Web of Science Pub Med B C Included 31 Excluded 13 Source: Rocha39. Of the works found on this subject, most were carried out in China (17 articles), fol- lowed by India (4), France and Iran (2 each); and Brazil, Spain, Finland, Israel, Malaysia and Portugal (1 each), as shown in figure 2. From the categorization of effluent treatment processes, 9 articles that addressed biological processes were found: where 4 referred to strictly biological processes – 3 for activated sludge and 1 for anaerobic reactors; 4 inte- grated the biological with the physical using membranes; and 1 associated the biological system with the chemical by biocatalysis, as shown in figure 3. SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 ((“antibiotic”) AND (wastewater treatment” OR “industrial efuent treatment” OR “industrial waste”) AND (“recycling” OR “reuse”)) ((“antibiotic”) AND (was treatment” OR “industrial treatment” OR “indus waste”) AND (“recycli BVS 26 Science Direct 33 Scopus Recovery of references 134 Where: Title, abstract and Subject Period: 2007 to 2017 Language: English Type of Document: Original articles or of Review Where: Title, abstract and Keywords Period: 2007 to 2017 Language: English Type of Document: Original articles or of Review Where: Title, abstract and Keywords Period: 2007 to 2017 Language: English Type of Document: Original articles or of Review Web of Science 29 Where: Topic Period: 2007 to 2017 Language: Portuguese, English and Spanish Type of Document: Original articles or of Review Pub Med 178 Where: All Field When: 2007 to Languag Portugu and spa Type of Origina or of Re A A Full Reading of Articles 44 Articles C Included 31 Excluded 13 Screening: Reading of Titles and Abstracts Duplicate removal 07 19 12 21 03 26 12 166 25 109 BVS Science Direct Scopus 44 Articles Web of Science Pub Med B Source: Rocha39. C Source: Rocha39. Of the works found on this subject, most were carried out in China (17 articles), fol- lowed by India (4), France and Iran (2 each); and Brazil, Spain, Finland, Israel, Malaysia and Portugal (1 each), as shown in figure 2. From the categorization of effluent treatment processes, 9 articles that addressed biological processes were found: where 4 referred to strictly biological processes – 3 for activated sludge and 1 for anaerobic reactors; 4 inte- grated the biological with the physical using membranes; and 1 associated the biological system with the chemical by biocatalysis, as shown in figure 3. SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 171 Overview of research on the treatment and reuse of effluents from the antibiotics industry Figure 2. Geographical distribution of the 31 articles that were reviewed after the application of all screening tools Figure 2. Geographical distribution of the 31 articles that were reviewed after the application of all screening tools Finland 1 Article Spain 1 Article France 2 Articles Iran 2 Articles Portugal 1 Article Brazil 1 Article Israel 1 Article China 17 Articles Malaysia 1 Article India 4 Articles Source: Rocha39. Figure 3. SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 Categorization according to the types of treatment systems applied in the article found by process: biological, physical, chemical, oxidative advanced and oxidative Finland 1 Article Source: Rocha39. Figure 3. Categorization according to the types of treatment systems applied in the article found by process: biological, physical, chemical, oxidative advanced and oxidative Figure 3. Categorization according to the types of treatment systems applied in the article found by process: biologica physical, chemical, oxidative advanced and oxidative Categorization of approaches 31 articles Biological Process Activated Sludge Method 03 Adsorption Method 08 Osmosis Method 01 Membrane Bioreactor Method (MBR) 02 Separation Method with Ionic Solution 01 Biocatalysis Method 01 Adsorption and Foaming Agent Separation Method 01 Anaerobic Stabilization Method in Fluidized Bed 01 Membrane Bioreactor (MBR) and Nanoﬁltration Method 02 Potassium Ferrate Association with Ultrasound Method 01 Photo Method – Fenton 04 Photocatalysis Method 04 Fenton Method 02 Photo Method - Fenton Associated with IBR 01 Fenton Method Associated with Coagulation and Sedimentation 01 Fenton Method with H2O2 and O3 Association 01 Polychromatic UV Oxidation Method 01 Physical Process Chemical Processes Advanced Oxidative Processes Oxidative Processes Source: Rocha39. Source: Rocha39. SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 Rocha ACL, Kligerman DC, Oliveira JLM 172 Of the other works, only 1 article used exclusively physical process by reverse osmosis, 10 articles used physicochemi- cal processes, 8 of adsorption, 1 of sepa- ration by foaming agent and adsorption and 1 of separation using ionic solution. Of the articles found that used the oxidative processes, 1 was by the potassium ferrate oxidation method with ultrasound and 1 by the polychromatic UV oxidation. Among the 13 articles that tested Advanced Oxidative Processes (POA) in their work, 11 were ex- clusive to POA, of these, 2 by the Fenton method, 4 by photo-Fenton, 1 by hydrogen peroxide and 4 by photocatalysis. In addi- tion, 2 articles used POA associated with other types of processes: one used Fenton associated with coagulation/sedimentation and the other applied photo-Fenton with immobilized biomass biological reactor. and the United States have shown the im- portance of monitoring these residues26. In addition, rules and procedures reinforce the care these nations require to prevent their water resources from containing these types of waste43,44. However, even on this subject, there is little research in Brazil26. SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 The POA, followed by membranes isolated or integrated to biological systems (Membrane Bioreactors – MBR), were the most cited in the articles found. The purely biological pro- cesses appeared in four works found. In fact, the most widely used treatment processes, whether for domestic or industrial wastewa- ter, are biological ones. While the activated sludge system serves most of the Brazilian population, anaerobic reactors are the most used in the industrial sector. However, there is doubt as to whether the biological process is effective in removing/degrading antibiot- ics. Mechanisms for the elimination/removal of effluent antibiotic residues by biological processes may occur by incorporation of the compounds into the sludge biomass (sorption) or by biodegradation45. It is also noteworthy that only four studies were obtained that addressed the possibility of reuse of these effluents. All of these studies found used POA as an alternative treatment. Abassi et al.46 observed that the presence of ampicillin, amoxicillin and ciprofloxacin wastes in the industrial effluent caused a re- duction in the concentration of microorgan- isms in the activated sludge process. Discussion Most of the work on treating effluent from antibiotic production occurred in China. This result may express a marked economic expan- sion after its commercial opening, besides the need to adapt to patent laws that allowed the reproduction of various industrialized products, including drugs. This economic evolution covered the pharmaceutical sector, mainly in the field of active pharmaceutical production40-42. Other data that draw at- tention refer to the fact that 25 works were developed in Asia, 5 in Europe and only 1 in the Americas. Considering the level of pro- duction and consumption of drugs that has been reflected worldwide, there should be more studies on this approach, especially in Brazil and the United States. On the other hand, studies on the occurrence of drugs in surface water and drinking water in Europe The acclimatization or prolonged exposure of antibiotic residues to microorganisms may increase their resistance to these compounds, improving their ability to degrade these sub- stances, as compared to Industrial and Sanitary Effluent Treatment Plants containing fluoro- quinolone-based antibiotics45-47. However, this procedure can be a risk, as these micro- organisms present in this sludge can carry this resistance to the environment. It is also em- phasized that this acclimatization is not a guar- antee that these microorganisms can develop the biodegradability of these compounds. Saravanne and Sundararaman48 used MBR with acclimatized biomass to treat cephalexin residues, and this resulted in the generation of 7 7-amino-3-desacetoxy cephalosporanic acid SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 173 Overview of research on the treatment and reuse of effluents from the antibiotics industry The phase transfer of the contaminant can also be observed in oxidation processes using medium support, such as in the application of photocatalysis in support medium for the removal of oxytetracycline63, tetracycline64 and deoxytetracycline65. This process did not present the complete elimination of antibiot- ics, however, photocatalysis without medium support has degraded 100% of antibiotic resi- dues65. On the other hand, the protolithic oxi- dation requiring only the light source was not efficient for sulfamethoxazole, oxytetracycline and ciprofloxacina66 elimination. and phenylacetic acid metabolites. Another alternative tested in the MBR study was the addition of enzymes to favor the degradation of antibiotics. However, besides the high cost, this procedure may not be effective for a broad spectrum of antibiotics45. Discussion The degradation of drug wastes by biologi- cal processes presents better performance when associated with other processes, such as oxidative or physical. Sistori et al.49 had an effective reduction in the concentration of na- lidixic acid of the effluent using Immobilized Biomass Reactors associated with photo-Fen- ton. However, this process generated metabo- lites compounds with higher toxic potential and could be a risk to aquatic ecosystems. Wang et al.50 used MBR with nanofiltration and observed a decrease in the concentra- tion of spiramycin, being related to the mass transfer from the liquid phase to solid phase retained in the membrane, which also occurs in the osmosis treatment system. Despite the good efficiency observed by studies using membrane processes, a transfer of these wastes is observed, so, there is a demand for the proper treatment of these membranes50-52. Oxidative processes may also be integrated with physical or chemical agents, such as the study by Zhang et al.67 who tested potassium ferrate with ultrasound for the degradation of sulfadiazine, sulfamazine and sulfamethoxa- zole. However, this process was not as effec- tive for the complete elimination of these compounds. Many authors cite that POA are the most promising for drug waste degradation because they are capable of generating free radicals (●OH) that have high oxidative potential. However, many of the articles found in this search show that these processes and their associations did not achieve a 100% reduc- tion in antibiotic concentration. The following methods were found: Fenton for removal of amoxicillin56, cefpirome, latamofex, aztreo- nam, cefoperazone, cefatrizine, propylene glycol, ceftazidime68 and sulfamethoxazole69; photo-Fenton associated with an iron and cerium heterogeneous catalyst in the presence of hydrogen peroxide under the irradiation of ultraviolet light for the removal of tetracycline in effluent70; and electro-photo-Fenton, elec- tro-Fenton ultraviolet irradiation to degrade tetracycline71. It is also worth mentioning the application of Fenton associated with hydro- gen peroxide and ozone that contributed to the complete degradation of sulfamethoxazole, sulfadimethoxine, sulfamethazine, erythromy- cin and tylosin tartrate72. All these processes, in addition to not completing the complete min- eralization of antibiotics, cite the formation of Extraction processes adopting nonpolar or foam phases that favor the removal by liquid-liquid processes were also found in this search. Almeida et al.53 analyzed the po- tential for removal of fluoroquinolones from the addition of an ionic solution to the effluent. Discussion The difference in polarity between these solu- tions (extractor and effluent) caused antibiotic residues to be removed from the effluent to the extractor solution. Kou et al.54 used flo- tation coupled with adsorption separation for streptomycin removal (recovery). Other authors have also tested adsorption processes for the removal of antibiotic residues such as dioxicycline55; amoxicillin56; gatifloxacin57; cephalexin, cephradine58, sulfamethoxazole59; tetracycline60,61 and ciprofloxacin62. The en- vironmental and financial cost of transferring phase contaminants can be extremely high due to the need for other processes that effectively degrade waste in these new matrices. SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 Rocha ACL, Kligerman DC, Oliveira JLM 174 treated by the pharmaceutical industry. This fact may be related to the sanitary require- ments for water use in this industrial typol- ogy75. However, other studies that apply the reuse of effluents in various industrial types are observed, being at the discretion of each manufacturing unit the best way to apply the reuse practice. Effluent reuse is reported in industrial activities that, due to quality criteria, are not incorporated into the product, and effluent reuse is more frequent after specific treatment in cooling towers and boilers76-79. other compounds (degradation by-products) that may have toxic characteristics or express similar resistance to the original compound73. Of the 31 articles recovered, only 2 pre- sented techniques capable of completely elimi- nating the antibiotic residues tested56,73, 27 demonstrated some level of removal and 2 were not efficient. The studies that carried out the association between the biological system by sludge activated with other processes presented good efficiency in the removal of antibiotics, which shows that this path can be quite promising. The plastic recycling industry is able to reuse 100% of the generated effluents. These effluents from the prewash and washing of plastics and sanitary sewage are directed to the wastewater treatment system, and the treated effluent is added to rainwater precipitated over the industry, which feeds the production process80. Likewise, the textile industry is also able to reuse its effluents from the ef- fluent aftertreatment by advanced oxidative process81. Effluent reuse is already practiced even in the food production industry82-84. Discussion In addition to industrial processes, other seg- ments also already perform the practice of reuse, as the International Airport of Rio de Janeiro, in which the treated effluents are di- rected to the cooling towers, reducing monthly consumption of up to 33 thousand m³ of water. Therefore, further studies are needed on the reuse of effluents mainly treated in the phar- maceutical industry. One noteworthy observation is that only one article was performed in a full-scale treat- ment system, demonstrating that some of the methods tested by other studies may undergo considerable variations when submitted to larger scales. From the analysis of these results, there is a need for urgent standardization measures for the release of effluents containing antibiotics. In addition, greater encouragement should be given to research and implementation of effective methods to reduce the risk of en- vironmental contamination by these wastes in alignment with the emergency situation presented by WHO. It is noteworthy that, in 2015, the WHO called on all countries to draw up National Action Plans to Contain Antimicrobial Resistance; and, in 2018, Brazil published its plan with the collaboration of the Ministry of Health and Welfare. It is emphasized that this plan is close to the current environmen- tal demand of the implementation of reverse logistics for antibiotics, not addressing the problem of liquid effluent from industrial pharmaceutical production74. Final considerations From this review, it can be concluded that, in these ten years of research, few studies have been found on treatment of effluents from the antibiotic industry, especially in countries with higher drug consumption and production. While China is emerging in research on this subject, Brazil, which rises in the ranking of pharmaceutical industries, generated little knowledge with this approach. Of the 31 references retrieved by the inte- grative review, only 4 evaluated the possibility of reuse of treated effluents from antibiotic production. These studies used as a treatment method the Fenton/coagulation/sedimenta- tion68 association, photolysis66 and MBR with nanofiltration50,51. Therefore, there are a small number of works seeking the reuse of effluents It is noteworthy that less than 5% of the SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. ESPECIAL 3, P. 165-180, DEZ 2019 175 Overview of research on the treatment and reuse of effluents from the antibiotics industry discarded as solid residues in the membranes. This same observation should be made to the processes of adsorption by activated carbon. studies found were developed in full-scale systems, which is a negative result since only in this way it is possible to understand the behavior of antibiotics in treatment systems already installed. This review shows that research on the use of effluents in pharmaceutical industries is still in its infancy; especially on antibiotic producers. The 21st century will be marked by the challenge of water scarcity; and one of the ways to alleviate this problem is the reuse of water and effluent in producing activities such as industrial. The most tested processes for antibiotic removal/degradation were the advanced oxidative processes (35%) followed by the physical-chemical processes (32%) and, lastly, the strictly biological ones (13%). It is notewor- thy that many works used combined processes, including obtaining good efficiencies in rela- tion to the isolated systems. The systems with the worst performance were strictly biological, while the advanced oxidative processes were the best. Few works used systems strictly of membranes (3%), but, yes, with the combina- tion to the biological system (MBR) in which they had good removal efficiencies. Finally, in Brazil, there are no legal limits for the release of effluents with antibiotic residues in the environment. However, this practice poses a risk to environmental health and may favor the development of multi-re- sistant bacteria. Orcid (Open Researcher and Contributor ID). Collaborators One of the problems for biological systems is the impact of these pollutants on sludge microfauna, because, once acclimatized, they can carry this resistance to antibiotics to the environment. When well adjusted, advanced oxidative processes promote good efficiency in the degradation of antibiotics, however, they can generate unknown byproducts or even with higher toxic potential than the original pollutant. Thus, an assessment of the toxicity and possible effects of these by-products on aquatic ecosystems is required. On the other hand, membrane systems are efficient in re- moving these compounds that are retained in the membrane; with this, a transfer of these pollutants is observed, which are then Rock ACL (0000-0001-7243-7832) contrib- uted substantially to the design, planning, data analysis and interpretation, drafting and critical review of the content, as well as the approval of the final version of the manu- script. Kligerman DC (0000-0002-7455-7931)* contributed substantially to the design, plan- ning, analysis and interpretation of the data and revision of the manuscript. Oliveira JLM (0000-0002-0361-3457)* contributed sub- stantially to the design, planning, analysis and interpretation of the data and revision of the manuscript. s SAÚDE DEBATE \| RIO DE JANEIRO, V. 43, N. 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https://openalex.org/W1514118629	https://hal.archives-ouvertes.fr/hal-01204198/file/2015_Draeger_BMC%20Plant%20Biology_1.pdf	English	null	Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth	BMC plant biology	2,015	cc-by	9,734	Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth Christian Draeger, Tohnyui Ndinyanka Fabrice, Emilie Gineau, Gregory Mouille, Benjamin M. Kuhn, Isabel Moller, Marie-Therese Abdou, Beat Frey, Markus Pauly, Antony Bacic, et al. Christian Draeger, Tohnyui Ndinyanka Fabrice, Emilie Gineau, Gregory Mouille, Benjamin M. Kuhn, Isabel Moller, Marie-Therese Abdou, Beat Frey, Markus Pauly, Antony Bacic, et al. To cite this version: Christian Draeger, Tohnyui Ndinyanka Fabrice, Emilie Gineau, Gregory Mouille, Benjamin M. Kuhn, et al.. Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth. BMC Plant Biology, 2015, 15, 10.1186/s12870-015-0548-8. hal-01204198 RESEARCH ARTICLE Open Access © 2015 Draeger et al. 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. HAL Id: hal-01204198 https://hal.science/hal-01204198v1 Submitted on 28 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Draeger et al. BMC Plant Biology (2015) 15:155 DOI 10.1186/s12870-015-0548-8 Abstract Background: Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. Results: The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. Conclusions: LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development. Keywords: Arabidopsis, Cell wall development, Cell wall structures, LRR-extensin, LRX Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth Christian Draeger1,8, Tohnyui Ndinyanka Fabrice1, Emilie Gineau3,4, Grégory Mouille3, Benjamin M. Kuhn2, Isabel Moller5,7, Marie-Therese Abdou1, Beat Frey6, Markus Pauly2, Antony Bacic5 and Christoph Ringli1* * Correspondence: chringli@botinst.uzh.ch 1Institute of Plant Biology, University of Zürich, Zollikerstr. 107, Zürich 8008, Switzerland Full list of author information is available at the end of the article Results LRR-extensin (LRX) proteins are extracellular proteins found in different plant species [17, 18]. LRX proteins contain an N-terminal LRR domain with 10 complete LRRs, and a C-terminal extensin domain with (Ser- Hyp4)-containing repetitive motifs typical for this class of HRGPs [19, 20]. While the LRR domain is well con- served among LRX proteins, the extensin domain is vari- able [17]. Many structural cell wall proteins, including extensins, are able to covalently crosslink in the cell wall and thereby influence mechanical properties [21–23]. For LRX1 of Arabidopsis thaliana, it was shown that the extensin domain crosslinks to cell wall components to anchor/insolubilize the protein and possibly properly position the LRR domain in the extracellular matrix [24, 25]. Expression of a truncated version of LRX1 lacking the extensin domain induces a dominant negative effect, resulting in an lrx1-like defect in root hair development. This suggests that the LRR domain binds and titrates out the interaction partner of the endogenous LRX1 and, hence, implies a binding partner of the LRR domain [24], the nature of which remains elusive. LRX3, LRX4, and LRX5 are conserved LRR-extensin proteins LRX3, LRX4, and LRX5 proteins show the typical structure of leucine-rich repeat (LRR)-extensins (LRXs), including an N-terminal LRR domain and a C-terminal extensin (HRGP) domain. The N-terminal LRR domain is preceded by a domain that is variable amongst LRX proteins, while a Cys- rich hinge region separates the LRR and the extensin do- mains (Fig. 1a). An alignment of the LRR domains under- lines the high degree of similarity between the three LRX proteins with 95% (LRX3 compared to LRX4), 86% (LRX3 compared to LRX5), and 85% (LRX4 compared to LRX5) (Additional file 1). As expected, the proteins encoded by the paralogous genes LRX3 and LRX4 [17] show the highest level of identity. By contrast, the extensin domain is much more variable amongst the three proteins, with a length of only 90 amino acids in LRX4 versus 367 and 445 amino acids in LRX3 and LRX5, respectively. The last 30 amino acid residues in all three proteins, however, show 75% iden- tity (Additional file 1). The extensin domain contains the Ser-Hyp4 motif characteristic for this HRGP family. The tremendous length differences amongst the extensin do- mains are not necessarily indicative of functional differ- ences since the extensin domains of LRX1 and LRX2 are functionally interchangeable despite limited homology [26]. Background structures by integrating new material, modifying linkages, and adjusting the composition of the cell wall [1]. Interfer- ing with either the biosynthesis or post deposition modifi- cation of cell wall components causes changes in cell wall structure/organisation, which can influence cell growth processes, as exemplified by a number of cell wall mutants of Arabidopsis thaliana that show changes in cell morph- ology (for review, see [2]). Characteristic features of plants are cell walls that not only protect each individual cell but also strongly influence plant development. While cell growth is driven by turgor pressure, expansion of the primary cell wall, which resists turgor pressure, is the rate limiting step. The primary cell wall is composed of complex interlinked networks, mainly composed of cellulose, non-cellulosic and pectic polysac- charides such as xyloglucan and heteroxylans, and also structural cell wall proteins. Cell wall expansion therefore requires a constant rearrangement of these supramolecular Plants have developed a sophisticated system to monitor cell wall formation in order to respond to changes in cell wall composition [2–5]. Genetic approaches have led to the identification of a number of receptor-like transmembrane proteins that perceive signals from the cell wall and trans- duce them to the cytoplasm. Wall-associated kinases have a cytoplasmic kinase domain and an extracellular domain * Correspondence: chringli@botinst.uzh.ch 1Institute of Plant Biology, University of Zürich, Zollikerstr. 107, Zürich 8008, Switzerland Full list of author information is available at the end of the article Draeger et al. BMC Plant Biology (2015) 15:155 Page 2 of 11 changes in cell wall structures and composition induced by mutations in LRX genes. Root hairs present a sub- optimal cell type for these analyses due to their low abundance and atypical (for plant cells) tip growing mode of expansion. LRX3, LRX4, and LRX5, in contrast, are all expressed in roots and shoots, and cluster together in a phylogenetic tree based on amino acid homology of the encoded LRR domain. LRX3 and LRX4 are paralogs and share an almost identical expression profile [17]. To- gether, it can be hypothesized that these three LRX pro- teins have similar functions in overlapping tissues. that can bind pectin, and serve functions in pathogen re- sponse as well as regulation of osmotic pressure [6–9]. Background THESEUS1 encodes a CrRLK-like receptor kinase that monitors changes in the cell wall caused by a reduced cellu- lose content and induces secondary changes in the cell wall such as lignin deposition [10, 11]. Leucine-rich repeat (LRR) proteins have been identi- fied in a number of systems to act as interaction part- ners in either a signaling cascade or as modulators of protein activity. Polygalacturonase inhibitors (PGIPs) specifically bind polygalacturonases, thereby inhibit their enzymatic function, and thus influence the turnover of pectic polysaccharides [12]. Pathogen-recognizing dis- ease resistance proteins often contain an LRR domain which is thought to interact with a pathogen-induced molecule [13]. On the other hand, the brassinosteroid and auxin binding proteins BRI and TIR1 harbour LRR domains [14, 15], revealing the broad chemical spectrum of potential binding partners of LRR domains. Out of over 200 LRR-receptor proteins encoded in Arabidopsis, some have been shown to be important for cell wall de- velopmental processes. FEI1 and FEI2 influence cell wall function and cell growth properties by affecting cell wall composition [16]. In this work, the characterization of LRX3, LRX4, and LRX5 is described. Single, double, and triple mutants established using T-DNA insertion mutants reveal syner- gistic mutant phenotypes, suggesting a similar function of these three LRX genes. The changes in cell wall com- position observed in the mutant lines compared to the wild type indicate that LRX proteins indeed have a func- tion in cell wall formation. The lack of these proteins in- duces not only changes in cell wall structures but also strongly affects plant development implying that LRX proteins have an important function during cell (wall) development. Results The Arabidopsis genome codes for a family of eleven LRX-type proteins. LRX1 and LRX2 are paralogous genes and are predominantly expressed in root hairs where they function synergistically during cell development. lrx1 lrx2 double mutants show a severe defect in root hair cell wall structures and growth, suggesting a role of LRX1 and LRX2 in cell wall formation [24, 26]. To bet- ter understand the function of LRX proteins during cell wall development, it is desirable to characterize the Mutations in LRX genes cause alterations in plant development Mutations in LRX genes cause alterations in plant development Mutations in LRX genes cause alterations in plant development The importance of LRX3, LRX4, and LRX5 was investigated by the identification and analysis of T-DNA insertion Draeger et al. BMC Plant Biology (2015) 15:155 Page 3 of 11 Fig. 1 Protein structure of LRX3, LRX4, and LRX5 and gene expression. a Leucine-rich repeat extensin (LRX) proteins consist of a signal peptide for protein export (black), followed by a variable domain, nine complete leucine-rich repeats (LRR, grey), a Cys-rich hinge region (dotted), and a C-terminal extensin domain (dark grey) that show the typical Ser-Hyp4 motifs of hydroxyproline-rich glycoproteins. Numbers indicate amino acid positions, arrowheads the positions corresponding to the T-DNA insertions in the identified mutants. b RT-PCR on total RNA extracted from wild-type and mutant seedlings with gene-specific primers for each of the three LRX genes and ACTIN2 as an internal control Fig. 1 Protein structure of LRX3, LRX4, and LRX5 and gene expression. a Leucine-rich repeat extensin (LRX) proteins consist of a signal peptide for protein export (black), followed by a variable domain, nine complete leucine-rich repeats (LRR, grey), a Cys-rich hinge region (dotted), and a C-terminal extensin domain (dark grey) that show the typical Ser-Hyp4 motifs of hydroxyproline-rich glycoproteins. Numbers indicate amino acid positions, arrowheads the positions corresponding to the T-DNA insertions in the identified mutants. b RT-PCR on total RNA extracted from wild-type and mutant seedlings with gene-specific primers for each of the three LRX genes and ACTIN2 as an internal control of the triple mutant suggests a synergistic activity of all three LRX genes during plant development. This syner- gistic effect was particularly detectable in root develop- ment where the lrx3 lrx4 double mutant and the lrx5 single mutant showed a root length similar to the wild type whereas the lrx3 lrx4 lrx5 triple mutant roots were much shorter (Fig. 2a). The increasingly severe impair- ment of plant growth was observed also at older stages of development of the lrx3 lrx4, and lrx3 lrx4 lrx5 mu- tants compared to the wild type, exemplified by the re- duction in rosette leave size (Fig. 2b; Additional file 2B). Scanning electron microscopic analysis revealed a sinu- ous cotyledon surface in the double mutant that was not observed in the wild type. This effect was even more pronounced in the triple mutant lines, where crater-like structures developed with occasional cracks in the epi- dermal cell layer (Fig. 2c). Mutations in LRX genes cause alterations in plant development The same phenotype in the epidermal surface was also observed in rosette leaves (Additional file 2C). It is important to note that the lrx5 single mutant was comparable to the wild type at all stages of development and only in combination with the lrx3 lrx4 mutations conferred an exaggerated growth de- fect indicative of a synergistic interaction between the LRX genes. mutants. One mutant line was identified for each of the LRX genes (Salk_094400, GABI_017A08, and Salk_013968 for LRX3, LRX4, and LRX5, respectively) in which the cod- ing region is interrupted (Fig. 1a). Since LRX3 and LRX4 are highly homologous paralogs [17] and thus likely to be functionally redundant, mutant analysis mainly focused on the lrx3 lrx4 double mutant and the lrx3 lrx4 lrx5 triple mutant. Homozygous mutants were obtained in all cases. To assess whether the mutant lines indeed fail to express the mutated genes, RT-PCR was performed on total RNA isolated from homozygous mutant seedlings. No RT-PCR product was obtained from any of the three mutant loci (Fig. 1b), confirming that the T-DNA insertions block gene expression and that the mutants most likely represent knock-out alleles. Since all three genes are expressed in seedlings and mature plants [17], both developmental stages were ana- lyzed to identify potential mutant phenotypes. At the seedling stage, both double and triple mutant seedlings had smaller cotyledons. This phenotype was more pro- nounced in the triple mutant, where cotyledons were also less epinastic than in either the wild type or the lrx3 lrx4 double mutant (Fig. 2a). Later during seedling de- velopment, the difference between the wild type, lrx3 lrx4 double, and lrx3 lrx4 lrx5 triple mutant was readily detectable in terms of size and shape, excluding that the mutants just grow slower than the wild type (Additional file 2A). Since the lrx5 single mutant development was comparable to the wild type, the aggravated phenotype Complementation of the lrx mutants To confirm that the aberrant growth phenotypes ob- served in the lrx3 lrx4 and lrx3 lrx4 lrx5 mutants were induced by the T-DNA insertions, complementation Page 4 of 11 Draeger et al. BMC Plant Biology (2015) 15:155 Fig. 2 Mutations in LRX genes cause aberrant plant growth. a Cotyledons of 7 days-old seedlings are gradually smaller in the lrx3 lrx4 double mutant and the lrx3 lrx4 lrx5 triple mutant compared to the wild type (Col). Roots of the same seedlings are significantly shorter in the lrx3 lrx4 lrx5 triple mutant. Error bars shown in the graph represent standard errors. Significance was tested by T-test; n ≥14, : P < 0.05. b Mature plants of double and triple mutants reveal a reduction in growth compared to wild-type plants, whereas the lrx5 single mutant grows comparable to the wild type. c Sinuous structures were observed in double and triple mutant cotyledons with occasional cracks (arrows) in the epidermis of the triple mutant. Bars: A = 0.5 mm; B = 10 mm; C = 100 μm Fig. 2 Mutations in LRX genes cause aberrant plant growth. a Cotyledons of 7 days-old seedlings are gradually smaller in the lrx3 lrx4 double mutant and the lrx3 lrx4 lrx5 triple mutant compared to the wild type (Col). Roots of the same seedlings are significantly shorter in the lrx3 lrx4 lrx5 triple mutant. Error bars shown in the graph represent standard errors. Significance was tested by T-test; n ≥14, : P < 0.05. b Mature plants of d bl d i l l d i i h d ild l h h l 5 i l bl h Fig. 2 Mutations in LRX genes cause aberrant plant growth. a Cotyledons of 7 days-old seedlings are gradually smaller in the lrx3 lrx4 double mutant and the lrx3 lrx4 lrx5 triple mutant compared to the wild type (Col). Roots of the same seedlings are significantly shorter in the lrx3 lrx4 lrx5 triple mutant. Error bars shown in the graph represent standard errors. Significance was tested by T-test; n ≥14, : P < 0.05. b Mature plants of double and triple mutants reveal a reduction in growth compared to wild-type plants, whereas the lrx5 single mutant grows comparable to the wild type. c Sinuous structures were observed in double and triple mutant cotyledons with occasional cracks (arrows) in the epidermis of the triple mutant. Complementation of the lrx mutants Bars: A = 0.5 mm; B = 10 mm; C = 100 μm experiments were performed. To this end, it was attempted to clone the wild-type copies of LRX3, LRX4, and LRX5. All three DNA constructs, however, were unstable in several different E. coli strains grown under different conditions. Most frequently, the extensin-coding region was truncated and could therefore not be propagated. Previous experiments had shown that extensin domains are interchangeable between different LRX proteins. The lrx1 root hair mutant phenotype can be complemented with chimeric proteins containing the extensin domain of either LRX1 or LRX2 [26]. Therefore, the genomic clones of LRX3 and LRX4 containing the sequences of the promoter to the Cys-rich hinge domain-coding sequence (located in between the LRR- and extensin domains; Fig. 1a) were amplified and fused with the extensin-coding sequence and the terminator of LRX1. The resulting chimeric complementation constructs were referred to as LRX3:LRR3-EXT1 and LRX4:LRR4-EXT1, respectively. For Draeger et al. BMC Plant Biology (2015) 15:155 Page 5 of 11 reduced content in Rha and Gal in both mutants whereas Man increased in the triple mutant compared to the wild type. Similar to Man, lignification is in- creased in the triple mutant (Fig. 4a). In extracts of stem tissue, most sugars showed a significant (T-test, P < 0.05) increase in the double but not the triple mutant com- pared to the wild type (Fig. 4b). Even though the in- crease in lignin content in the mutants did not fulfill our criteria of significance, we still visualized lignification on stem sections. In agreement with the observed reduction in overall plant growth, cross-sections of the mutants were smaller in diameter than the wild type, which was also reflected by smaller cell sizes, and this effect was stronger in the triple mutant (Table 1). Compared to the wild type, the lignin-staining ring of (inter-)fascicular cells of the mutants covered a larger fraction of the en- tire stem section (Fig. 4c). To quantify this observation, the thickness of the interfascicular lignified ring was measured and compared to the full diameter. This con- firmed that the lignified ring in relation to the stem diameter is larger in the mutants than in the wild type (Table 1). LRX5, this alternative cloning approach was also unsuc- cessful and thus, we abandoned complementation of the lrx5 mutation. LRX3:LRR3-EXT1 and LRX4:LRR4-EXT1 were success- fully transformed into the lrx3 lrx4 double mutant. CoMPP analysis of lrx mutants Based on previous work, LRX proteins are assumed to have a role in cell wall development [24, 26]. Therefore, the composition and structures of wild-type and mutant cell walls were analyzed using various approaches. In a first step, the neutral sugar monosaccharide composition of cell wall material of the mutants and the wild type was performed. Analysis of rosette leaf alcohol insoluble residue (AIR) revealed a significantly (T-test, P < 0.05) y To analyze the polysaccharide composition of the cell wall of the double and the triple mutant in more detail, a high-throughput characterization using Comprehen- sive Microarray Polymer Profiling (CoMPP; [27]) on ros- ette leaf and stem extracts was performed. Cell walls were sequentially extracted with diamino-cyclo-hexane- tetra-acetic acid (CDTA) and NaOH, to extract pectins and non-cellulosic polysaccharides, respectively, spotted on microarrays, and the intensity of the spot signals was quantified. CoMPP revealed significant changes in the relative intensities of JIM13 (recognizes arabinogalactan- proteins [AGPs]), JIM20 (recognizes extensins), LM11 (rec- ognizes xylan), and LM13 (recognizes arabinan) (Table 2, Additional file 3). Samples extracted with CDTA and la- beled with JIM13, JIM20, and LM13 revealed a reduc- tion by up to one third in epitope detection levels. Leaf samples extracted with NaOH showed a reduced LM11 epitope intensity close to one half in the double mutant and a somewhat less in the triple mutant (Fig. 4a). LM13 (arabinan) epitopes were reduced up to 74% in lrx3 lrx4 plants and up to 90% in lrx3 lrx4 lrx5 mutants. The differential labelling observed between the wild type and the mutants does not systematically fit with the altered monosaccharide composition observed (Fig. 4)—for example in the case of some epitopes such as LM13 arabinan, this antibody will bind to only a sub- set of all arabinose-containing polymers in the cell walls. Together, these analyses confirm that the muta- tions lead to a modification of cell wall compositions in different organs. Fig. 3 Mutations in LRX genes cause dwarfism. As exemplified by the lrx3 and lrx4 mutations, single mutants grow similar to the wild type whereas the lrx3 lrx4 double mutant shows severely reduced growth that is alleviated by the LRX3 and LRX4 complementation constructs. Bar = 10 mm. Fig. 3 Mutations in LRX genes cause dwarfism. Complementation of the lrx mutants By contrast, transformation of the lrx3 lrx4 lrx5 triple mu- tant resulted in poor seed set from which no transgenic seed could be recovered. Hence, complementation ex- periments were limited to the double mutant. Trans- genic lines segregating in the T2 generation 3:1 for the selectable marker were analyzed using plant height as the parameter for complementation. While the lrx3 lrx4 double mutant develops shorter inflorescences compared to the wild type or either of the single mutants, comple- mentation with either LRX3:LRR3-EXT1 or LRX4:LRR4- EXT1 resulted in alleviation of the reduced plant height phenotype (Fig. 3), suggesting that the chimeric comple- mentation constructs encode functional proteins and that the double mutant phenotype is indeed caused by the mutations in LRX3 and LRX4. CoMPP analysis of lrx mutants As exemplified by the lrx3 and lrx4 mutations, single mutants grow similar to the wild type whereas the lrx3 lrx4 double mutant shows severely reduced growth that is alleviated by the LRX3 and LRX4 complementation constructs. Bar = 10 mm. Draeger et al. BMC Plant Biology (2015) 15:155 Page 6 of 11 Fig. 4 Cell wall composition analysis. Cell wall material was extracted from rosette leaves a and stem tissue b and monosaccharides were quantified. Error bars shown in the graph represent standard errors. Significance was tested by T-test; n = 3, P < 0.05. c The degree of lignification was visualized in stem cross section using Wiesner staining. Bar = 500 μm. Fig. 4 Cell wall composition analysis. Cell wall material was extracted from rosette leaves a and stem tissue b and monosaccharides were quantified. Error bars shown in the graph represent standard errors. Significance was tested by T-test; n = 3, P < 0.05. c The degree of lignification was visualized in stem cross section using Wiesner staining. Bar = 500 μm. Fig. 4 Cell wall composition analysis. Cell wall material was extracted from rosette leaves a and stem tissue b and monosaccharides were quantified. Error bars shown in the graph represent standard errors. Significance was tested by T-test; n = 3, P < 0.05. c The degree of lignification was visualized in stem cross section using Wiesner staining. Bar = 500 μm. LRX3, LRX4, and LRX5 support this proposed function since mutations in these genes lead to aberrant cell wall composition. The alterations ultimately impact cell growth properties, resulting in generally reduced plant growth. The repetitive nature of the extensin-coding region of LRX genes made the cloning strategy difficult, in particu- lar of LRX5, which we were unable to clone and utilize for complementation experiments. For LRX3 and LRX4, chimeric constructs containing the LRX1 extensin-coding region had to be used. This resulted in functional proteins, consistent with the previous domain swap experiments, which showed that the extensin domains of LRX1 and LRX2 are interchangeable [26]. The complementation of the lrx3 lrx4 double mutant with LRX3 and LRX4 com- bined with the analysis of the double/triple mutants pro- vide good evidence that the mutant phenotypes observed Discussion Compensatory changes might depend on the severity of defects in cell wall for- mation, which would explain the observation of certain changes in stem tissue only in the lrx3 lrx4 double mu- tant but not the lrx3 lrx4 lrx5 triple mutant. Reducing cellulose content by the procuste1 or fei1 fei2 mutants causes an increase in lignin content [16, 33]. Other changes than a reduction in cellulose content may also induce increased lignification, as the level of cellulose is not affected by the lrx mutations, yet lignification is ele- vated in rosette leaves. In stems, the fraction of the lig- nin ring relative to the stem diameter is increased. This, however, is not reflected by an altered lignin content, suggesting that the distribution of lignin is modified. Xy- lan is associated with cellulose and mainly found in sec- ondary cell walls. In CoMPP experiments, the binding of the anti-xylan antibody LM11 is decreased in leaf tissue. The LM11 antibody recognizes β1-4 linked Xyl and also arabinoxylan [34]. The xylan backbone is frequently substituted with side chains of either glucuronic acid (GlcA) or methyl-GlcA, and a reduction in these substi- tutions changes xylan structure and can cause a dwarf growth phenotype [35]. Modified antibody labelling might be induced by either changes in the overall xylan structure or epitope accessibility. Future experiments with a variety of anti-xylan antibodies might provide bet- ter information on the potential changes in the xylan structure induced by the lrx mutations. are indeed caused by the mutations in these three LRX genes. Even though complementation of the lrx5 mutant was not possible due to technical difficulties, the synergis- tic effect of lrx5 and lrx3 lrx4 in the context of the lrx3 lrx4 lrx5 triple mutant implies comparable activities of the affected proteins. p Several different cell wall structures are modified in the lrx3 lrx4 double and lrx3 lrx4 lrx5 triple mutants. Also, these changes differ between rosette leaves and stem tissue. Rosette leaves show reduced levels of the monosaccharides Rha and Gal. In stem tissue, an in- crease in most neutral sugars was found for the lrx3 lrx4 double mutant. Since Rha and Gal are found in pectin, tissues with a high proportion of pectin-rich primary cell walls [28], such as rosette leaves, are more likely to re- veal a difference in pectin content. Discussion Leucine-rich repeat extensins (LRX) are extracellular proteins involved in cell wall assembly. This assumption was based on the previous characterization of mutants of the Arabidopsis LRX1 and LRX2 that revealed a defect in the cell wall ultrastructure [24, 26]. The analysis of Table 1 Quantification of stem diameter and degree of lignification in stem cross-sections Table 1 Quantification of stem diameter and degree of lignification in stem cross-sections Genotype Stem diameter [mm] Lignin ring [mm] % of ring to diameter col 1.81 0.11 6 lrx3 lrx4 1.34 * 0.10 7.5 * lrx3 lrx4 lrx5 1.01 * 0.09 * 9 * Sections at the base were used for analysis. Significance was tested by T-test; n ≥15, : significantly different from the wild type (col), P < 0.01 Sections at the base were used for analysis. Significance was tested by T-test; n ≥15, : significantly different from the wild type (col), P < 0.01 Draeger et al. BMC Plant Biology (2015) 15:155 Page 7 of 11 Table 2 Glycome profiling of selected cell wall polysaccharide epitopes performed on cell wall extracts Wash-ing Mutant Organ JIM13 (AGP) JIM20 (extensin) LM11 (xylan) LM13 (arabinan) CDTA wt stem 90.04 ± 8.62 85.59 ± 8.41 97.44 ± 2.55 lrx3 lrx4 stem 78.78 ± 0.48 75.97 ± 2.22 92.50 ± 2.05 lrx3 lrx4 lrx5 stem 66.16 ± 13.46 58.93 ± 6.98 66.14 ± 4.35 NaOH wt leaf 95.54 ± 3.86 lrx3 lrx4 leaf 40.20 ± 2.80 lrx3 lrx4 lrx5 leaf 58.68 ± 16.16 wt stem 49.91 ± 16.50 lrx3 lrx4 stem 13.41 ± 7.81 lrx3 lrx4 lrx5 stem 5.22 ± 5.17 Only significant differences between wild type and mutants are shown. Values are mean signal intensities ± standard deviation of three independent extracts. Detailed data is shown in the Additional file 3 ng of selected cell wall polysaccharide epitopes performed on cell wall extracts Organ JIM13 (AGP) JIM20 (extensin) LM11 (xylan) LM13 (arabinan provide an explanation for the observed divergence in structural adaptation of cell walls of leaf and stem tissues to mutations in lrx3, lrx4, and lrx5. Alternatively, changes in the composition of the cell wall can trigger a number of additional, secondary modifications as an at- tempt of the plant to directly compensate for alterations in the extracellular matrix [11]. Discussion Interestingly, LM13, which binds longer oligoarabinosides thought to be part of the pectin fraction, gives a strongly reduced signal in stems but not rosette leaf extracts. Since this is not reflected in a reduced Ara content, Ara-containing structures not detected by LM13 are probably also modified. In stems, the LM13 epitope is mostly abun- dant in epidermal walls and implicated in responses to mechanical stress [29, 30]. It is likely that the changes in cell wall structures affect the mechanical properties in the stem, which in turn also modifies the accumulation of LM13-detected pectic arabinan. In rosette leaf cell walls of the mutants, mannose is the only neutral sugar that shows an increase in abundance in lrx mutants. Mannose forms several types of mannan polymers which represent hemicelluloses that are particularly abundant in secondary cell walls and woody tissue [31, 32]. In the lrx3 lrx4 lrx5 triple mutant, however, a change in man- nan and lignin is not found in stem but only in rosette leaves. Whether the increase of mannose and lignin in rosette leaves is based on a common regulation remains to be shown. The anti-extensin antibody JIM20 also shows a reduc- tion in labelling in the mutants compared to the wild- type. The Arabidopsis genome contains over 20 genes that encode extensin proteins [36], a number of which are expressed in stem tissue. The contribution of each of Depending on the cellular function, tissues respond differently to changes in growth properties. This would Draeger et al. BMC Plant Biology (2015) 15:155 Page 8 of 11 these proteins to JIM20 labelling is not known. Hence, it is possible that the absence of the three LRX proteins causes a reduction in labelling since they all represent potential epitopes of JIM20. Extensins belong to the class of HRGPs and are known to influence physical properties of cell walls by their ability to form an insolu- bilized, covalent network in cell walls [37–39]. Root hair formation appears to be particularly sensitive to changes in this protein network since interfering with proline hydroxylation of extensins causes defects in root hair de- velopment [40]. RSH/EXT3 has been shown to be im- portant for cell wall formation during cytokinesis. With its propensity to self-aggregate, RSH/EXT3 has been proposed to form a scaffold for the deposition of new cell wall material [36, 41]. Conclusions The numerous changes in cell wall structures found in the lrx mutants reflects a general problem in the analysis of cell wall mutants: the difficulty to distinguish between primary effects of the mutations under investigation and the secondary effects induced by the plant with the aim of compensating the primary changes. In the cesa6 mutant procuste1, at least some of the compensatory changes in cell wall structures are induced by the receptor-kinase THESEUS1, which plays a role in cell wall integrity sensing. The the1 mutation blocks com- pensatory modifications, resulting in a partial suppres- sion of the procuste1 mutant phenotype [10]. It will be interesting to see whether some of the cell wall modifi- cations observed in the lrx mutants are also induced by THESEUS1 and thus whether the introduction of the the1 mutation in the lrx mutant background might help identifying the primary changes induces by the muta- tions in LRX genes. LRX3, LRX4, and LRX5 are homologous LRR-extensin proteins found in overlapping tissues and have similar functions. Mutations in these lrx genes induce growth phenotypes implying that LRX proteins are indispensable for proper plant development. Since LRX proteins localize to the cell wall, the identification of numerous changes in cell wall structures of lrx3, lrx4, and lrx5 mutant lines sup- port the assumption that LRX proteins are involved in cell wall development. Additional analyses are necessary to unravel the exact function of LRX proteins in this process. Discussion In the context of LRX pro- teins, however, the extensin domain appears to have an anchoring function. The insolubilization of LRX1 is not a default reaction but developmentally regulated. Also, overexpression of the extensin domain of LRX1 does not alleviate the lrx1 root hair formation defect, suggesting that LRX proteins are not primarily reinforcing the cell wall but the extensin domain might rather position the protein within the cell wall [24, 25]. component and thus serve in establishing a membrane-cell wall signaling continuum with the goal of controlling the deposition of new cell wall material. This would provide an alternative explanation for the pleiotropic changes in cell wall structure and composition observed in the lrx mutants. Alternatively, the LRR domain of LRX proteins might influence extracellular enzymatic activity. One class of LRR-containing proteins known to bind and inhibit cell wall degrading enzymes are endopolygalacturonase- inhibiting proteins (PGIPs) which are involved in plant de- velopment as well as pathogen defense [42, 43]. The LRR domain of these proteins contains eleven repeats, which is very similar to the ten and a half repeats of LRX proteins [17]. Whether this reflects a functional similarity is not yet clear, particularly since the LRR domains of LRXs and PGIPs share limited sequence homology. Hence, LRX pro- teins might be involved in either the regulation of enzym- atic activities in the cell wall or the recruitment of enzymes to the appropriate location in the cell wall. It can be specu- lated that the function of LRX proteins is related to pec- tins, since mutations modifying pectin structures were found to suppress the lrx1 root hair phenotype [44, 45]. Plant material and growth ld For experiments wild-type Arabidopsis thaliana (ecotype Col-0) and the mutants lrx3 (At4g13340; Salk_094400), lrx4 (At3g24480; GABI_017A08), and lrx5 (At4g18670; Salk_013968) were used. T-DNA insertions are located 752 bp, 1173 bp and 898 bp downstream of the start codon in lrx3, lrx4, and lrx5, respectively. The changes in cell wall structures observed in the lrx3 lrx4 and lrx3 lrx4 lrx5 mutants do not lead to a conclusive picture on the function of LRX proteins dur- ing cell wall formation. The interaction partner of LRX proteins would be very informative in this context but remains currently unknown. LRX proteins do not appear to serve as transmembrane receptor proteins since they lack a transmembrane domain. On the other hand, they are anchored in the cell wall via their extensin domain [24, 25]. The identification of the interacting partner(s) should provide a better picture on the orientation of the LRR domain of LRX proteins. Hypothetically, the LRR domain might interact with a membrane-bound Seeds were obtained from the NASC European Arabi- dopsis stock center. Seeds were surface sterilized (1% sodium hypochlorite, 0.03% TritonX-100) and stratified 2–4 days at 4 °C. Seeds were plated on 1/2 Murashige and Skoog medium (0.6% phytagel (Sigma), 2% sucrose) and grown in growth chambers with 16 h light, 8 h dark cycles at 22 °C. Pavement cell analysis and SEM analysis y y Imprinting of pavement cells was done following Horigu- chi et al. [49] and observed with a Zeiss AX10 microscope. Scanning Electron Microscope (SEM) analysis of cotyle- dons and rosette leaves was done as described by Baum- berger et al. [26]. Additional files Additional file 1: Alignment of LRX3, LRX4 and LRX5 proteins. Alignment of the full-length proteins by ClustalW revealed high Additional file 1: Alignment of LRX3, LRX4 and LRX5 proteins. Alignment of the full-length proteins by ClustalW revealed high homology in the N-terminal variable domain as well as the leucine-rich repeat domain (flanked by arrowheads). The extensin domains are very different between the proteins, except the last 35 amino acids which are again well conserved. The Pro residues encoded in the extensin domain are posttranslationally modified to Hyp. Identical, conserved, and similar positions in the alignment are indicated by asterisks, colons, or single dots, respectively. Accession numbers The accession numbers of the genes analyzed in this study are as follows: LRX3: At4g13340; LRX4: At3g24480; LRX5: At4g18670. DNA primers, constructs and plant transformation For the complementation constructs LRX3:LRR3 EXT1 and LRX4:LRR4 EXT1, the promoter and coding sequence Page 9 of 11 Draeger et al. BMC Plant Biology (2015) 15:155 described in [50]. Quantification was done with biological triplicates and the average values and standard errors are indicated in the graphs. encoding the hinge region of LRX3 and LRX4 were ampli- fied by PCR with the following primers to introduce a PstI site at the 3′ end coding for the hinge region: LRX3F: 5′-TC ATATGTGCTGTAGATGATTGGG-3′; LRX3R: 5′-CTG CAGTTTACCGGCGGACGAGACAAAAACG-3′; LRX4F: 5′-ACCCTCTAGCCTTTATATATTTATAG-3′; LRX4R: 5′-CTGCAGTCCACCGAAGGCCGTGACAAGAAAG-3′. The PCR fragments were cloned into the pSC PCR cloning vector (Stratagene) and correct clones were cut out by ApaI/PstI and cloned into a vector containing the LRX1 extensin-coding region and terminator [26] opened with the same enzymes. For plant transform- ation the constructs were cloned into pBART27 [46, 47] with NotI and plants were transformed and T1 transfor- mants selected as described [48]. T2 seeds obtained from the primary transformants were sterilized and sown on BASTA plates and resistant seedlings were transferred to soil for propagation. encoding the hinge region of LRX3 and LRX4 were ampli- fied by PCR with the following primers to introduce a PstI site at the 3′ end coding for the hinge region: LRX3F: 5′-TC ATATGTGCTGTAGATGATTGGG-3′; LRX3R: 5′-CTG CAGTTTACCGGCGGACGAGACAAAAACG-3′; LRX4F: 5′-ACCCTCTAGCCTTTATATATTTATAG-3′; LRX4R: 5′-CTGCAGTCCACCGAAGGCCGTGACAAGAAAG-3′. The PCR fragments were cloned into the pSC PCR cloning vector (Stratagene) and correct clones were cut out by ApaI/PstI and cloned into a vector containing the LRX1 extensin-coding region and terminator [26] opened with the same enzymes. For plant transform- ation the constructs were cloned into pBART27 [46, 47] with NotI and plants were transformed and T1 transfor- mants selected as described [48]. T2 seeds obtained from the primary transformants were sterilized and sown on BASTA plates and resistant seedlings were transferred to soil for propagation. Comprehensive microarray polymer profiling (CoMPP) and monosaccharide analysis Preparation of AIR (alcohol insoluble residue) was done as follows: Plant materials (stem and rosette leaves of adult plants) were ground in liquid nitrogen, washed with 80% EtOH three times and subsequently washed in 100% acetone, and CoMPP was performed as described in Moller et al. [27]. Quantification was done with bio- logical triplicates and the average values and standard deviations are indicated in the tables. Monosaccharide composition and linkage analysis of polysaccharides The analyses of polysaccharides were performed on an AIR prepared as follows. One hundred mg (FW) of ground rosette leaf or stem tissue of adult plants were washed twice in 4 volumes of absolute ethanol for 15 min, then rinsed twice in 4 volumes of acetone at room temperature for 10 min and left to dry under a fume hood overnight at room temperature. Neutral monosaccharide composition analysis was performed on 5 mg of dried AIR after hydrolysis in 2.5 M TFA for 1.5 h at 100 °C as described in [50]. To determine the cellulose content, the residual pellet obtained after the monosaccharide analysis was rinsed twice with ten vol- umes of water and hydrolysed with H2SO4 as described [51]. The released glucose was diluted 500 times and then quantified using an HPAEC-PAD chromatography as Lignin quantification and visualization One hundred mg (FW) of dry ground leaf were washed twice in 3 mL of water at 80 °C for 15 min, twice in 3 mL of ethanol at 80 °C for 15 min and once in 3 mL acetone at room temperature for 10 min and left to dry under a fume hood overnight at room temperature. The following protocol is adapted from Fukushima and Hat- field [52]. Lignin from the prepared cell wall residue was solubilized in 1 mL of acetyl bromide solution (acetyl bromide/acetic acid (1/3, V/V)) in a glass vial at 55 °C for 2.5 h under shaking. Samples was then let to cool down at room temperature and 1.2 mL of NaOH 2 M/ Acetic acid (9/50 V/V) was added in the vial. One hun- dred μL of this sample was transferred in 300 μL of 0.5 M hydroxylamine chlorhydrate and mixed with 1.4 mL of acetic acid. The A280 absorbance of the sam- ples was measured. Lignin content was calculated using the following formula: %lignin = 100 x (A280 x V reac- tion x V dilution) / (20 x V sample solution x m sample in mg). In stem cross-sections lignin was visualized by Wiesner staining. Quantification was done with bio- logical triplicates and the average values and standard errors are indicated in the graphs. References References 1. Cosgrove DJ. Growth of the plant cell wall. Nature Rev Mol Cell Biol. 2005;6(11):850–61. 2. Wolf S, Hematy K, Hoefte H. Growth control and cell wall signaling in plants. In: Merchant SS, editor. Annual Review of Plant Biology, Vol 63, vol. 63. 2012. p. 381–407. 3. Seifert GJ, Blaukopf C. Irritable walls: The plant extracellular matrix and signaling. Plant Physiol. 2010;153(2):467–78. 4. Ringli C. Monitoring the Outside: Cell Wall-Sensing Mechanisms. Plant Physiol. 2010;153(4):1445–52. 5. Tsang DL, Edmond C, Harrington JL, Nuehse TS. 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Tsang DL, Edmond C, Harrington JL, Nuehse TS. Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway. Plant Physiol. 2011;156(2):596–604. 30. Competing interests 14. He ZH, Wang ZY, Li JM, Zhu Q, Lamb C, Ronald P, et al. Perception of brassinosteroids by the extracellular domain of the receptor kinase BRI1. Science. 2000;288(5475):2360–3. The authors declare that they have no competing interests. Author details 1 Wolf S, Hematy K, Hoefte H. Growth control and cell wall signaling in plants. In: Merchant SS, editor. Annual Review of Plant Biology, Vol 63, vol. 63. 2012. p. 381–407. 3. Seifert GJ, Blaukopf C. Irritable walls: The plant extracellular matrix and signaling. Plant Physiol. 2010;153(2):467–78. 4. Ringli C. Monitoring the Outside: Cell Wall-Sensing Mechanisms. Plant Physiol. 2010;153(4):1445–52. 5. Tsang DL, Edmond C, Harrington JL, Nuehse TS. Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway. Plant Physiol. 2011;156(2):596–604. 6. Wagner TA, Kohorn BD. Wall-associated kinases are expressed throughout plant development and are required for cell expansion. Plant Cell. 2001;13(2):303–18. 7. Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G. A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci U S A. 2010;107(20):9452–7. 8. Kohorn BD, Kobayashi M, Johansen S, Riese J, Huang LF, Koch K, et al. An Arabidopsis cell wall-associated kinase required for invertase activity and cell growth. Plant J. 2006;46(2):307–16. Additional file 2: Growth defect phenotypes of the lrx mutants. (A) Additional file 2: Growth defect phenotypes of the lrx mutants. (A) While the wild type and lrx5 single mutant are comparable in size, the double and triple mutant seedlings grow gradually smaller, indicating a synergistic interaction between the lrx mutations. (B) Rosette leaves of the mutants also show a gradually reduced leave area compared to the wild type. Error bars shown in the graphs represent standard errors. Significance was tested by T-test; n=6, *: P<0.05. (C) Compared to the even surface of wild-type (Col) rosette leaves, double and triple mutants frequently developed uneven, sinuous surfaces with occasional cracks (arrows) in the epidermis of the triple mutant. Bars: A= 5 mm; B= 10 mm; C= 300 μm. Additional file 3: Glycome profiling of selected cell wall polysaccharide epitopes. Biological triplicates of cell wall extracts of the wild type, the lrx3 lrx4, and the lrx3 lrx4 lrx5 mutants were washed with Page 10 of 11 Draeger et al. BMC Plant Biology (2015) 15:155 9. He ZH, He DZ, Kohorn BD. Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response. Plant J. 1998;14(1):55–63. either CDTA or NaOH and epitope abundance was measured by quantification of antibody binding and expressed here as relative spot intensity (corrected to 100). The monoclonal antibodies used and bound epitopes are listed. The upper half of the table represents the mean values of technical triplicates of each extract, the lower half lists the standard deviation in the technical triplicates. either CDTA or NaOH and epitope abundance was measured by quantification of antibody binding and expressed here as relative spot intensity (corrected to 100). The monoclonal antibodies used and bound epitopes are listed. The upper half of the table represents the mean values of technical triplicates of each extract, the lower half lists the standard deviation in the technical triplicates. 10. 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Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis. Plant Cell. 2008;20(11):3065–79. 17. Baumberger N, Doesseger B, Guyot R, Diet A, Parsons RL, Clark MA, et al. Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice: a conserved family of cell wall proteins form a vegetative and a reproductive clade. Plant Physiol. 2003;131:1313–26. Funding 19. Cassab GI. Plant cell wall proteins. Annu Rev Plant Physiol Plant Mol Biol. 1998;49:281–309. g Australian Research Council through a grant to the ARC CoE in Plant Cell Walls to AB and IM; Swiss National Science Foundation Grants Nr. 122157 and 138472 and the Swiss SystemsX/MechanX initiative to CD, TNF, and CR. 20. Lamport DTA, Kieliszewski MJ, Chen Y, Cannon MC. Role of the extensin superfamily in primary cell wall architecture. Plant Physiol. 2011;156(1):11–9. 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BMC Plant Biology (2015) 15:155 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W2049636954	https://www.scielo.br/j/rbpm/a/MnT7kQxTZdvyyH3L89KwRrs/?lang=pt&format=pdf	Portuguese	null	Potencialidades fitoquímicas do melão (Cucumis melo L.) na região Noroeste do Rio Grande do Sul - Brasil	Revista brasileira de plantas medicinais	2,013	cc-by	3,114	Recebido para publicação em 30/08/2001 Aceito para publicação em 26/04/2012 Potencialidades fitoquímicas do melão (Cucumis melo L.) na região Noroeste do Rio Grande do Sul – Brasil. Key words: Cucumis melo L., phytochemical analysis, juice content. Potencialidades fitoquímicas do melão (Cucumis melo L.) na região Noroeste do Rio Grande do Sul – Brasil. MULLER, N.G. 1; FASOLO, D.2; PINTO, F.P 1;BERTÊ, R.1; MULLER, F.C. 1 1URI, Departamento de Ciências Biológicas, C. postal 464, 98802- 470, Santo Ângelo-Brasil; 2URI Depto. de Ciências da Saúde; nil@urisan.tche.br MULLER, N.G. 1; FASOLO, D.2; PINTO, F.P 1;BERTÊ, R.1; MULLER, F.C. 1 1URI, Departamento de Ciências Biológicas, C. postal 464, 98802- 470, Santo Ângelo-Brasil; 2URI Depto. de Ciências da Saúde; nil@urisan.tche.br RESUMO: O melão (Cucumis melo L.) é uma fruta muito apreciada por suas qualidades e sua produção vem crescendo e ganhando espaço no mercado nacional e internacional. Em regiões como o Noroeste do Rio Grande do Sul, destaca-se como uma nova alternativa de renda para vários agricultores. Neste contexto, o presente trabalho teve como objetivo analisar o potencial fitoquímico de alguns cultivares de melão da região Noroeste do Rio Grande do Sul. A análise fitoquímica utilizando como farmacógeno as folhas, foi realizada para a verificação da presença de metabólitos secundários, tais como: saponinas, cumarinas, cardiotônicos, cianogenéticos, alcalóides, taninos, antraquinonas, flavonoides, e óleos voláteis. Também foi avaliado o teor de suco a partir dos frutos. Dentre os cinco cultivares analisados, Gaúcho, Imperial, Hy Mark, Magelan, e Cantaloupe, o cultivar Gaucho apresentou a maior variedade em metabólitos secundários. Na avaliação do teor de suco a cultivar Magelan se destacou em comparação às demais cultivares testadas. Palavras-chave: Cucumis melo L., análise fitoquímica, teor de suco. ABSTRACT: Phytochemical potentialities of melon (Cucumis melo L.) in the northwest region of Rio Grande do Sul - Brazil. The melon (Cucumis melo L.) is a fruit highly appreciated for its qualities and its production has been growing and gaining space in the national and in the international market. In regions like the northwest of Rio Grande do Sul - Brazil, it stands out as a new income alternative for farmers. In this context, this study aimed to analyze the phytochemical potential of some melon cultivars in the northwest region of Rio Grande do Sul. The phytochemical analysis, using the leaves as pharmacogen, was performed to verify the presence of secondary metabolites such as saponins, coumarins, cardiac glycosides, cyanogenetic glicosides, alkaloids, tannins, anthraquinones, flavonoids and volatile oils. The juice content from the fruits was also evaluated. Among the five analyzed cultivars, Gaucho, Imperial, Hy Mark, Magelan and Cantaloupe, cultivar Gaucho had the greatest variety of secondary metabolites. In the assessment of the juice content, cultivar Magelan was superior compared to the remaining tested cultivars. 194 194 MATERIAL E MÉTODO MATERIAL E MÉTODO sendo atualmente uma das atividades que mais gera emprego e contribui com o superávit da balança comercial no Brasil (Oliveira et al., 2005). O período de realização deste trabalho contemplou os meses de janeiro e fevereiro de 2008, na Região Noroeste do Rio Grande do Sul. As diversas cultivares de melão foram coletadas em diferentes municípios da região, selecionados aleatoriamente. As amostras representam dois grupos: Cucumis melo L. var. cantaloupensis Naudin – cultivar do tipo Gaúcho e Cucumis melo L. var. reticulatus - cultivares do tipo Cantaloupe, Imperial, Magelan e Hy Mark, as quais foram coletadas na fase em que o fruto encontrava-se maduro, com a casca amarelada. Atualmente, a produção de frutíferas como o meloeiro, vem crescendo e ganhando espaço em várias regiões. No Noroeste do Rio Grande do Sul, por exemplo, diversos tipos de agricultores familiares encontram dificuldade de viabilizar suas unidades produtivas num prazo mais ou menos curto (Dudermel et al., 1993; Noronha, 2005). Nesse contexto, a fruticultura constitui uma alternativa com elevado potencial de retorno econômico para estas famílias e consequentemente para região, destacando a produção de melão por parte de alguns produtores. Após a coleta, as amostras foram depositadas em sacos de papel Kraft, devidamente etiquetadas e identificadas, e encaminhadas ao Laboratório de Botânica da Universidade Regional Integrada do Alto Uruguai e das Missões (URI), campus de Santo Ângelo, para identificação das espécies. As exsicatas foram preparadas com material in natura recém colhido, ainda antes da formação do fruto, quando em plena fase de floração. O ramo florido foi colocado em uma prensa com papel jornal por sete dias, efetuando-se a substituição por outro papel diariamente. Decorrido o período de desidratação, o material foi fixado com agulha e linha em cartolina, identificado e depositado no HPBR - Herbário Balduíno Rambo da Universidade Regional Integrada do Alto Uruguai e Missões (URI) – Campus Erechim. Neste as exsicatas foram registradas com os seguintes números 11.271, 11.272, 11.273, 11.307 e 11.308. Os frutos produzidos possuem forma, tamanho e coloração variáveis, contendo de 200 a 600 sementes, possuindo casca lisa ou levemente enrugada. O meloeiro é uma planta anual, herbácea, com número de hastes ou ramificações variáveis dependendo da cultivar, e considerado um fruto não- climatério; as folhas são alternadas e simples. Sua polinização é feita por insetos, principalmente por abelhas. O sistema radicular é bastante ramificado e pouco profundo. INTRODUÇÃO No que se refere às exportações, o Brasil é o sexto maior do mundo; foi introduzido pelos imigrantes europeus, inicialmente cultivado no Rio Grande do Sul (Grangeiro et al., 2002). Originário da região central da Ásia e da África, o melão (Cucumis melo L.) pertence à família das Cucurbitáceas. Fruto tropical de alto valor comercial, apreciado por suas características peculiares, tanto no mercado interno, quanto no externo, os melões nobres (“cantaloupes”) são os preferidos pelo mercado norte americano devido ao sabor, coloração, valor nutritivo (fonte de vitamina A) e uniformidade (Boas et al., 1998; Silva, 2001). O destaque da produção do melão na Região Nordeste do Brasil, acompanha o desenvolvimento da fruticultura que vem ao longo das últimas duas décadas substituindo as atividades agrícolas tradicionais no sertão, como o cultivo de feijão, algodão e milho. Isto se dá pela produção de frutos proporcionar maiores lucros, ocasionando outras oportunidades de crescimento para os produtores, O melão é uma das olerícolas mais populares do mundo, sendo a China o maior produtor responsável por 35% da produção mundial. Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.194-198, 2013. 195 MATERIAL E MÉTODO Sendo originário de regiões tropicais, as condições de clima quente e umidade relativa baixa favorecem o melhor desenvolvimento das plantas propiciando a alta produtividade e frutos de excelente qualidade (Grangeiro et al., 2002). Composto basicamente de água e sendo pouco calórico, o melão é uma fruta muito refrescante, podendo ser indicado para vários tipos de dietas alimentares, como para regimes de emagrecimento e para o restabelecimento de doentes em convalescença (Silva, 2001). Assim, a recomendação da introdução do consumo de frutas na dieta alimentar e na cura de determinadas doenças requer maiores pesquisas acerca de suas potencialidades fitoquímicas, fornecendo importantes parâmetros do material vegetal e auxiliando na descoberta de compostos bioativos em frutíferas. As folhas e frutos foram retirados em diversos exemplares situados ao longo da área de cultivo, objetivando formar uma amostragem de maior representatividade de cada cultivar (Bôas et al., 2002). As folhas foram submetidas à secagem à sombra em ambiente arejado e protegido de insetos. A análise fitoquímica através de métodos clássicos de caracterização (Tabela 1), utilizando como farmacógeno as folhas, e a avaliação do teor de suco, obtido a partir dos frutos, foram realizadas no Laboratório de Farmacognosia da Universidade Regional Integrada do Alto Uruguai e das Missões (URI) – Campus Santo Ângelo. Os componentes bioativos presentes na biodiversidade podem fornecer produtos de grande importância econômica. Dentre eles destacam-se os fitoterápicos e os fitofármacos originados dos recursos genéticos vegetais (Simões et al., 2007). Para determinar o teor de suco, os frutos foram medidos, pesados e posteriormente triturados em um liquidificador. Filtrou-se o suco em um tecido duplo de algodão, para obter-se apenas o líquido, livre da presença da polpa. Utilizou-se um fruto maduro e inteiro, selecionado aleatoriamente como amostra. O teor de suco foi obtido pela equação abaixo (Boâs et al., 2002): Os princípios ativos são compostos químicos naturalmente produzidos pelas plantas para protegê-las contra vírus, fungos e podem ser benéficas quando ingeridas pelos humanos através da alimentação de frutas e verduras, por exemplo, prevenindo determinadas doenças. Neste contexto, o presente trabalho teve por objetivo avaliar o potencial fitoquímico e o teor de suco de diferentes cultivares de melão produzido na região Noroeste do Rio Grande do Sul (Simões et al., 2007). Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.194-198, 2013. 196 Metabólitos Secundários Metodologia utilizada Referência Óleos voláteis Extração em aparelho de Clevenger. Farm. Bras. IV (1988) Flavonóides Reações: Shinoda, Hidróxidos Alcalinos e UV. MATERIAL E MÉTODO Simões et al. (2004) Alcalóides Reagentes: Mayer, Dragendorff e Bouchardat. Costa (2002) Heterosídeos Cardiotônicos Reações: Kedde, Baljet, Liebermann-Burchard e Keller-Kiliani. Macek et al. (1972) Cumarinas Fluorescência no UV Costa (2002) Saponinas Determinação do índice de espuma. Farm. Bras. IV (1988) Taninos Reações: Gelatina 2,5%, Acetato de Chumbo, Sulfato de Quinina e Acetato de Cobre. Simões et al. (2004) Antraquinonas Reação de Bornträger Costa (2000) Cianogenéticos Teste com papel picrossódico Costa (2000) TABELA 1. Métodos utilizados para a análise fitoquímica. Metabólitos Secundários Metodologia utilizada TABELA 1. Métodos utilizados para a análise fitoquímica. Simões et al. (2004) Costa (2000) Costa (2000) RESULTADOS E DISCUSSÃO reações com acetato de chumbo e acetato de cobre para todas as cultivares avaliadas (Tabela 2). Estes compostos estão relacionados principalmente por sua propriedade adstringente. Além disso, promovem efeitos antidiarréico, antisséptico, antimicrobiano e protegem as plantas contra ataques de herbívoros e microrganismos; são utilizados na tanagem (curtimento) do couro, de onde surgiu a nomenclatura desta classe de metabólitos (Monteiro, 2005). Dentre as cultivares testadas nenhuma indicou a presença de óleos voláteis. Não existem relatos na literatura quanto à presença ou ausência de óleos voláteis nas folhas do melão para que se pudesse fazer uma análise comparativa. Os óleos voláteis são misturas complexas de substâncias voláteis, geralmente odoríferas e líquidas, também chamados de óleos essências ou essências. Sua principal característica é a volatilidade (Simões et al., 2002). Alguns óleos essenciais são conhecidos por suas propriedades antioxidantes, sendo de grande aplicação na indústria farmacêutica, de cosméticos e na produção de alimentos (Yunes & Cechinel Filho, 2007). Em relação aos alcalóides, nenhuma das cultivares analisadas neste estudo apresentou positividade nos testes realizados (Tabela 2), possivelmente, por serem substâncias de distribuição restrita. As antraquinonas, além de serem consideradas corantes naturais também apresentam propriedades laxativas. Após realizada a reação de Bornträger, pôde-se observar a ausência destas substâncias em todo material amostrado (Tabela 3). Em relação à determinação de saponinas, as cultivares Gaúcho e Magelan apresentaram resultados positivos, destacando a maior quantidade destes compostos na cultivar Gaúcho (Tabela 2). As saponinas apresentam propriedades detergentes e emulsificantes, e também são empregadas na indústria farmacêutica por suas propriedades expectorante, diurética e antiinflamatória (Simões et al., 2002). Dentre as cultivares analisadas, a do tipo Cantaloupe apresentou uma positividade mais acentuada em relação à presença de compostos flavonoídicos (Tabela 3) em comparação com as outras, que também demonstraram possuir estes compostos. Os flavonóides são substâncias que apresentam inúmeras propriedades, dentre elas Amplamente encontrados no reino vegetal, os taninos apresentaram resultado positivo nas Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.194-198, 2013. Cultivar Saponinas Taninos Alcalóides Acetato de Sulfato de Gelatina Acetato de Mayer Bouchardat Dragendorf chumbo Quinina cobre Gaúcho ++ + - - + - - - Imperial - + - - + - - - Cantaloupe - + - - +- - - - Magelan + + - - +- - - - Hy Mark - + - - + - - - TABELA 2. Avaliação da presença de saponinas, taninos e alcalóides. RESULTADOS E DISCUSSÃO * (+) Presença do metabólito; (-) Ausência do metabólito; (+-) Presença reduzida do metabólito; (++) Presença acentuada do metabólito. TABELA 2. Avaliação da presença de saponinas, taninos e alcalóides. Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.194-198, 2013. 197 podemos citar a anti-inflamatória, antimicrobiana, antifúngica e antioxidante (Longhini, 2007). l Os compostos cianogenéticos e os cardiotônicos são encontrados com maior facilidade em plantas que apresentam potencial tóxico, sendo mais difíceis de serem encontrados em frutíferas justamente por sua toxicidade. Os resultados mostraram ausência destes compostos para todas as cultivares (Tabela 4) com apenas uma pequena exceção em relação ao teste de Liebermann- Burchard, o qual apresentou positividade reduzida para a cultivar Gaúcho. Entretanto, como a reação de Liebermann-Burchard é característica para detectar núcleos esteroidais e triterpênicos, este resultado possivelmente está relacionado à presença do núcleo das saponinas, detectadas anteriormente para esta cultivar. Os flavonóides são compostos amplamente distribuídos no reino vegetal, representados por diferentes classes de substâncias, sendo as flavonas um dos mais abundantes, possuindo atividades anticancerígenas, além de atuarem em processos reguladores do metabolismo. Encontram- se em abundância em várias espécies de frutas e hortaliças, sendo assim, a sua ingestão pode ser benéfica na proteção contra a incidência de diferentes tipos de câncer (Chitarra, 2005). O interesse econômico dos flavonóides também é decorrente da sua propriedade corante, pois são considerados os pigmentos naturais mais abundantes no reino vegetal, contribuindo no que se refere à atratividade dos alimentos utilizados na dieta (Simões et al., 2002). O maior teor de suco foi obtido com a cultivar Magelan, seguido da Imperial e Cantaloupe (Figura 1). O melão Gaúcho, com menor representatividade, ainda demonstrou proporcionar maior quantidade de suco que a cultivar Hy Mark, sendo este último mais indicado para o consumo in natura, em pedaços. Os melões Gaúcho e Imperial possuem entre os seus compostos fitoquímicos as cumarinas (Tabela 3), substâncias encontradas predominantemente em angiospermas. Possivelmente não foi possível identificar a presença de cumarinas em todas as cultivares, por estas serem pertencentes à família Cucurbitácea, a qual não possui destaque na literatura dentre as famílias que apresentam maior número de relatos de compostos cumarínicos. Estas substâncias, pelo seu odor, são muito utilizadas nas indústrias de domissanitários e cosméticos (Simões et al., 2002). Com base na análise fitoquímica, a cultivar que apresentou maior variedade em metabólitos secundários foi o Melão Gaúcho, com presença de saponinas, taninos, flavonóides, cumarinas e cardiotônicos. AGRADECIMENTOS NORONHA, A.D.H. O desenvolvimento da agricultura de Eugênio de Castro: Análise e recomendações de políticas. 2005. Dissertação (Mestrado – Programas de Pós-graduação da CAPES) UNIJUÍ, Ijuí. Ao CNPq pelo auxílio financeiro, a URI – Campus Santo Ângelo pela disponibilidade dos laboratórios e aos produtores pelo apoio na doação das cultivares. Ao CNPq pelo auxílio financeiro, a URI – Campus Santo Ângelo pela disponibilidade dos laboratórios e aos produtores pelo apoio na doação das cultivares. OLIVEIRA, A.M.; GURGEl, A.F.; LIMA, L.C.R. Diagnóstico do agronegócio do melão (Cucumis melo L.) produzido em Mossoró/RN: estudo de caso em três empresas produtoras. Holos. v.2, p.27-36, 2005. RESULTADOS E DISCUSSÃO V.28, n.5, p.892-96, 2005. RESULTADOS E DISCUSSÃO Neste contexto, deve-se levar em consideração alguns fatores que podem influenciar na composição qualitativa e quantitativa dos metabólitos secundários, como a colheita, o clima e a época da realização do experimento (Oliveira, Cultivar Antraquinonas Flavonóides Cumarinas Shinoda Hidróxidos Alcalinos UV Gaúcho - - +- +- + Imperial - - + +- ++ Cantaloupe - - + + - Magelan - - + +- - Hy Mark - - +- +- - TABELA 3. Avaliação da presença de antraquinonas, flavonóides e cumarinas. * (+) Presença do metabólito; (-) Ausência do metabólito; (+-) Presença reduzida do metabólito; (++) Presença acentuada do metabólito. TABELA 3. Avaliação da presença de antraquinonas, flavonóides e cumarinas. Cultivar Cianogenéticos Cardiotônicos Liebermann-Burchard Kedde Baljet Keller-Kiliani Gaúcho - +- - - - Imperial - - - - - Cantaloupe - - - - - Magelan - - - - - Hy Mark - - - - - TABELA 4. Avaliação da presença de cianogenéticos e cardiotônicos. *(-) Ausência do metabólito; (+-) Presença reduzida do metabólito. TABELA 4. Avaliação da presença de cianogenéticos e cardiotônicos. Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.194-198, 2013. Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.194-198, 2013. Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.194-198, 2013. 198 198 FIGURA 1. Comparação do teor de suco das diferentes cultivares analisadas. Teor de suco Cultivar Teor (%) Hy Mark Gaúcho Cantaloupe Imperial Magelan 85 80 75 70 65 60 FIGURA 1. Comparação do teor de suco das diferentes cultivares analisadas. 1998). Como exemplo desses fatores podemos citar perdas quantitativas como na redução no peso do alimento, por perda de água ou perda de matéria seca, manuseio inadequado e perdas acidentais. Já nas perdas qualitativas, verificamos os padrões de qualidade, incluindo perdas no sabor, deterioração na textura e aparência (Chitarra, 2005). 1988. 1320p. GRANJEIRO, L.C.; CECÍLIO FILHO, A.B.; BRAZ, L.T.; GONÇALVES, F.C. Cultivo de melão amarelo. Jaboticabal: Cecílio Filho, A. B., 2002. 30p. LONGHINI, R.; RAKSA, S.M.; OLIVEIRA, A.C.P.; SVIDZINSKI, T.E.; FRANCO, S.L. Obtenção de extratos de própolis sob diferentes condições e avaliação de sua atividade antifúngica. Revista Brasileira de Farmacognosia. V.17, n.3, p.388-95, 2007. Novos experimentos fitoquímicos são necessários em frutíferas, como o melão, por exemplo, pois poucas informações sobre determinadas espécies são encontradas na literatura. MACEK, K. Pharmaceutical Applications of Thin-Layer and Paper Chromatography. Amsterdam: Elsevier Publishing Company, 1972. 744p. MONTEIRO, J.M.; ALBUQUERQUE, U.P.; ARAÚJO, E.L. Taninos: uma abordagem da química a ecologia. Química Nova. REFERÊNCIAS OLIVEIRA, F. Farmacognosia. São Paulo: Editora Atheneu, 1998. 412p. BOAS, E.V.B.V.; CHITARRA, A.B.; MENEZES, J.B. Modificações dos componentes de parede celular do melão Orange Flesh submetido a tratamento pós-colheita com cálcio. Brazilian Archives of and Tecnology, v.41, n.4, p.467-74, 1998. SILVA, S.P. Frutas no Brasil. São Paulo: Nobel, 2001. 230p. SIMÕES, C.M.O.; SCHENKEL, E.P.; GOSMANN, G.; MELLO, J.C.P.; MENTZ, L.A.; PETROVICK, P.R. Farmacognosia: da planta ao medicamento. 4.ed. Porto Alegre/Florianópolis: UFRGS Editora/ Editora da UFSC, 2002. 833p. BÔAS, R.L.V.; MORAES, M.H.; ZANINI, J.R.; PAVANI, L.C.; CAMARGO, D.A.; DUENHAS, L.H. Teores de nutrientes na folha, qualidade do suco e massa seca de raízes de laranja “valência” em função da irrigação e fertirrigação. Revista Brasileira de Fruticultura, v.24, n.1, p.231-35, 2002. SIMÕES, C.M.O.; SCHENKEL, E.P.; GOSMANN, G.; MELLO, J.C.P.; MENTZ, L.A.; PETROVICK, P.R. Farmacognosia: da planta ao medicamento. 5ª. ed. Porto Alegre/ Florianópolis: UFRGS Editora/ Editora da UFSC, 2004. CHITARRA, M.I.F. Pós-colheita de frutas e hortaliças: fisiologia e manuseio. Lavras: UFLA, 2005. 785p. SIMÕES, C.M.O.; SCHENKEL, E.P.; GOSMANN, G.; MELLO, J.C.P.; MENTZ, L.A.; PETROVICK, P.R. Farmacognosia: da planta ao medicamento. 6ª. ed. Porto Alegre/ Florianópolis: UFRGS Editora/ Editora da UFSC, 2007. 1104p. COSTA, A.F. Farmacognosia. Lisboa: Fundação Calouste Gulbenkian, 2000. 992p. COSTA, A.F. Farmacognosia. Lisboa: Fundação Calouste Gulbenkian, 2002. 1117p. p DUDERMEL, T.; BASSO, D.; LIMA, A.P. A Política Agrícola e Diferenciação da Agricultura no Noroeste do RS. Ijuí: UNIJUÍ. 1993. YUNES, R.A.; CECHINEL, F.V. Química de produtos naturais, novos fármacos e a moderna farmacognosia. Itajaí: UNIVALI, 2007. 303p. FARMACOPÉIA Brasileira. 4. ed. São Paulo: Atheneu, Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.194-198, 2013.
https://openalex.org/W1945175822	https://link.springer.com/content/pdf/10.1007/s10584-015-1506-z.pdf	English	null	Toward a club of carbon markets	Climatic change	2,015	cc-by	8,630	Climatic Change (2017) 144:81–95 DOI 10.1007/s10584-015-1506-z Climatic Change (2017) 144:81–95 DOI 10.1007/s10584-015-1506-z Toward a club of carbon markets N. Keohane1 & A. Petsonk1 & A. Hanafi1 Received: 16 March 2015 /Accepted: 11 September 2015 /Published online: 15 October 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract This paper proposes the creation of a club of carbon markets (CCM), to promote deep reductions in greenhouse gas emissions by supporting the development, harmonization, and increased ambition of domestic carbon markets. To achieve its aims, the club would establish common or reciprocal standards for environmental market infrastructure, transpar- ency and environmental integrity; offer mutual recognition of members’ emissions units; allow participating jurisdictions to share experience and gain assistance in building institutional capacity; and promote domestic and cross-border investment in low-carbon development. Using a suite of incentives, including some from the trade arena, a club of carbon markets could serve as a powerful attractive nucleus for broadening the participation of jurisdictions in climate mitigation, much as the General Agreement on Tariffs and Trade (GATT) served as the nucleus for broadening trade in products and services. A carbon markets club could be launched under UNFCCC auspices, but a more promising avenue might be to pursue the creation of the CCM as a complement to but outside the UN talks. This article is part of a Special Issue on “Alternate Structures for Global Climate Action: Building Blocks Revisited” edited by Richard B. Stewart and Bryce Rudyk. 1 Environmental Defense Fund, New York, NY, USA * A. Hanafi ahanafi@edf.org N. Keohane nkeohane@edf.org A. Petsonk apetsonk@edf.org 1 We use the term “carbon markets” to refer generally to greenhouse gas emissions trading (or “cap and trade”) programs. 2 See, e.g., Van Asselt (2014), Victor (2007, 2013), Stewart et al. (2013), Oppenheimer and Petsonk (2004), and Pizer (2007). 3 For early focus on markets, see Stewart et al. (1996, UNCTAD/GDS/GFSB/Misc.1); UNCTAD (1996, UNCTAD/GDS/GFSB/Misc.2). For more recent discussion of linking emission trading systems, see, e.g., Bodansky et al. (2014); Green et al. (2014). 4 Prof. William Nordhaus has recently proposed a similar “carbon pricing club” approach for harmonized carbon taxes (Nordhaus 2015). 1 Introduction With the impacts of climate change already evident and atmospheric concentrations of greenhouse gases continuing to rise, the scientific and economic case for climate action is overwhelming (IPCC 2014). Because it is a global challenge, international cooperation to curb emissions is needed. Indeed, an extensive climate change “regime complex” has emerged comprising a wide range of initiatives and institutions aimed at reducing emissions (Keohane This article is part of a Special Issue on “Alternate Structures for Global Climate Action: Building Blocks Revisited” edited by Richard B. Stewart and Bryce Rudyk. * A. Hanafi ahanafi@edf.org N. Keohane nkeohane@edf.org A. Petsonk apetsonk@edf.org 82 Climatic Change (2017) 144:81–95 and Victor 2011), including the United Nations Framework Convention on Climate Change (UNFCCC), ratified by 196 countries; “minilateral” efforts such as the Climate and Clean Air Coalition to Reduce Short-Lived Climate Pollutants, launched in 2012, with 45 country partners and 56 non-state partners; and bilateral efforts such as the November 2014 United States-China announcement. Yet these efforts have yielded neither emission reductions nor climate finance at the scale and pace required. While a growing number of jurisdictions are implementing emission trading programs to cap and reduce greenhouse gas emissions, no part of the current regime complex has capitalized on these efforts to promote the broader expansion and coordination of such policies. It is a premise of this paper that mobilizing markets much more broadly, through well- designed emission trading programs that place durable, declining limits on emissions, will be critical to drive large-scale emissions reductions, spur the development and deployment of clean technologies, and channel necessary investment into low carbon development. To that end, we propose a club of carbon markets (CCM).1 The club’s goal would be to promote deep reductions in emissions by supporting the development, harmonization, and increased ambition of domestic carbon markets, including in fast-growing economies. Mem- bers would establish harmonized or reciprocal standards for monitoring and reporting emis- sions, accounting, transparency, and environmental integrity; create a shared market infrastructure to support the mutual recognition of emissions units; share experience and cooperate in building institutional capacity; and work jointly to further the ability of these markets to promote domestic and cross-border investment in low-carbon technologies. 1 Introduction Our proposal joins a rich and growing literature on plurilateral climate governance in a fragmented or “bottom-up” world.2 Within that literature, a number of commentators have contemplated linking carbon markets.3 What distinguishes our proposal is the focus on carbon markets as a means of boosting ambition and participation in climate action, and the close attention to the formation of a plurilateral club to foster them. 4 While club members would enjoy the intrinsic benefits of linkage, we see the CCM as much more than a set of linking arrangements. We envision a CCM in the mold of plurilateral institutions, in areas such as trade and security, which jurisdictions have joined because the benefits of membership justified the disciplines that membership imposed. Just as the General Agreement on Tariffs and Trade (GATT) served as the nucleus for broadening participation and ambition in the multilateral system of trade in products and services, the CCM could serve as a crucial and powerful attractive nucleus for broadening participation and ambition in climate mitigation. By establishing a stable institutional structure with harmonized rules and procedures, the CCM could provide greater incentives for climate policy durability over time and encourage greater investment in low-carbon development in participating jurisdictions. Through mutual provision of technical support and capacity-building, along with a ready-made market infra- structure, the CCM could reduce new members’ transactions costs in setting up emission trading programs. The reputational benefits of membership could provide incentives for newly Climatic Change (2017) 144:81–95 83 joining jurisdictions to be more ambitious in their mitigation efforts. And perhaps most importantly, since club members would have an incentive to ensure credible carbon “curren- cies” among participants and avoid “carbon inflation,” the CCM could generate mutual pressure for greater ambition in future commitments. joining jurisdictions to be more ambitious in their mitigation efforts. And perhaps most importantly, since club members would have an incentive to ensure credible carbon “curren- cies” among participants and avoid “carbon inflation,” the CCM could generate mutual pressure for greater ambition in future commitments. Although in principle a CCM could be formed inside the UNFCCC, creating the club outside the UN climate talks would likely be nimbler and more efficient, and could invite participation not only by national and regional jurisdictions, but also subnationals (e.g. states and provinces). 1 Introduction The relevant qualification would be a jurisdiction’s capacity to ensure com- pliance with its domestic emissions limits and required carbon market infrastructure. While a club approach appears to be a more promising avenue than the UNFCCC for plurilateral cooperation on carbon markets—and indeed for greater ambition on climate—it is not a panacea. Each carbon market must first learn to “walk” before it can “run,” let alone run a relay race; particularly in developing countries, the initial focus must be on building institutional capacity to implement markets successfully, rather than quickly attempting to link with other markets. Differences in jurisdiction-specific political, financial, regulatory, technical, and cultural contexts will complicate club formation. Despite these obstacles, however, it would surely be easier to reach agreement on the infrastructure and standards for a common carbon market among the subset of jurisdictions with a keen interest in carbon markets. With over 50 jurisdictions implementing or beginning to implement carbon markets, such a club’s time may have arrived. The next section briefly reviews the rationale for our focus on carbon markets. Section 3 presents key functions and provisions of a CCM. Section 4 considers participation and incentives: who would join, and why? Section 5 explores antecedents, drawing particularly on trade and security. Section 6 explores the potential relationship of the CCM to the UNFCCC. Section 7 concludes. 5 Keohane 2009. See also Wiener (1999). 2 Why carbon markets The economic rationale for market-based instruments, including emission trading, is well- established (Baumol and Oates 1988). Market-based instruments are cost-effective: in theory, they achieve a given level of emissions reduction at least cost. By putting a price on emissions (whether directly as a tax or fee, or indirectly through an emissions market), market-based instruments force individual actors to internalize the cost of their pollution, which would otherwise be external to their decision-making. These instruments give all polluters an economic incentive to reduce emissions, spurring innovators to come up with better, cheaper, faster ways of reducing emissions, and rewarding less and less pollution. And systems that allow firms to “bank” reductions for later use provide an incentive for early reductions. A standard result from economic theory is that if the benefits and costs of abatement were known and fixed, “price” instruments (such as carbon taxes) and “quantity” instruments (emission trading) would be essentially equivalent; a robust academic economics literature considers the theoretical case when abatement costs are uncertain (Weitzman 1974; Newell and Pizer 2003; but see Keohane 2009). Practical experience, meanwhile, suggests a number of advantages for emission trading over a carbon tax, especially in a “bottom-up” international context.5 Climate policy objectives are often framed in terms of limiting emissions, facilitating their translation into quantity 84 Climatic Change (2017) 144:81–95 targets (caps). Emission trading also provides a readier basis for cost-effective emissions reductions in a “bottom-up” world. Efficient allocation of abatement effort requires that the marginal abatement cost—that is, the cost of the last ton of emissions reduction—be equal across jurisdictions. (Otherwise, the same amount of abatement could be achieved at lower cost by shifting effort from high-marginal-cost jurisdictions to those with lower marginal costs.) Under emission trading, this “equimarginal” condition can be achieved directly, through linking: trading will equate the carbon price, and therefore the marginal abatement cost, across any two fully-integrated, linked markets (in the absence of exogenously introduced limits on trading). In contrast, equalizing the marginal abatement cost in a tax system requires explicit coordination—“top-down” multilateral negotiation—to set a common tax. Such coordination has been unavailing even in the economically integrated European Union. g y g p Emission trading systems have worked well in practice. Perhaps the best-studied market- based policy is the U.S. sulfur dioxide trading program created by the 1990 Clean Air Act Amendments (Ellerman et al. 6 See United States Environmental Protection Agency, “NOx Budget Trading Program,” http://www2.epa.gov/ airmarkets/nox-budget-trading-program. 7 The jurisdictions include the 28 Member States of the EU plus Norway, Lichtenstein, and Iceland; Switzerland; New Zealand; Kazakhstan; Korea; California; Quebec; the nine states in the Regional Greenhouse Gas Initiative; Tokyo; and seven Chinese cities and provinces with pilot emission trading systems in place. 8 See, e.g., Dudek et al. (1998). 9 See Pizer and Yates (2014). 2 Why carbon markets 2000); other successful applications include the phasedown of leaded gasoline in the United States (Newell and Rogers 2007), the control of nitrous oxides in the eastern U.S.,6 and the implementation of the Montreal Protocol on the Ozone Layer (Stavins 2003). More recently, the European Union’s Emission Trading System (EU-ETS) has achieved its targets and successfully reduced emissions, although a deep recession, a host of complemen- tary energy policies, and the lack of demand from other jurisdictions for offset credits has contributed to lower-than-expected allowance prices (Convery and Redmond 2013). Meanwhile, California’s carbon market has performed well since it began operating in 2013, and has established a linkage with Quebec. All told, more than 50 national and subnational jurisdictions with nearly one billion inhabitants have launched emission trading programs.7 The experience of the 1997 Kyoto Protocol is instructive—both for its failings and for its positive lessons. The Protocol placed legally binding caps on the emissions of more than thirty industrialized countries for the years 2008–2012 and established international market-based flexibility mechanisms to aid their achievement. The Protocol’s failure to include all major emitters under its emissions caps, and the hurdles it erected to broadening participation, contributed to opposition in the U.S. Senate (it was never submitted to that body for consent to U.S. ratification), and ultimately undermined its effectiveness in reducing global emissions. Nonetheless, the Protocol provided positive lessons on policy design. Key system design elements,8 many of which are also incorporated in the EU-ETS, include: & Absolute, legally binding multi-year emissions budgets; & Transactions limited to approved types of emissions units; & Standardized, serialized, vintaged, fungible emissions units denominated in tonnes of CO2-eq; & Transparent reporting of emissions; & Transparent tracking and reporting of emission unit transactions, including explicit re- quirements that transfers of emissions allowances be subtracted from the transferor’s emissions budget account and added to the transferee’s; & Transparent tracking and reporting of emission unit transactions, including explicit re- quirements that transfers of emissions allowances be subtracted from the transferor’s emissions budget account and added to the transferee’s; & Accountability for emissions as compared with units held; Climatic Change (2017) 144:81–95 85 & “Banking,” i.e., the ability to carry surplus reductions forward from one commitment period to a subsequent period; and & “Banking,” i.e., the ability to carry surplus reductions forward from one commitment period to a subsequent period; and & Stringent, specified consequences for non-compliance. 2 Why carbon markets While the Kyoto Protocol was the archetype of a “top-down” multilateral climate treaty, these design features holds useful lessons for market-based policies in a “bottom-up” world. 3 A new infrastructure: forming the club of carbon markets In a “bottom-up” climate architecture, individual jurisdictions establish their own domestic climate policies, including emission trading programs. A core purpose of the CCM would be to fulfill the functions necessary to link carbon markets across member jurisdictions, encourage new jurisdictions to join, and ultimately increase ambition. In particular, the CCM would need to create the conditions for mutual recognition of emission units among members; maintain the market infrastructure necessary for trading; establish clear criteria for membership; and inform assessments of mitigation effort and ambition among prospective and current members. 3.2 A shared carbon market infrastructure A second core function of the CCM would be to maintain the shared carbon market infrastructure necessary for trading. Thus the CCM would establish either a common registry of emissions units, or a formal means of communication among member registries, in order to facilitate the tracking of emissions units from their creation (either as allowances under a cap or as offset credits) to their retirement. To avoid “double-claiming” of reductions, the CCM would establish double-entry bookkeeping rules to ensure that all units, whether offset credits or allowances, transferred from one jurisdiction, are subtracted from the transferor’s unit account. In addition, the CCM (or a third party designated by the CCM to act on behalf of its members) could perform other market functions such as administering common allowance auctions, maintaining allowance accounts for regulated entities, and even overseeing and operating a “reserve” of allowances to be released into the market if prices rise above certain thresholds. While these functions could in principle be provided through bilateral linkages, the CCM would take advantage of economies of scale in establishing a common carbon market infrastructure. 3.1 Mutual recognition on the basis of harmonized standards At the core of the CCM would be mutual recognition of carbon emissions units among members. By joining the CCM, each jurisdiction would agree to allow entities with compli- ance obligations under its domestic carbon market to meet those obligations using emission units issued by any other member jurisdiction in good standing. To promote the fungibility of emission units, the CCM would establish and maintain harmonized standards among its members. For example, the CCM would provide a common emissions accounting framework to ensure that “a tonne is a tonne,” including mutually agreed procedures for monitoring, reporting, and verification (MRV) of emissions. The CCM would also ensure the harmonization of rules governing the creation and use of “offset credits” for verified reductions outside of capped sectors. Mutual recognition need not require that members adopt identical regulations. Even California and Quebec, which have linked their markets to an unprecedented degree, are developing separate offset protocols. To ensure flexibility while also providing clear guide- lines, club members might agree on a “minimum list” of regulatory design features or parameters that were essential for membership; a “negative list” of design features or param- eters that would be ruled out; a “positive list” of features that would be automatically accepted; and perhaps a final category of design features that would require case-by-case judgment by existing members based on an agreed set of high-level principles. The CCM could provide routine, periodic review of member jurisdictions to ensure that they observed the agreed standards and guidelines. Such a review process could draw on precedents for policy surveillance in the IMF, OECD, and other institutions (Aldy 2014). Anticipating that political and economic circumstances might alter the willingness of member jurisdictions to stay in the CCM, the CCM would also need to identify rules for exit, including the treatment of banked allowances. 9 Climatic Change (2017) 144:81–95 86 3.3 Criteria for membership Since the CCM would not be a treaty, participants could include sub-national jurisdic- tions such as states and provinces, as well as nations. The key requirement would not be “capacity to treat” but capacity to administer a market—i.e., establish an emissions cap, issue and accept emissions units, require compliance, and penalize non- compliance. Minimum eligibility criteria for membership in the CCM could include: & Emission targets defined as emissions budgets over some minimum duration, e.g. a decade, covering all or a significant portion of a jurisdiction’s emissions of one or more greenhouse gases, denominated in tonnes of CO2 or CO2-eq.; & Emission targets defined as emissions budgets over some minimum duration, e.g. a decade, covering all or a significant portion of a jurisdiction’s emissions of one or more greenhouse gases, denominated in tonnes of CO2 or CO2-eq.; & The use of emission trading to meet the cap; & The use of emission trading to meet the cap; & Jurisdictional law requiring covered entities to comply; clear, sufficient penalties for non- compliance; and institutional capacity to enforce the cap; and & A commitment to transparent monitoring, reporting, and verification of emissions and transactions. That the CCM would not be structured as a formal treaty might also increase the potential for eventual U.S. participation. In the postwar period from 1946 to 2006, 94 % of international agreements negotiated by the United States were “executive agreements,” implemented under existing authorities and never submitted to the Senate for advice and consent under Article II of the Constitution (Krutz and Peake 2009). In line with this trend, U.S. participation (including by U.S. states) might be more likely if a CCM were constituted not as a treaty, but as a voluntary association among jurisdictions with the capacity to administer their own internal markets, specifying obligations congruent with authorities afforded to the U.S. Executive Branch and the U.S. states by virtue of the Constitution and already-enacted legislation. Climatic Change (2017) 144:81–95 87 3.4 Mitigation effort and ambition To assure the environmental integrity of emissions units, CCM members would need to tackle the thorny challenge of assessing the stringency of each other’s emission caps—both at the time a prospective member applied to join (or, for founding members, at the time of the CCM’s creation), and periodically thereafter to ensure that participating jurisdictions remained “in good standing.” For prospective members, a natural approach might be to conduct a case-by-case review (and negotiation) of the emission targets proposed by the jurisdiction, much as applicants to the WTO must negotiate a schedule of tariff bindings.10 Another model of case-by-case review is the process established by the UNFCCC’s 2013 Warsaw Framework for Reducing Emissions from Deforestation and Degradation (REDD), under which Parties may propose REDD reference levels, submit those for independent technical expert review, and respond to the published results of the review. In either case, the CCM could oversee the process of gathering and disseminating information on prospective members. Information relevant to an assessment of ambition would include mitigation policies and accounting rules, including the treatment of direct and indirect emissions from capped sectors and procedures to address potential emis- sions leakage. To encourage broad membership, the CCM might offer consultations to jurisdictions whose initial applications were rejected. Once having joined, member jurisdictions could be subject to periodic assessments to ensure that they maintained or increased the ambition of their targets, as part of the routine policy surveillance mentioned above. Such a routine review would help to guard against the perverse incentive that might otherwise exist for jurisdictions—having linked with each other—to loosen their targets in order to improve their emission units “balance of trade.”11 To help inform both ex ante and ex post reviews, the CCM might develop a set of metrics for assessing “comparability of effort.” A range of approaches has been suggested in the literature, including evaluating targets on the basis of percentage reductions below historical or projected baselines, taking into account differing national circumstances; comparing targets to modeled emission trajectories consistent with a specified level of overall ambition; and estimating mitigation costs.12 4 Incentives to participate Crucial to the effectiveness of the CCM approach is the creation of incentives to encourage jurisdictions to join—and having joined, to comply with the obligations of membership. We envision the CCM as a true “club” in the economic sense: that is, a voluntary group whose members share a set of benefits from which non-members are excluded (Buchanan 1965). _ _ _ _ p _ 11 See, e.g., Green, Sterner, and Wagner, supra. 12 12 See, e.g., Aldy and Pizer (2014); Aldy et al. (2015); and Climate Action Tracker, http://climateactiontracker. org/indcs.html. 10 See World Trade Organization, Handbook on Accession to the WTO, chapter 4, https://www.wto.org/engl thewto_e/acc_e/cbt_course_e/c4s6p1_e.htm. 11 10 See World Trade Organization, Handbook on Accession to the WTO, chapter 4, https://www.wto.org/english/ thewto_e/acc_e/cbt_course_e/c4s6p1_e.htm. 11 See, e.g., Green, Sterner, and Wagner, supra. 12 See, e.g., Aldy and Pizer (2014); Aldy et al. (2015); and Climate Action Tracker, http://climateactiontracker. org/indcs.html. 10 See World Trade Organization, Handbook on Accession to the WTO, chapter 4, https://www.wto.org/english/ thewto_e/acc_e/cbt_course_e/c4s6p1_e.htm. 11 See, e.g., Green, Sterner, and Wagner, supra. _ _ _ _ p _ 11 See, e.g., Green, Sterner, and Wagner, supra. 12 13 Republic of South Africa National Treasury, “South Africa’s Policy Interaction Experience,” Presentation to the Partnership for Market Readiness (March 2013), www.thepmr.org/system/files/documents/Policy% 20Mapping%20II_South%20Africa_0.pdf 14 See Aldy, “The crucial role of policy surveillance in international climate policy,” supra. 4.1 Benefits from linkage By enabling trading of carbon units among a set of jurisdictions, the CCM would secure a range of benefits associated with linkage, including lower overall abatement costs (the 88 Climatic Change (2017) 144:81–95 broader trading area provides greater heterogeneity, lowering the cost of emissions reduc- tions), potentially greater price stability (assuming that price shocks are at least partially uncorrelated across jurisdictions), and greater market liquidity. These benefits are likely to be particularly attractive for “small” jurisdictions, or those dominated by a handful of emitters. While a small economy could face significant allowance price uncertainty in an independent domestic market, it could be more certain of prices with plurilateral linkages. Access to a larger market could also be significant for jurisdictions with only a handful of major emitters. For example, South Africa’s public utility, Eskom, generates over 90 % of the country’s electricity—making it a near-monopolist in South Africa, but a price taker in a potential CCM.13 As in any trade relationship, other benefits of linkage would vary. Jurisdictions with relatively abundant low-cost abatement opportunities would value the access to jurisdictions where demand for those is strong. At the same time, higher-cost jurisdictions might be attracted by the opportunity to meet compliance obligations by financing lower-cost abatement opportunities elsewhere—while being assured by the cap that the resulting emissions reduc- tions would be “real and additional.” 4.2 Reduced barriers to policy adoption The CCM could help prospective and existing members by lowering barriers to estab- lishing carbon markets or improving the efficacy of existing systems. Jurisdictions with less experience in designing and implementing environmental markets could access capacity-building assistance, technical expertise and analytical support. Jurisdictions without capacity or infrastructure to meet CCM membership criteria might be able to gain recognition as “Observer” or “Affiliated” members with the intention of joining later. pp g _ _ p 14 See Aldy, “The crucial role of policy surveillance in international climate policy,” supra. 4.4 Information exchange, institutional capacity-building, and policy coordinatio Visits to observe market infrastructure and implementation, periodic membership meetings, and other information-sharing programs would enable CCM members to share experiences and deepen market ties. Prospective members might be invited to participate in some of these, which might also be facilitated by coordination with other complementary capacity building initiatives, including the World Bank’s Partnership for Market Readiness. 4.5 Enhanced access to low-carbon investment capital CCM membership could help participating jurisdictions attract low-carbon investment. That is because the key entry requirement for CCM membership—a well-designed cap and trade program—also enhances the returns from investments in emissions reductions. Mutual recog- nition and fungibility of emission units could help participating firms access capital in other member jurisdictions and manage risk. CCM membership would also provide a strong signal not only of a jurisdiction’s commitment to reducing emissions, but also its institutional capacity to provide a stable environment for low-carbon investment. Indeed, political leaders focused on strengthening climate action might make membership a criterion for investment decisions by export–import banks, overseas development institutions, pension or sovereign wealth funds, or other large public investors. 4.6 Reputational benefits Enhanced international recognition, strengthened strategic alliances, improved interaction with regional partners, greater credibility in economic transactions, or better negotiating leverage in the UNFCCC might motivate new jurisdictions to join the CCM.15 Cultural and/or political affinities (e.g., the opportunity to learn from high-profile China or Cali- fornia carbon markets) might also lead jurisdictions to perceive it in their self-interest to join the CCM. 15 See Hathaway (2007); Rose and Spiegel (2009). 4.3 Enhanced transparency and MRV Scholars have long highlighted the need for transparent and rigorous monitoring, reporting, and verification (MRV) of emissions. Improved MRV helps jurisdictions understand the scope of the climate challenge, develop strategies to address it, and assess the extent to which policy interventions are succeeding. Robust MRV boosts public and private actors’ confidence in low-carbon investment—particularly when supported with a long-term governmental commit- ment to keep reducing emissions. The CCM could offer policy and technical support to improve members’ and prospective members’ domestic MRV. Such assistance would be in the self-interest of all members, since it would bolster market integrity, enhancing the value of club membership (even for those not receiving such support). Again, useful analogs can be found in the work of the IMF and WTO to assist members in building technical capacity and improving standards for data collection and analysis.14 Climatic Change (2017) 144:81–95 89 4.7 Safe harbor from potential trade measures on carbon-intensive products Just as Parties to the Montreal Protocol on the Ozone Layer prohibited trade in ozone- depleting substances (ODS) with non-Parties, various jurisdictions that have adopted or are considering carbon emissions limits have considered how to address leakage of emissions. “Border carbon adjustments” (BCAs)—requiring importers of carbon- intensive products (e.g., steel or aluminum) from jurisdictions without comparable emis- sions limits to purchase emissions allowances comparable to those required for domestic producers—have been discussed in the EU and elsewhere, and were included in proposed U.S. climate legislation in 2009. While a number of technical issues would arise in determining the exact form of any BCA, a growing body of legal analysis argues that appropriately-designed BCAs could be consistent with WTO rules (Horn and Mavroidis 2010; WTO and UNEP 2009). If jurisdictions with emissions limits decide to impose BCAs on carbon-intensive imports, they would likely make exceptions for jurisdictions with comparably stringent climate policies. 90 Climatic Change (2017) 144:81–95 In such a scenario, it would be natural for CCM members to exempt each other from BCAs, on the grounds that participation in the CCM met the condition of “comparability.” Such safe harbor could represent a powerful additional “club benefit” to joining the CCM. And as the CCM expanded, it would not only increase trade in carbon emissions units and reduce cross- border leakage, but also contribute to freer trade in other goods and services by widening the circle of countries that could claim exemption from BCAs. 4.9 Exclusion of non-members To ensure that these benefits are exclusive to jurisdictions in the CCM “club,” members would agree not to accept emission units from, or allow the transfer of units to, any jurisdiction outside of the CCM (unless all CCM members agreed to accept units from that jurisdiction). Such an approach would create strong incentives for jurisdictions with domestic market-based programs to conform to or exceed minimum CCM mem- bership criteria, to attract greater interest in linkage and investment from other jurisdictions. Again, the Montreal Protocol provides a model. Its Parties barred trade in ODS with non- Parties unless the non-Parties had adopted comparable measures. That provision created market-pull for more ratifications, better MRV, and a strong market force for the development of low-ODS alternatives (Petsonk 1990; See also Shepherd 2014)—exactly the dynamic that the CCM would seek to replicate in carbon markets. Such a “free-trade area for carbon” should not raise any tensions with the WTO: we agree with the prevailing view that carbon emission units are neither products nor services and thus are not directly subject to WTO disciplines.16 4.8 CCM secretariat A small CCM secretariat might strengthen general coordination, facilitate exchange of infor- mation, and provide a clearinghouse for institutional capacity-building. Considering analogies in the trade field among others, the secretariat might also house analytical capacity to undertake expert review of the policies in member jurisdictions, facilitate access to indepen- dent expertise, assess conformance with the membership criteria, and promote transparency in monitoring, reporting, and verification. As the CCM developed, the secretariat might also facilitate deeper coordination among CCM members—for example through centralized trans- action logs, model contracts, uniform contract codes, harmonized registries, shared “strategic allowance reserves” that could be tapped in the event of unexpectedly high prices, or common mechanisms for settling disputes. 16 For a full discussion of the trade implications of our CCM proposal, see Keohane and Petsonk (2015). See also UNCTAD (1996, UNCTAD/GDS/GFSB/Misc. 2) at 11; Petsonk (1999). This discussion of the origins of the GATT draws on Irwin et al. (2009). 18 We are indebted to Prof. Robert Keohane for suggestions on this section, and to Ms. Lindsay Brewer for research and suggestions. Materials in this section are drawn in part from MCTR Frequently Asked Questions, http://www.mtcr.info/english/FAQ-E.html. 19 See Zaborsky (2004). 5.1 First lesson: start small, and use market access as an attractant “Start small” emerges from the history of the modern trade regime.17 The origins of the GATT can be traced to the bilateral negotiations between the United States and the United Kingdom over the Atlantic Charter, issued in August 1941, and the Mutual Aid Agreement of the following year. In 1945, as part of negotiations on a postwar American loan, the two countries hammered out a joint commercial policy agreement on a range of issues including tariffs, subsidies, and import quotas, to serve as the basis for multilateral negotiations on tariff reductions. Following a suggestion by Canada, those negotiations were to start among a “nuclear” group of fifteen countries. In parallel, the U.S. proposed a larger UN conference to establish an International Trade Organization (ITO). By October 1947, the “nuclear” group—now numbering 23 countries, and meeting in Geneva—reached a General Agreement on Tariffs and Trade (GATT), even as they and others prepared to negotiate a charter for the larger organization. The failure of the United States to ratify the 1948 Havana Charter in effect doomed the ITO. But because the smaller GATT included provisions for it to take effect even if the ITO didn’t, the GATT was able to form an economic nucleus, using market access to attract, over time, broad multilateral participation in trade liberalization and tariff reduction. The “start small” theme also emerges in the development of the Missile Technology Control Regime (MTCR), which started in 1987 as an informal political agreement among G-7 countries to limit the risks of proliferation of weapons of mass destruction (WMD).18 The MTCR provides guidelines for export license controls on missile and space technologies and products. Although the MTCR, being voluntary, lacks penalties for noncompliance, under U.S. law the U.S. government may (and in some cases must) impose sanctions on individuals, companies, or governments that export controlled items in violation of the guidelines. Sanc- tions include cutting off bilateral aid or arms sales for up to two years (Arms Control Association 2012). The MCTR has grown to some 34 members, with participating nations gravitating to it in part because of the perception that adherence brings market access.19 5 Antecedents The development of the CCM might draw on lessons from other plurilateral regimes. Here we draw three lessons for club development from the fields of trade and security. 91 Climatic Change (2017) 144:81–95 5.1 First lesson: start small, and use market access as an attractant 17 This discussion of the origins of the GATT draws on Irwin et al. (2009). 18 20 See “Market and Non-Market Mechanisms,” UNFCCC, http://unfccc.int/cooperation_support/market_and_ non-market_mechanisms/items/7551.php; Lima Call for Climate Action, Decision 1/CP.20 para. 19(a)(v) (December 2014). 5.2 Second lesson: choose a legal architecture that facilitates participation The GATT, as an “agreement” among “contracting parties” (rather than a treaty), could be implemented by the U.S. Executive Branch, using its existing legal authorities. While the GATT eventually provided the basis for the much broader and more formal WTO, it might never have gotten off the ground in the first place had it not enabled immediate U.S. participation by working within executive authorities. (The very broad Havana Charter likely would have necessitated Congressional action, but was never submitted to the Congress precisely because such approval was unlikely.) Similarly, the MTCR is an informal “under- standing” based on voluntary participation, which allowed it to start promptly with U.S. participation and has facilitated broader membership since. The history of both regimes thus suggests that a CCM could start promptly and attract eventual U.S. national participation if it were formed as a voluntary association of jurisdic- tions, with obligations that could be met by the U.S. Executive Branch under contemporaneous 92 Climatic Change (2017) 144:81–95 domestic law, and thus not require ratification under Article II of the U.S. Constitution. In the case of the CCM, such a voluntary approach would also facilitate a prompt start by allowing subnational jurisdictions to participate. 5.3 Third lesson: seek critical mass The GATT 1947, and the MTCR, both started small but with a critical mass of relevant actors (producers and consumers) in their respective fields. That “critical mass” enabled them to exert further market pull to broaden membership. The recent history of multilateral trade negotiations offers similar lessons. In the trade arena, prior to 1994, trade negotiations proceeded along the approach of a “Single Undertaking”, in which nothing is agreed until everything is agreed— multilaterally. (The UNFCCC largely follows that approach.) The result has been that in recent times, those least interested in trade liberalization have exercised hold-out power. Multilateral trade liberalization has sputtered. In contrast, more progress has been made through the WTO’s de facto embrace of “less-than-all” agreements on information technology products, telecom- munications, and financial services, where entry into force is contingent on accession by participants constituting “critical mass” (e.g. 90 % of covered trade.) These recent successes in trade negotiations demonstrate the power of starting small, while seeking critical mass. The importance of reaching critical mass can be seen in environmental agreements as well. For example, the Montreal Protocol on the Ozone Layer, which froze and phased out consumption of ODS, entered into force when eleven nations representing two-thirds of global calculated ODS consumption had ratified the Protocol—making it economically unattractive for the remaining one-third of ODS consumers to ramp up their own production of ODS. Following this model, the CCM might focus on securing the participation of jurisdictions comprising a significant share (perhaps a majority?) of suppliers and demandeurs in extant cap-and-trade markets. The goal would be to limit the opportunity for a country outside the club to act as a large supplier of low-quality emission units, undermining the environmental integrity of the club. p g g y _ g _ g g p 23 The November 2014 U.S.-China joint climate announcement and September 2015 joint presidential statement both illustrate this dynamic. See “U.S.-China Joint Announcement on Climate Change,” White House Office of the Press Secretary, https://www.whitehouse.gov/the-press-office/2014/11/11/us-china-joint-announcement- climate-change; “U.S.-China Joint Presidential Statement on Climate Change,” White House Office of the Press Secretary, https://www.whitehouse.gov/the-press-office/2015/09/25/us-china-joint-presidential-statement- climate-change. 21 Examples include the International Carbon Action Partnership (ICAP) the Asia-Pacific Carbon Markets Roundtable; the “Under 2 MOU” initiative launched by the State of California; the Governors Forest and Climate Task Force C40 Cities Climate Leadership Group; and the World Bank’s Partnership for Market Readiness. 22 6 The CCM and the UNFCCC In principle, a CCM could be formed under the auspices of the UNFCCC, either through negotiations under the “Framework for Various Approaches” or by building on the 2014 decision of the Conference of the Parties to organize technical expert meetings to “[s]upport the accelerated implementation of policy options and enhanced mitigation action, including through international cooperation.”20 In this scenario, the UNFCCC itself could provide some of the functions of the CCM (e.g., a common market infrastructure), and might “take note” of a deeper plurilateral agreement on carbon markets reached by a subset of like-minded jurisdic- tions. In practice, however, the COP may not be able to agree more than minimal accounting standards to avoid double-claiming of transferred units. Alternatively, the CCM could develop wholly outside the UNFCCC, as a free-standing building block that complements the UNFCCC’s high-visibility platform. Here, the examples Climatic Change (2017) 144:81–95 93 of the GATT, which developed in parallel with efforts to create a larger, more ambitious International Trade Organization, and the MTCR, whose initial membership of seven countries has grown to more than 30, might be instructive. Building on these models, the convening could be done by states, jurisdictions, or non-state actors. It could build on efforts already undertaken by national and subnational jurisdictions, particularly those interested in market- based environmental policy.21 The “platform for a strategic dialogue” on carbon markets included in the 2015 G7 Leaders’ Communique could also provide a basis for initial discussions around a CCM.22 In turn, the CCM could facilitate both the achievement of members’ UNFCCC mitigation obligations and the UNFCCC’s transparency objectives. Since CCM membership would be conditional upon robust MRV commitments from members, the CCM would create strong incentives for jurisdictions with domestic market-based programs to ensure rigorous and transparent measurement and reporting in order to attract greater interest in linkage and investment from other jurisdictions. 22 See https://www.g7germany.de/Content/DE/_Anlagen/G8_G20/2015-06-08-g7-abschluss-eng.pdf. 23 6.1 Role of the UNFCCC in enhancing market coordination Even if the CCM were formed outside of the Framework Convention, individual nations’ ongoing work to draft, and later finalize, their post-2020 mitigation commitments as part of any Paris agreement could provide a boost to the CCM’s genesis and illustrate a potential “middle way” forward. Buttressed by the technical expert meetings on international cooper- ative approaches established by the 2014 Lima Call for Climate Action, the submission of national commitments provides an opportunity for any market-interested country to indicate the preliminary criteria that external emission units must meet to be accepted for compliance in its domestic system. Such public transparency could also aid other nations keen to design their own compatible domestic systems, and thus could help guide the development and future membership of a carbon markets club. Indeed, among those “intended nationally determined contributions” for the post-2020 period submitted by June 2015, Switzerland, Mexico, Nor- way, and Lichtenstein indicated their desire to use carbon markets to fulfill their pledges and articulated preliminary criteria for emissions units they would consider eligible for trading. Of course, the CCM would need to be consistent with Parties’ obligations under the UNFCCC, including any agreement reached under UNFCCC auspices in Paris in 2015. The Paris talks, and the UNFCCC itself, will continue to have important roles to play. The UNFCCC gives a voice and standing to virtually every country in the world—even the least developed and most vulnerable—on a critical global issue. It also creates a platform (and an obligation) for countries to announce commitments, helping to drive ambition at the national level.23 94 Climatic Change (2017) 144:81–95 In this context, it is important to note that UNFCCC Parties retain full authority to use domestic emissions markets to fulfill any commitments they might make. Moreover, existing UNFCCC provisions make clear that UNFCCC Parties also retain full authority to link their markets with similar markets created and administered by other Parties.24 In other words, no additional multilateral authority would be needed for UNFCCC Parties to fulfill their com- mitments under a Paris agreement through a CCM In this context, it is important to note that UNFCCC Parties retain full authority to use domestic emissions markets to fulfill any commitments they might make. 6.1 Role of the UNFCCC in enhancing market coordination Moreover, existing UNFCCC provisions make clear that UNFCCC Parties also retain full authority to link their 24 In this context, it is important to note that UNFCCC Parties retain full authority to use domestic emissions markets to fulfill any commitments they might make. Moreover, existing UNFCCC provisions make clear that UNFCCC Parties also retain full authority to link their markets with similar markets created and administered by other Parties.24 In other words, no additional multilateral authority would be needed for UNFCCC Parties to fulfill their com- mitments under a Paris agreement through a CCM. 7 Conclusion Market mechanisms such as emission trading systems can play a key role in driving emissions reductions and low carbon investment at a scale and pace needed to meet the urgent challenge of climate change. To that end, this paper has proposed a new addition to the existing “regime complex” on climate change: a Club of Carbon Markets. By establishing a common market infrastructure based on mutual recognition of carbon emission units, with standards to promote transparency and environmental integrity, the CCM could facilitate ambitious emissions reductions and mobilize significant low-carbon investment. Properly designed, the CCM could attract participation by offering carbon market linkage, informational and reputational benefits, and increased trade and investment flows. While the club could conceivably be formed under the auspices of the UNFCCC, it need not be. We envision the CCM emerging out of plurilateral agreement among carbon market jurisdictions, following the model of the GATT and recent arms control agreements. The CCM seeks to emulate a lesson from those and other examples: To encourage ambition and cooperation in the multilateral arena, create an institution that jurisdictions want to join. Acknowledgments We are grateful to Frank Convery, Jos Cozijnsen, Janneke de Vries, Jill Duggan, Dan Dudek, Ruben Lubowski, Robert Keohane, Michael Oppenheimer, Bryce Rudyk, José Eduardo Sanhueza, Dick Stewart, Maja Valstar, Gernot Wagner, three anonoymous referees, members of the E15 Expert Working Group on Climate Change and Trade, and participants at workshops hosted by NYU, Columbia, Harvard, Duke, and OECD/IDDRI/ICTSD. Lindsay Brewer and Daniel Francis provided able research and administrative assistance. 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https://openalex.org/W3164337474	https://www.emerald.com/insight/content/doi/10.1108/QROM-01-2020-1884/full/pdf?title=when-cooperation-turns-ugly-exploring-the-dark-side-of-social-capital	English	null	When cooperation turns ugly: exploring the dark side of social capital	Qualitative research in organizations and management	2,021	cc-by	12,090	Abstract Purpose – This article explores theoretical assumptions regarding negative consequences of social capital in the empirical case of a failed cooperation project, and how these consequences are related to processes involving people, structures and environments. Design/methodology/approach – The article is based on a case study of a cooperation project within municipal labor market services. The methodology followed a theorizing process, where data were collected through ethnographical methods and analyzed in relation to existing concepts from theories describing negative effects of social capital and shadow organizing. Findings – The results highlight how the development of negative social capital in the project can be understood through three relational processes, namely the social dynamics of insulation, homogenization and escalating commitment. The authors conclude that the quality of social capital is conditional upon complex interactions within social structures. Moreover, the results highlight the importance of studying organizing practices outside explicit structures, in order to identify the development of non-canonical practices and their consequences. q Practical implications – Organizing cooperation projects that aim to bridge professional competencies or organizational boundaries have to be attentive toward informal organizing practices which if remaining unrecognized may grow and threaten the original intentions. Originality/value – The study makes a theoretical contribution by combining a shadow organizing approach with literature on social capital. This combination proves especially useful for analyzing how organizational dynamics can influence the development of social capital into producing negative effects. Keywords Social capital, Shadow organizing, Cooperation, Social dynamics, Sweden h Keywords Social capital, Shadow organizing, Cooperation, Social dynamics, Sweden Paper type Research paper Paper type Research paper When cooperation turns ugly: exploring the dark side of social capital Received 27 January 2020 Revised 26 August 2020 4 December 2020 9 March 2021 Accepted 21 April 2021 Christian Stahl Department of Behavioural Sciences and Learning, Link€oping University, Link€oping, Sweden and HELIX Competence Centre, Link€oping University, Link€oping, Sweden HELIX Competence Centre, Link€oping University, Link€oping, Sweden The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/1746-5648.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/1746-5648.htm Qualitative Research in Organizations and Management: An International Journal Vol. 17 No. 5, 2022 pp. 1-18 Emerald Publishing Limited 1746-5648 DOI 10.1108/QROM-01-2020-1884 When cooperation turns ugly: exploring the dark side of social capital Lena Strindlund and Madeleine Abrandt Dahlgren Department of Medical and Health Sciences, Link€oping University, Link€oping, Sweden, and When cooperation turns ugly Exploring the dark side of social capital p g p Social capital has been discussed for decades with some variations in definitions and applications. The historical roots of the concept can be traced back to fundamental sociological notions of social cohesion, socialization and stratification. One of the first modern theorists that used social capital as a term was Bourdieu, who described it as a resource based on a person’s membership in social networks and groups and the gains this could bring in terms of recognition and social benefits (Bourdieu, 1986). Since then, social capital has become an increasingly popular concept and applied across a variety of disciplines and research fields (Rostila, 2010; Woolcock, 2010; Kwon and Adler, 2014). Most commonly, these modern adaptations use it as a positive characteristic of social groups, e.g. for studying the amount of support or cohesion, in many studies related to various forms of outcomes, such as health (Szreter and Woolcock, 2004; Oksanen et al., 2008). Empirical studies using the term in this way generally conclude that social capital is positively related to health outcomes, which is at least partly a consequence of how it is conceptualized and operationalized, leaving little room for exploring potentially negative effects (Szreter and Woolcock, 2004). For instance, in a widely used measure of social capital in workplace settings, the concept is operationalized in exclusively positive terms (Kouvonen et al., 2006). Portes (1998) argues that we tend to have a bias toward seeing good things coming out of sociability, but that we need to emphasize the negative sides in order not to promote networks, social control and collective sanctions as unequivocal blessings and in order to provide serious sociological analyses rather than moralizing statements. In this article, we theorize social capital as primarily related to groups and as a neutral concept which may have positive or negative effects. In this vein, we argue that for scholars taking a sociological perspective, the notion of a “dark side” to social capital should be quite uncontroversial, as this perspective proposes that the positive or negative consequences of social bonds vary according to the situation (Portes, 1998). Acknowledging potential negative effects of social capital are, hence, a way of returning the concept to its roots, rather than considering it as a genuinely positive resource and as a remedy for various problems. Introduction hi To achieve purposeful cooperation between professionals from different organizations, it is crucial to secure trusting relationships. The importance of trust and adequate structural prerequisites has been emphasized in multiple theoretical and empirical studies of interorganizational cooperation. (de Rijk et al., 2007; Stahl, 2010; Stahl et al., 2017) Theories on cooperation often point out the complexities in attaining such conditions, and some © Lena Strindlund, Madeleine Abrandt Dahlgren and Christian Stahl. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/legalcode € y p g y g Funding for this study was provided by the Coordination Association in Central €Osterg€otland. The funder had no influence over the study design, analysis or the conclusions. authors have even recommended against engaging in cooperative initiatives unless deemed unavoidable (Huxham, 2003). ( , ) In the research on trust and interorganizational relations, the extent of social capital within the relationships has been identified as a key aspect in explaining how well cooperation goals are achieved (Nahapiet and Ghoshal, 1998; Tsai and Ghoshal, 1998). The literature on social capital is vast, and it is commonly described as a positive resource in various interpersonal and community-based relationships (Szreter and Woolcock, 2004; Woolcock, 2010). However, a “dark side” of social capital has also been identified, i.e. how close relationships may also function to be excluding and destructive toward those not included in the group (Gargiulo and Benassi, 1999; Pillai et al., 2017). Such negative effects have received less focus in empirical research. 2 2 p The purpose of this article is twofold: (1) to contribute to the understanding of negative consequences of social capital in interorganizational cooperation between public organizations by exploring theoretical assumptions in the empirical case of a failed cooperation project, and (2) how these consequences are related to processes involving people, structures and environments. Exploring the dark side of social capital The potential negative effects of social capital seem to be more commonly found in closed or isolated networks, which may exclude outsiders, lead to unequal distribution of social resources (Rostila, 2010) and limit connections to outside influences which may result in intolerance and discrimination (van Deth and Zmerli, 2009). The “dark side” of social capital has been explored in empirical studies across a variety of settings in which researchers examine, for example, adolescent drinking (Martins et al., 2017), sports (Whittaker and Holland-Smith, 2016; Brown, 2018), finance (Carrera et al., 2017), or mental health problems and alienation in workplaces (Zhang et al., 2016; Sakuraya et al., 2017). Portes identifies at least four negative consequences of social capital: “exclusion of outsiders, excess claims on group members, restrictions on individual freedoms, and downward leveling norms” (Portes, 1998, p. 15) and gives examples of social contexts with high levels of social capital which have obvious negative consequences, such as Mafia families, prostitution rings and youth gangs, which are strongly embedded in social structures. Social capital is, hence, indifferent to the underlying values of a group (van Deth and Zmerli, 2009) and functions as a “sociological superglue” (Putnam, 2000, p. 23). 3 y ( , ) The “dark side” of social capital has been explored in empirical studies across a variety of settings in which researchers examine, for example, adolescent drinking (Martins et al., 2017), sports (Whittaker and Holland-Smith, 2016; Brown, 2018), finance (Carrera et al., 2017), or mental health problems and alienation in workplaces (Zhang et al., 2016; Sakuraya et al., 2017). Portes identifies at least four negative consequences of social capital: “exclusion of outsiders, excess claims on group members, restrictions on individual freedoms, and downward leveling norms” (Portes, 1998, p. 15) and gives examples of social contexts with high levels of social capital which have obvious negative consequences, such as Mafia families, prostitution rings and youth gangs, which are strongly embedded in social structures. Social capital is, hence, indifferent to the underlying values of a group (van Deth and Zmerli, 2009) and functions as a “sociological superglue” (Putnam, 2000, p. 23). , ) g p g ( , , p ) For the topic of the current article, we found a review of empirical evidence of negative effects of social capital in organizations and management (Pillai et al., 2017) to be particularly instructive. When cooperation turns ugly Exploring the dark side of social capital A third variety, linking social capital (vertical relationships between people interacting across gradients in society) was introduced later (Szreter and Woolcock, 2004). The potential negative effects of social capital seem to be more commonly found in closed or isolated networks, which may exclude outsiders, lead to unequal distribution of social resources (Rostila, 2010) and limit connections to outside influences which may result in intolerance and discrimination (van Deth and Zmerli, 2009). The “dark side” of social capital has been explored in empirical studies across a variety of settings in which researchers examine, for example, adolescent drinking (Martins et al., 2017), sports (Whittaker and Holland-Smith, 2016; Brown, 2018), finance (Carrera et al., 2017), or mental health problems and alienation in workplaces (Zhang et al., 2016; Sakuraya et al., 2017). Portes identifies at least four negative consequences of social capital: “exclusion of outsiders, excess claims on group members, restrictions on individual freedoms, and downward leveling norms” (Portes, 1998, p. 15) and gives examples of social contexts with high levels of social capital which have obvious negative consequences, such as Mafia families, prostitution rings and youth gangs, which are strongly embedded in social structures. Social capital is, hence, indifferent to the underlying values of a group (van Deth and Zmerli, 2009) and functions as a “sociological superglue” (Putnam, 2000, p. 23). dimension of social capital, while trust represents the cognitive dimension (Nahapiet and Ghoshal, 1998). Social resources, which are products of networks and trust and hence the actual “capital”, may be collective or individual and be expected to lead to different returns, either instrumental (such as economic, political or social gains), or expressive (such as physical or mental health benefits, based on the emotional support social capital may bring). Other scholars have suggested different typologies of social capital, where an influential distinction was made by Gitell and Vidal (1998) between bonding social capital (horizontal relations of trust and reciprocity between individuals with similar social identities at the same hierarchical level) and bridging social capital (horizontal relations between persons at the same hierarchical level from different backgrounds). A third variety, linking social capital (vertical relationships between people interacting across gradients in society) was introduced later (Szreter and Woolcock, 2004). Exploring the dark side of social capital Social capital may be argued to have both individual and collective dimensions (Harpham et al., 2002; Rostila, 2010; Kwon and Adler, 2014). Social capital fundamentally consists of formal or informal social networks (that may be open or closed), social trust (that may be strong or weak depending on the strength of the networks) and social resources, which are derived from the previous two aspects (Rostila, 2010). Networks represent the structural dimension of social capital, while trust represents the cognitive dimension (Nahapiet and Ghoshal, 1998). Social resources, which are products of networks and trust and hence the actual “capital”, may be collective or individual and be expected to lead to different returns, either instrumental (such as economic, political or social gains), or expressive (such as physical or mental health benefits, based on the emotional support social capital may bring). Other scholars have suggested different typologies of social capital, where an influential distinction was made by Gitell and Vidal (1998) between bonding social capital (horizontal relations of trust and reciprocity between individuals with similar social identities at the same hierarchical level) and bridging social capital (horizontal relations between persons at the same hierarchical level from different backgrounds). A third variety, linking social capital (vertical relationships between people interacting across gradients in society) was introduced later (Szreter and Woolcock, 2004). The potential negative effects of social capital seem to be more commonly found in closed or isolated networks, which may exclude outsiders, lead to unequal distribution of social resources (Rostila, 2010) and limit connections to outside influences which may result in intolerance and discrimination (van Deth and Zmerli, 2009). dimension of social capital, while trust represents the cognitive dimension (Nahapiet and Ghoshal, 1998). Social resources, which are products of networks and trust and hence the actual “capital”, may be collective or individual and be expected to lead to different returns, either instrumental (such as economic, political or social gains), or expressive (such as physical or mental health benefits, based on the emotional support social capital may bring). Other scholars have suggested different typologies of social capital, where an influential distinction was made by Gitell and Vidal (1998) between bonding social capital (horizontal relations of trust and reciprocity between individuals with similar social identities at the same hierarchical level) and bridging social capital (horizontal relations between persons at the same hierarchical level from different backgrounds). Exploring the dark side of social capital The review focuses primarily on the context of firms, and how social capital relates to several organizational outcomes, such as innovation, organizational cultures, organizational inertia, performance, decision effectiveness, and the acquisition, creation and transfer of knowledge. Pillai et al. identify six types of negative effects of social capital, which are all based on dysfunctional social identification processes: (1) dilution of the dialectical process; (2) inhibition of individual learning; (3) groupthink; (4) postponement of structural adjustments; (5) non-rational escalation of commitment; and (6) blurring of firms’ boundaries (10, p. 98). In their review and the following theoretical development of the concept, Pillai et al. argue that social identification processes are divided into structural, relational and cognitive aspects, where a strong social capital within a group will effectively block external impulses through shared sense-making and confirmation bias (Pillai et al., 2017). Such groupthink processes will isolate a group to become a closed information-processing unit that fuels uniformity of beliefs and exaggerates the group’s internal bonds, which may cause over-commitment and justification of previous decisions, as well as polarization in relation to other groups. This is also a reason, Pillai et al. argue, that managers tend to not shut down projects which they have invested in. Introducing new decision-makers at points where the future of a project is to be decided is hence an effective method to reduce commitment to failing projects (Pillai et al., 2017). Negative effects of social capital may appear only late in a social process, where it is possible to have a positive development which morphs into more intense and closed networks, thus shifting from the “bright” to the “dark” side. It is difficult to break strong social ties which may lead to a relational inertia where the same contacts are repeatedly used by habit, serving as a filter that isolates practices from new possibilities (Gargiulo and Benassi, 1999). The possible gains of social networks hence come with a risk of negative effects. This focus of social capital as a changing quality of social contexts also raises the question of the mechanisms involved where a situation characterized by mutual trust and consensus turns into its opposite. In such negative circumstances, particular mechanisms determine how conflicts occur and how these interact with social identification processes. While the review of Pillai et al. Exploring the dark side of social capital mainly focused on private business contexts, the types of social identifications and group processes described could be expected to translate well to other types of organizations, although it is likely that any kind of organizational context will have its own influence over how such processes occur and develop. Our study focuses particularly on how negative social capital develops in cooperative projects between public organizations. 4 4 To add to the theories of negative effects of social capital, we have also identified a complementarytheoretical metaphor,namely thatofshadow organizing. Thismetaphor enables exploring organizing as intra-relating processes and to focus on what happens in the interstices between intentional and unintentional organizing actions (Gherardi et al., 2017). The “shadow” is a symbol of what is obscured, and which can only be seen by looking past the obvious and focusing on what is hidden. In this article, we have used this metaphor as an organizing tool to guide the discussion of the results, which are related to three concepts from this theory: productivity, liminality and secrecy. Productivity is illustrated “through the image of the forest and its sheltered spaces in penumbra”; liminality is seen “through the image of a liminal, grey zone between canonical and non-canonical practices”; secrecy captures “the organizational secrecy kept in-between the sayable and the unsayable” (Gherardi et al., 2017, p. 7). Methods The methodological process for this article was inspired by Hammond’s (2018) and Swedberg’s (2012) perspectives on theorizing. Theorizing is described as a process driven by identification of interesting problems and a motivation to solve them (Hammond, 2018). The analysis builds on a rich ethnographic material, which provides an authentic contextualization of the theoretical assumptions. Empirical context: a Swedish cooperation project The empirical context for our analysis is a cooperation project within municipal labor market services in a medium-sized Swedish city. Sweden is internationally renowned for its consensus-driven policies, with processes for resolving conflicts in the labor market as a prime example. In Swedish studies of cooperation practices between authority actors, consensus has been identified as a starting point for actors when engaging in joint projects, although such preconceptions may be put to the test by interorganizational dynamics or systems not designed for joint action (Stahl et al., 2011). Sweden is an interesting empirical context for exploring the “dark side” of social capital, since there may be an expectation for actors to share a basic level of social capital in the drive for consensus, and that the people involved in projects organized by authorities could be expected to adhere to project guidelines. The project studied here was organized through a coordination association, which is an interorganizational structure that is co-owned by municipalities, county councils, the Swedish Social Insurance Agency and Public Employment Services (Stahl, 2010). The project aimed to support teenagers and young adults to participate in the labor market, and the original intention was for the project to function as an arena where the involved authorities could direct them to appropriate support. The project was staffed with personnel from each of the authorities who were meant to work part time in their regular organization and part time in the project to facilitate interorganizational contacts. It was started in 2015 and closed before its allotted time in 2017. The project was followed using an interactive research approach, which included a learning evaluation (Svensson et al., 2009) to inform the development of the project. p p j In a previous analysis (Strindlund et al., 2020) we outlined four organizational conditions that influenced the development of the project, namely communication, structure, trust and steering. Our previous results identified how flaws in these conditions shaped two different perceptions of the project, where a coordination rationality (i.e. When cooperation turns ugly Methods that the project should aim for increased interorganizational contacts and a steady flow of participants through the project) clashed with an empowerment rationality (i.e. that the project should focus on working closely with fewer participants). In this article, we focus on how the organizing in the project, viewed from a processual and relational perspective involving people, structures and environments, can be theorized (Czarniawska, 2008). For this analysis, we make use of theories from the literature on social capital and place this within a shadow organizing framework (Gherardi et al., 2017) in order to illuminate that which takes place underneath the explicit project organization. 5 Data collection Th fi b i The first basic step of theorizing suggested by Swedberg is to observe and choose something interesting. In order to theorize a phenomenon, one needs to observe the phenomenon and see it for oneself, preferable through field studies (Swedberg, 2012). The data consisted of ethnographical material collected by the first author from spring 2017 to November 2018 through observations, focus groups, interviews and document studies in line with recommendations for ethnographic studies (Hammersley, 2018). In total, more than 70 observations, nine focus group interviews and 34 interviews were carried out by the first author. Data from observations and informal interviews were documented in field notes and the interviews were recorded and transcribed verbatim. Table 1 presents a compilation of the data collection, comprising methods, interviewees, focus and structure, documentation strategy, timing and numbers. In summary, the data collection met the recommendations of theorizing, namely, to observe and stay in the context of discovery and develop tacit knowledge of the phenomenon by collecting strong and nuanced descriptions. Data analysis h 2017) No.1: 2 focus groups, 9 informants No. 2: 2 focus groups, 11 informants Focus group interviews with staff members external to the project Expectations and perceptions of the project and how cooperation and communication between the actors worked Transcribed verbatim Development phase (September– October 2016) 5 focus groups with a total of 22 informants Document studies Studies of various written documents such as project plan, project application, e-mail conversations etc. Written documents and e-mail conversations During all phases >40 documents Formal interviews with actors in the project organization (Project group members and Their perceptions of the project and reasons for closing down Transcribed verbatim Closing down phase (April– June 2017) 17 interviews (9 project group members and 8 steering group members) Table 1. QROM 17,5 6 Documenting meetings and developments in the project, who is doing and saying what, what questions are being raised, etc. 6 6 Everything related to the project, from smaller to bigger issues Informal interviews with actors in the project organization Everything related to the project, from smaller to bigger issues Written down field notes and reflections from various informal conversations During all phases >50 occasions Focus group interviews with project staff members at two occasions No. 1: Expectations related to the project and perceptions of the project’s aim and goal and their own role No. 2: Describing perceptions of the program theory of the project Transcribed verbatim No. 1: Beginning of development phase (Jan. 2016) No. 2: The end of development phase (Jan. 2017) No.1: 2 focus groups, 9 informants No. 2: 2 focus groups, 11 informants Focus group interviews with staff members external to the project Expectations and perceptions of the project and how cooperation and communication between the actors worked Transcribed verbatim Development phase (September– October 2016) 5 focus groups with a total of 22 informants Document studies Studies of various written documents such as project plan, project application, e-mail conversations etc. Written documents and e-mail conversations During all phases >40 documents Formal interviews with actors in the project organization (Project group members and Steering group members) Their perceptions of the project and reasons for closing down Transcribed verbatim Closing down phase (April– June 2017) 17 interviews (9 project group members and 8 steering group members) Table 1. Data analysis h The next step of theorizing (Swedberg, 2012) is to name and formulate the central concept. The three authors gathered in several analysis sessions all contributing with different perspectives and pre-knowledge of the project and the theories. The first author had first- hand experience of collecting the data, the second author contributed with the shadow organizing perspective and the third author with social capital theory. The analysis started from the six types of negative effects of social capital identified by Pillai et al. (2017): dilution of the dialectical process, inhibition of individual learning, groupthink, postponement of structural adjustments, non-rational escalation of commitment, and blurring of firms’ boundaries. These served as an entrance-point for analyzing the material. Thereafter, the data, consisting of short notes based on the total ethnographic material such as quotations from interviews and reflections formulated in field notes, were mapped onto the effects identified above by the first author using an Excel spreadsheet. The data were analyzed by all three authors together searching for patterns and common denominators. As a further analytical step, the material was analyzed and rearranged in relation to and cross-tabulated with the concepts of bonding, bridging and linking social capital. The general goal of theorizing is to come up with new ideas. This aspect, abduction, is defined as “observing a fact and then professing to say what idea it was that gave rise to that fact” (Swedberg, 2012). Abduction has both a logical and an innovative character and the quality lies in its “means-of-inferencing”. It is a sensible and scientific form of interference Data collection Focus Documentation Time point Numbers Weekly observations of project staff meetings and monthly observations of steering group meetings Documenting meetings and developments in the project, who is doing and saying what, what questions are being raised, etc. Continuous field notes and written down reflections During all phases >70 occasions Informal interviews with actors in the project organization Everything related to the project, from smaller to bigger issues Written down field notes and reflections from various informal conversations During all phases >50 occasions Focus group interviews with project staff members at two occasions No. 1: Expectations related to the project and perceptions of the project’s aim and goal and their own role No. 2: Describing perceptions of the program theory of the project Transcribed verbatim No. 1: Beginning of development phase (Jan. 2016) No. 2: The end of development phase (Jan. Focus group interviews with project staff members at two occasions Focus group interviews with staff members external to the project Data analysis h Overview of the empirical data intended to help researchers to be able to make new discoveries in a logically and methodologically ordered way (Reichertz, 2007). It is a reflexive and iterative process, where the theoretical concepts and the empirical study stimulate the next step in the theorizing process, namely, to build out the theory. This step aims to give form to the central concepts by outlining the structure of the phenomenon (Swedberg, 2012). In our study, this was done by analyzing how the negative effects of social capital unfolded, which we describe through three distinct social dynamics, namely: insulation, homogenization and escalating commitment. These dynamics comprise our main analytical contribution, which were identified inductively from the empirical material, where the labels were inspired by the descriptions of negative effects of social capital identified by Pillai et al. (2017). The labeling When cooperation turns ugly When cooperation turns ugly was hence done through an iterative process where theoretical concepts were adapted to suit the specific empirical context. The negative effects identified in the social capital literature where interleaved with these social dynamics; specific effects do not map into specific phases but come into all three dynamics in different ways, where these effects are described in the results as they appear in the social dynamics described. The metaphor of shadow organizing, finally, added a terminology for the organizational context and for describing the milieu in which negative effects of social capital could develop (Buch, 2020; Gherardi et al., 2017). An overview of the theoretical concepts used in the analytical process is presented in Figure 1. The purpose of the analysis was to explore how the theoretical concepts could make sense of the data from the empirical case and what the case could contribute to the existing theories. This is the last step in the theorizing process: complete the tentative theory, including the explanation (Swedberg, 2012) which is summarized in the conclusions, where we outline the development of negative effects as a highly dynamic and non-linear process. For this step, the theoretical metaphor shadow organizing was useful for contextualizing the analysis. Figure 1. Concepts used in the analysis, and the resulting three social dynamics Figure 2. The project idea, as described in documentation and interviews Results To understand the results and how the social dynamics developed, we will start with presenting the founder’s and the financier’s view of the project, as conveyed in project documentation and in interviews. In their view, illustrated in Figure 2, the project was intended to enhance collaboration and contribute to optimal use of the welfare system by creating an arena for coordinating services. The cooperative arena, i.e. the project, is here Figure 1. Concepts used in the analysis, and the resulting three social dynamics Figure 2. The project idea, as described in documentation and interviews Figure 1. Concepts used in the analysis, and the resulting three social dynamics illustrated as a circle in which representatives from each of the participating actors each represent their respective organization. The representatives would divide their work hours between the project and the home organizations (i.e. the organization that the person originally worked in), thereby having a central, linking function, acting as knowledge brokers. The idea was that this project structure would facilitate project staff to gain access to each actor’s resources and internal activities and also contribute to increase the knowledge in the home organization concerning the other actors’ legal frameworks and cultures. The project staff was to represent their home organizations and contribute with their specific professional competence. However, this original idea was not clearly communicated to the project team or to cooperative actors (i.e. the organizations that were to cooperate with the project). Instead, the project group developed an independent organization with the aim to empower individuals. 8 8 p This development became a hotbed for negative effects of social capital, where our analysis presents how this unfolded through three different intra-relational processes, namely the social dynamics of insulation, homogenization and escalating commitment. The social dynamics of insulation Early in the project, the project group began to distance themselves from their organizational environment and to focus on developing internal cohesion rather than fostering links with the surrounding organizations. This process served to insulate the group from external influence and strengthened the project group’s steering of the project in a different direction than that of the original idea. This development was driven by strong individuals in the project group wanting to build a project with an empowerment-inspired approach. g p j p p pp This insulation process pushed the focus of the group inward regarding the project group’s identification process, where this involved distancing themselves from their home organizations. This is illustrated in the first phase in Figure 3, insulation, as creating a bubble around the project group to protect and insulate themselves. The one-directional arrows illustrate the lack of information from the project to the surrounding actors, and the thick line illustrates how this barrier prevents external information or influences from reaching the project. Representatives from external organizations experienced how communication attempts failed, both concerning getting information from the project as well as reaching the project. No, it has been very quiet, there has been very little information (. . .) And so we have been standing here knocking on the door, failing to reach them. (Representative Municipal Labor Market Unit) Figure 3. The three social dynamics identified in the study Figure 3. The three social dynamics identified in the study Figure 3. The three social dynamics identified in the study When cooperation turns ugly When cooperation turns ugly When describing the insulation process retrospectively, actors in the steering group used the metaphor of a “bubble”. This corresponds with the picture of the project as an isolated environment. However, unlike a bubble, it is neither transparent nor easy to break. Instead, the bubble membrane grew thicker to become an insulation barrier against the cooperative actors. This bubble that this project group went into, it was like, it shut others out (...) it would have been easier to punch the bubble if it had been transparent and you could see how things were in day-to-day practice. (Steering group member) 9 The insulation process was enforced by a strong identification process where the project group focused on branding the project as special and identified themselves through this uniqueness. This involved spending excessive amounts of time and money on creating a logo, producing marketing materials such as brochures, pencils and sweaters and requesting project-specific e-mail addresses instead of their regular organizational addresses. All these activities served to highlight the uniqueness of the project. The bubble creation puts a lot of focus on the internal bonding process in the group and created a strong sense of “us and them”. The orientation toward empowerment methods also stressed the use of the group as a tool, rather than promoting coordination with other actors. This inward movement could be seen in the project group’s daily work, where they engaged in various group exercises to tighten the ties within the group, which also contributed to creating a distance from the home organizations. The insulation was strengthened further by structural aspects concerning location and time. The project group was placed in their own office with limited physical access to the home organizations. In this sense, the project group became both isolated from and insulated against their environment. Instead of enabling access points, the project group focused on creating a non-authority-like environment, both for the youths and themselves, further distancing the project from the home organizations. This process of creating a home-like environment was described by a representative from the steering group as a “cozy-cozy” process, interpreted as a strategy to distance the project from the alleged hard and stressful environments in the home organizations. The organization of work hours also contributed to the insulation, as some group members managed to abandon the original idea of sharing their hours between the project and the home organizations and instead spent all their work hours in the project. This limited knowledge exchange further increased the insulation effect. The social dynamics of homogenization The social dynamics of homogenization Gradually, the group members began to lose their original identities as representatives for each participating organization and to assimilate into the new project identity. Based on the original idea, the staff members were to contribute each participating actor’s unique competence and resources to the project. Instead, the project developed into an independent unit with group members striving for homogeneous views instead of diversity. This process is illustrated in the second phase in Figure 3, homogenization, with the group members (the small circles in the middle) losing their affiliation pattern and assimilating into a new common identity. The communication between the organizations is illustrated as a dotted line to indicate its sporadic character. The process is aptly described by a project group member as the project having created “a world of its own”. Just as in the social dynamic of insulation, the homogenization process was related to social identification. The project group sought homogeneity in the project concerning everything from identification attributes to roles. Work clothes with the project logo and the use of empowerment-oriented expressions strengthened the project members’ identification with the project and further enforced the “us and them” feeling. Instead of affirming differences and using the various competencies in the group, in line with the original project idea, they focused on developing work methods where everyone would do everything, regardless of profession and affiliation. This social identification process meant the group members relinquished their affiliation to their home organizations in favor of the project identity, which was strongly criticized by steering group representatives. differences and using the various competencies in the group, in line with the original project idea, they focused on developing work methods where everyone would do everything, regardless of profession and affiliation. This social identification process meant the group members relinquished their affiliation to their home organizations in favor of the project identity, which was strongly criticized by steering group representatives. And that is a little strange when one calls for different professions, and the business becomes so that one can almost go in and take over each other’s work. (Representative Steering Group) 10 Another aspect of the homogenization process was the pursuit of thinking similarly within the group. As the cohesion within the group increased, it became more insulated as an information-processing unit, thus diluting the dialectical process (illustrated as a dotted line in Figure 3). The social dynamics of homogenization The group used a consultant to train them in a specific empowerment method, which gave them a guideline for how to think and act in the “right” way in order to succeed with the task to empower youths, thus pressuring the group members to uniformity of beliefs. The project leader had no experience of the chosen method or of project management, which led the project members with more knowledge of the method to become informal leaders. These informal leaders were, due to their experience, regarded as experts and as such they gained power and were rarely contradicted. They prioritized meetings to settle group values and thereby directed the members into thinking and acting uniformly. The pressure to conform to the group’s beliefs also led to closed-mindedness. The project group developed a culture where information or ideas that were not in line with the endorsed way of thinking were questioned, ignored and sometimes even ridiculed. This also meant distancing from and minimizing dialog with surrounding organizations. The closed- mindedness meant that the project group was not susceptible to counterfactual thinking, and the devotion to the empowerment method limited the project group’s openness to new or opposing information. This inhibition of critical thinking convinced the group that their interpretation and choice of method was right and prevented them from seeing or acknowledging alternatives. At times when the project’s direction was questioned, the project group reacted strongly and focused on justifying their choice of the empowerment method. This further strengthened the bonding process and made the group members grow even closer by supporting one another, while weakening any cohesion with colleagues from the surrounding organizations or with their managers. And this, that it was going in the wrong direction, that was a really great thing for us, it became a great thing for us (. . .) and then you feel you’ve failed, you have worked, you are passionate about this and have worked hard for it and want the best for the project and everyone. (Project group member) 11 [The project leader] sided too much with the group, she became a group member (. . .) the group ruled her (...), [there were] strong personalities in the group who were allowed to take over. (Representative, Steering group) The expanding sense of importance was also reflected in a diminished role of the steering group. Due to the insulation and homogenization process, the group had become used to managing themselves and was convinced that they had both the mandate and capacity to do so. When the steering group, through feedback from the learning evaluation, became aware of the project deviation from the original idea, they began to question the project. This strengthened the group’s unyielding commitment even further, and they made every effort to question the steering group. The project group spent much time processing and trying to influence the outcome of managerial decisions by reaching out to the home organizations, to the media, to unions and to the young people enrolled in the project. In the end, this commitment only impaired the opportunities of the group to continue the project, as the steering group decided to shut it down. They had too much room for maneuver, and they got too much to work against and they tried to make us responsible for the closure. . . Of course, you can get angry for a while (. . .), but then I think you have to move on (. . .). Sometimes, it became very disproportionate. (Representative, Steering group) Another aspect of the non-rational escalation of the project group’s commitment was the development of downward leveling norms. These behaviors escalated in connection to the decision to close, and group members displayed behaviors that were not in line with how they acted in the past, e.g. rallying over the financier and making threats against the steering group. Apart from the negative effects of these dubious behaviors for the involved actors, interviews with the project group members indicate negative effects also on the ones who performed them. Yes, it is really bad. I mean, no one wants anything bad for anyone else, but it gets to that point, along the way. Through all the frustration, misconceptions and misunderstandings that arise along the way. And it’s really sad, but thats the way it is. (Project Group Member) The social dynamics of escalating commitment y f g The insulation and homogenization processes enforced groupthink and contributed to an overestimation of the group and a non-rational escalation of commitment. This process is illustrated in the third phase in Figure 3, escalating commitment, where the expanded circle in the middle indicates how the group members experienced a magnified sense of self- importance. The previous communication paths are now entirely removed to visualize the total breakdown in communication between the parties. The insulation and the dilution of the dialectical process contributed to the group overestimating themselves. This became evident in the surrounding organizations not sharing their sense of the project’s excellence. Instead, representatives from these organizations questioned the project’s image of having a unique function and relevance and especially the perception of being the only solution for the target group. This resulted in the group being criticized as having delusions of grandeur. When cooperation turns ugly When cooperation turns ugly I think that the team felt that they were superior, to the degree that they did not need the other professions in our organizations: they should do everything (. . .) I think they are way off track, because there are many who are experienced in having conversations with and reaching young people. It is not that this is a completely unique skill. I do not think that’s good, because then you place yourself above the others and say that the others are not as good. (Representative, Steering Group) The overestimation of the group also meant the project leader’s role was diminished. The project leader, according to the original idea, was supposed to have a bridging function between the project members and management in the surrounding organizations. However, the insulation and homogenization processes affected the project leader in gradually beginning to identify herself with the project group rather than as a representative of the management team. As the polarization increased between the groups, she was drawn to the project group where she felt loyalty. The project leader was later dismissed as the steering group did not trust her to be able to fulfill her task. At this point, the project group was convinced that they did not need a project leader, and that the project could be run by the group and its informal leaders. Discussion Th i f The aim of this study was to explore theoretical assumptions of negative consequences regarding social capital in the empirical case of a failed cooperation project between public organizations. In so doing, we focused explicitly on social processes within the project, where the organizing of the actual practice was given centrality. By studying such processes, we could identify three distinct phases characterized by three corresponding social dynamics, namely insulation, homogenization and escalating commitment. In this section we will relate these processes and dynamics to theories of shadow organizing and the development and consequences of negative social capital. organizations. In so doing, we focused explicitly on social processes within the project, where the organizing of the actual practice was given centrality. By studying such processes, we could identify three distinct phases characterized by three corresponding social dynamics, namely insulation, homogenization and escalating commitment. In this section we will relate these processes and dynamics to theories of shadow organizing and the development and consequences of negative social capital. 12 Shadow organizing By studying the three social dynamics in light of the “shadow organizing” metaphor, we can contribute new understandings and perspectives of the negative consequences of social capital (Gherardi et al., 2017). We use this metaphor for interpreting the interacting elements, making it possible to illustrate dynamics of organizing in the interstices of a formal project structure. The different perceptions of the project may be seen as a tree and its shadow. The tree stands for the initially communicated image of the project as a cooperative arena. This view is based on the original idea of the project and illustrates what was expected from the outside. The shadow is a symbol of what is “betwixt and between”, i.e. representing the actual development of the project. The metaphor is used to look past the obvious and focus on the hidden, thereby capturing the social dynamics appearing within the project. First, the social dynamics of insulation can be understood as a process of secrecy. The project group’s inward focus and distancing from cooperative actors was described as the “creation of a bubble” and the limiting of transparency into the project relates to the use of the organizational shadow as a secluded environment enabling processes to develop unnoticed. Discussion Th i f A relatively absent steering group and strong group members created possibilities for the group to act without insight, which made the project develop for a relatively long time before anyone noticed any deviation from the original idea. Second, the social dynamics of homogenization can be understood through the concept of liminality, which describes the grey zone between canonical and non-canonical practices. There is usually a discrepancy between what is prescribed and what is actually practiced. According to Gherardi et al. (2017), professional discretion is a good example of how organizing and working emerge in a grey zone which leaves room in which professionals can maneuver. The social dynamics of homogenization can be understood as a consequence of this liminality, where the project group was given room to design their own roles and assignments. Third, the social dynamics of escalating commitment can be understood through the perspective of performativity. Operating under the shadow of group seclusion, the group created a productive and protective space. This penumbra, or the half-lit environment of the project, enabled unintentionally favorable growth for strong social identification processes, which resulted in overestimation of the group and downward leveling norms. Building on this self-perceived strength, the group tried to use its social capital to act against external impulses to make the project change direction, by various actions, such as turning to the media. The penumbra of the project organization hence created a specific “ecology” for the group, where the shady organizational undergrowth bred a specific performativity. Negative effects of social capital While shadow organizing is a g ff f p While shadow organizing is a useful metaphor for describing and illustrating social dynamics, the theories of social capital and its potential negative consequences are valuable in understanding these dynamics. The development within the studied project will in this section be related to the negative effects of social capital as outlined by Pillai et al. (2017) and Portes (1998) and will further be related to the distinction between bonding, bridging and linking social capital (Szreter and Woolcock, 2004). The three social dynamics described in the results are highly relational in character and can be understood as unfolding through several phases. Figure 2 illustrates the starting point of the group dynamics, where the original idea of the project was for the project staff to act as representatives for their home organizations and to stimulate cooperation between the project and cooperative actors. This corresponds to the idea of fostering bridging social capital by focusing on establishing horizontal relations between persons at the same hierarchical level from different backgrounds (Szreter and Woolcock, 2004). As shown in the results, this idea was not fulfilled in the studied practice. The project saw a rapid development from heterogeneity to homogeneity. While the project design and staffing were based on the idea of placing different professions in the same space and fostering communication with their home organizations through a part-time staffing structure, the project soon evolved into a singular information-processing unit, where differences between the professionals were gradually erased. The staffing structure changed into promoting full-time project employees rather than sharing work hours between the project and the home organizations. 13 g p j g While bridging social capital is based on the combination of differences, bonding social capital celebrates similarity and focuses horizontal relations of trust and reciprocity between individuals with similar social identities at the same hierarchical level (Szreter and Woolcock, 2004). This was enforced in the project by the social dynamics of insulation and homogenization, as illustrated in Figure 3, where the project group distanced themselves from the surrounding organizations, which enabled them to focus on internal group and communication processes. Pillai et al. (2017) describes such group insulation processes as one aspect of groupthink, which serves to exclude others through promoting introversion and disinterest in communication with actors outside the group. This broken connection to the home organizations induced the group to strive for a common project identity. When cooperation turns ugly Negative effects of social capital While shadow organizing is a The following homogenization phase saw a further increased inward focus and limiting of external impulses which resulted in shared sense-making and confirmation bias within the group, with uniformity of beliefs as a consequence. These negative communication effects of social capital can be understood in relation to the negative effects described by Pillai et al. (2017) as dilution of the dialectical process, inhibition of individual learning and postponement of structural adjustments which may be needed to function in relation to the project’s environment. Af h i l d i f i l i d h i i h d f l d h d l f j y p j After the social dynamics of insulation and homogenization had fueled the development of bonding social capital in the group, a social dynamic of escalating commitment commenced. The lack of communication and mistrust between the parties solidified a polarization between those inside and outside the project and resulted in a power struggle. Pillai et al. (2017) describe such escalating commitment as non-rational and as a breeding ground for downward leveling norms which escalate negative behavior. As a result, the project experienced a communication collapse and established an alternative hierarchical order. This hampered the potential for developing linking social capital, i.e. vertical relationships between people interacting across gradients in society (Szreter and Woolcock, 2004). Linking social capital did occur, but as the project actors polarized, so did the relationships between organizational levels; linking social capital was evident among the allied in the respective fractions. The project group developed vertical relationships with the project leader during the insulation and homogenization processes, which added to the project group’s feeling of independence by having representatives from a higher organizational level on their side. The steering group however, being officially the highest hierarchical level in the project organization, was perceived as a threat to the project. Bonding social capital is discussed in the literature as the type of social capital that has perhaps the strongest inherent risk of producing negative effects, as it tends to develop the formation of closed networks and groupthink among members (Pillai et al., 2017). This assumption was supported by our data: the bonding social capital identified had strong negative effects that in the end forced external actors to dissolve the group. Pillai et al. Negative effects of social capital While shadow organizing is a (2017) discuss whether there is a threshold effect of social capital where, up to a certain level, it would mainly have positive consequences, but after the threshold is passed it will produce negative effects. Our results indicate that this process is not linear, but affected by dynamic interactions within social structures, where attention should be placed both on the personal relationships and the organizational or structural levels. In the studied practice, the project was organized to promote bridging and linking social capital but was effectively usurped by strong informal leaders who steered it toward a strongly bonded social group which promoted similarity and resistance to the organizational environment. 14 Although this bonding process unfolded through different phases, this does not imply that what is described here necessarily illustrates a typical trajectory of cooperative projects. The phases in this case were influenced by vague instructions, weak management, strong informal leaders and external influences by a consultant which, when combined, shifted the project from its initial design toward another development and another rationality (Strindlund et al., 2020). The organizing of the actual practice took place in the shadows, making it immune to insight from the steering group. As described in the shadow organizing literature (Gherardi et al., 2017), secrecy and liminality play important parts in allowing for alternative non-canonical practices to develop, where the bonding social capital described here can be explained as a non-canonical practice. In this sheltered space, the project staff could perform their organizing activities and initially even be unaware that they were taking non-sanctioned courses of action. This was revealed only later, after the strong bonds had already been established, causing polarization and communication breakdown and in the end the closure of the project. What grows in the shadows? By interleaving the theoretical assumptions of negative social capital with that of shadow organizing in the context of an empirical study, we can begin to make out the contours of how these are related. The literature emphasizes how social capital is a complex concept which involves both individual and collective dimensions: from cognitive to interpersonal structures (Rostila, 2010; Pillai et al., 2017). Our study illustrates how organizational conditions, organizing practices, relational structures and social identification processes interacted with cognitive structures, e.g. through the retrospective self-reflection from previous group members on how they got carried away and behaved in ways which previously would have been alien to them. The negative effects of social capital thus had both its causes and its consequences in such dynamic interactions, where it would prove difficult to identify explicit and causal relationships. Rather, the shadow organizing metaphor provides a vocabulary to make sense of the ecology in which social capital may grow into weeds rather than flowers. By focusing more specifically on the non-canonical practices within an organizational structure, we can identify processes that develop in the penumbra and which defies insight from external points of view. In such protective spaces, relationships can be established which result in strong cognitive biases toward specific views, which in turn may prove difficult to reconcile with those views championed in the formal organization. 15 The credibility of the study was strengthened by the broad data collection (comprising observations, interviews, focus groups and documentation) and the inclusion of perspectives from several different actors in the analysis. The transferability of the study results is limited to similar cooperative projects between authorities, which means the results should be interpreted according to the specific context. The dependability of the study was strengthened by the joint analysis process where the authors collaborated and contributed with different perspectives; further, they had varying closeness to the empirical context, where the first author knew the context well, while the other authors could take more of a critical outside view. The trustworthiness of the study was strengthened by the interactive approach, where the results were reported back to the organizations and where the results have been acknowledged and confirmed. When cooperation turns ugly Methodological considerations Methodological considerations We found the theorizing process described by Swedberg (2012) to be a valuable methodological tool in structuring and guiding the data analysis. In combination with the ethnographic longitudinal approach which allowed a study of the social interactions within the project and in relation to cooperative actors, the theorizing process enabled the identification of the social dynamics which our results are based on. The research design was interactive in character, meaning that the first author was observing the project and also reported back observations to the project and the When cooperation turns ugly When cooperation turns ugly management. This can be seen both as a strength and a limitation, where the strength is that this facilitated proximity to the project and insight into the everyday developments. A potential limitation with this strategy is that the researcher becomes involved in this development. Reports from the researchers informed management decisions, including the decision to shut down the project, although this was not a recommendation from the researchers. We have throughout the research process tried to manage a balance of closeness and distance, which included refraining from offering specific recommendations for decisions and to clearly communicate that the researchers had an observing role. Conclusions Thi i l h It also emphasizes the importance of prioritizing communication, structure and steering, and building trustful relations between involved actors when organizing new interorganizational projects in order to avoid cooperation to turn ugly. A theoretical conclusion from the study is that the effects of social capital are conditional upon complex interactions within social structures. In this study, we have focused particularly on conditions leading to negative effects, which do not imply that we consider social capital as such to have negative connotations – rather, the concept is theoretically neutral and may be used for analyzing both positive and negative consequences of social organization. As for the negative effects, one interesting finding from our study was that social capital did not develop in a linear fashion where a threshold was reached when its consequences turned from positive to negative. Rather, the effects of social capital were more unpredictable and dependent on a dynamic interplay between people, structures and environments, which influenced the trajectory of the organizing processes. The case described here is illustrative of this point, and we can reasonably assume it is not unique in this respect. 16 We also combined social capital theory with the metaphor of shadow organizing, which proved to be a useful tool to identify unrecognized organizational practices in which social capital may develop. The combination of these two perspectives can offer explanatory value for determining the organizational and interpersonal dynamics which may lead to negative effects of social capital. The metaphor of shadow organizing lends itself well to the context of interorganizational cooperation between public organizations, as there are strong canonical practices often prescribed in legislation. The metaphor allows for an analysis of what happens when such regulated organizations jointly creates a space between them, where the regulations have less reach. This space, which using the metaphor can be described as a penumbra, is only partly lit and hence obscured from insight. The social dynamics described in our analysis serve to explain how this process resulted in a group strong enough to try to resist the dominant organizational logics of their surroundings. A practical conclusion from the study is that anyone organizing cooperation projects that aim to bridge professional competencies or organizational boundaries have to be attentive toward the processes within an organization that takes place beyond the canonical and illuminated practices and designs. Conclusions Thi i l h This article has shown how dysfunctional group processes in a cooperation project can be understood from theories addressing the dark side of social capital (Pillai et al., 2017) and the theoretical metaphor shadow organizing (Gherardi et al., 2017). By analyzing a failed cooperation project between Swedish public organizations, we have shown how negative social capital can be a consequence of poor organization and its interaction with different social dynamics. Hampering of communication, transparency and cooperation, in combination with a homogenized group identity, led to polarized relations which effectively blocked any attempts at communicative or cooperative action. These dynamics occurred in the dark and remained unrecognized by management until late in the project. Our study showcases how these processes lead to deleterious consequences, where strong internal cohesion in a project group caused upper management to shut the project down. The study was carried out in the context of the Swedish public sector, which could, depending on one’s preconceptions, be expected to involve relatively consensus-oriented bureaucrats. This was however not the case. Instead, our results point to high levels of conflict between people grouping together to form tight bonds, and the official organization of the project was hi-jacked and used as a vehicle for non-intended ends. We may hence conclude that when it comes to public services, a Swedish setting is no guarantee for consensus or organizational compliance. The social relations described in this study reek of contempt and hostility, and the theories of negative effects of social capital and shadow organizing serve well as tools for understanding the dynamics that created the milieu for such relations to develop. The contribution of our analysis is how negative effects of social capital are realized in practice through social dynamics. We specifically identify three such dynamics: insulation, homogenization and escalating commitment. These can be used as a terminology identifying dysfunctional identification processes which can characterize interorganizational projects. Here, the boundaries of the project became increasingly non-permeable due to the tight relationships within the group, which made influence from the external milieu nearly impossible. Knowledge of negative effects of social capital and the risks of letting projects become both isolated from and insulated against their environment can increase the understanding of involved actors’ different perspectives and driving forces in interorganizational cooperation. Conclusions Thi i l h In this project, protective spaces were created in the penumbra which became a breeding ground for unexpected practices. If such practices are allowed to grow without insight, they may end up threatening the original intentions and organizational goals. 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(2017), “Workplace social capital and the onset of major depressive episod among workers in Japan: a 3-year prospective cohort study”, Journal of Epidemiology an Community Health, Vol. 71 No. 6, pp. 606-612, doi: 10.1136/jech-2016-208561. Stahl, C. (2010), Cooperation We Trust: Interorganizational Cooperation in Return-To-Work an Labour Market Reintegration, Link€oping University, Link€oping. Stahl, C., Svensson, T. and Ekberg, K. (2011), “From cooperation to conflict? Swedish rehabilitation professionals’ experiences of interorganizational cooperation”, Journal of Occupationa Rehabilitation, Vol. 21 No. 3, pp. 441-448. Stahl, C., Andersen, A., Anderzen, I. and Larsson, K. (2017), “Process evaluation of an interorganizational cooperation initiative in vocational rehabilitation: the Dirigo project” BMC Public Health, Vol. 17 No. 1, doi: 10.1186/s12889-017-4357-x. Strindlund, L., Dahlgren, M.A. and Stahl, C. (2020), “Zooming in on labour market cooperation: a study of a failed project to support unemployed young people”, Social Policy and Administration Vol. 54 No. 3, pp. 410-426. Svensson, L., Brulin, G., Jansson, S. and Sj€oberg, K. (2009), Learning through Ongoing Evaluation Studentlitteratur, Lund. Swedberg, R. (2012), “Theorizing in sociology and social science: turning to the context of discovery” Theory and Society, Vol. 41 No. 1, pp. 1-40, doi: 10.1007/s11186-011-9161-5. Szreter, S. and Woolcock, M. (2004), “Health by association? Social capital, social theory, and th political economy of public health”, International Journal of Epidemiology, Vol. 33 No. 4 pp. 650-667. Tsai, W. and Ghoshal, S. (1998), “Social capital and value creation: the role of intrafirm networks” Academy of Management Journal, Vol. 41 No. 4, pp. 464-476, available at: http://www.scopus com/inward/record.url?eid52-s2.0-0032220427&partnerID540&md5539f35e755fc66c55 4bdac3e4c233d89. van Deth, J.W. and Zmerli, S. (2009), “Introduction: civicness, equality, and democracy—a ‘dark side of social capital?”, American Behavioral Scientist, Vol. 53 No. 5, pp. 631-639, doi: 10.1177 0002764209350827. Whittaker, C.G. and Holland-Smith, D. Corresponding author Christian Stahl can be contacted at: christian.stahl@liu.se
https://openalex.org/W2116480472	https://www.scielo.br/j/csp/a/Cx7CyZDx5qGwyQpyLSkDsbT/?lang=pt&format=pdf	Portuguese	null	Baixo peso ao nascer no Brasil de acordo com as informações sobre nascidos vivos do Ministério da Saúde, 2005	Cadernos de Saúde Pública	2,008	cc-by	5,292	1 Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz, Rio de Janeiro, Brasil. 2 Instituto de Comunicação e Informação Científica e Tecnológica em Saúde, Fundação Oswaldo Cruz, Rio de Janeiro, Brasil. 3 Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brasil. ARTIGO ARTICLE 2564 ARTIGO ARTICLE 2564 ARTIGO ARTICLE 2564 Baixo peso ao nascer no Brasil de acordo com as informações sobre nascidos vivos do Ministério da Saúde, 2005 Low birth weight in Brazil according to live birth data from the Ministry of Health, 2005 Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Correspondência C. L. T. Andrade Departamento de Administração e Planejamento em Saúde, Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz. Rua Leopoldo Bulhões 1480, Rio de Janeiro, RJ 21041-210, Brasil. carlamv@ensp.fiocruz.br Carla Lourenço Tavares de Andrade 1 Celia Landmann Szwarcwald 2 Euclides Ayres de Castilho 3 Abstract Although the Brazilian Information System on Live Births (SINASC) has expanded its coverage and improved its data quality since the system was implemented, the live birth count in Brazil is still not complete. The current study analyzes inequalities in low birth weight in Brazil in 2005, based on geographic factors, municipality of resi- dence, maternal schooling, and prenatal care. An analysis of all live births showed a low birth weight paradox, namely high percentages in ar- eas with the highest socioeconomic development. The main explanation for these paradoxical find- ings involves shorter neonatal survival and inad- equate recording of premature births in poorer municipalities. Considering at-term live births from non-multiple gestations, inequalities were found in maternal schooling. Comprehensive, quality prenatal care could have a greater im- pact by reducing negative outcomes of gestation and decreasing the socioeconomic inequalities of perinatal health in Brazil. O Sistema de Informação sobre Nascidos Vivos (SINASC), implantado no ano de 1990 pelo Mi- nistério da Saúde, tem como base a Declaração de Nascido Vivo, documento cuja emissão é obri- gatória no serviço de saúde onde ocorreu o parto. Diferentemente do sistema de nascimentos do Registro Civil, cujo objetivo principal é a conta- gem do número de registros de nascimentos, o SINASC tem como propósito caracterizar as con- dições de nascimento, de acordo com alguns fa- tores como peso ao nascer, duração da gestação, tipo de parto, idade da mãe e paridade que, re- conhecidamente, influenciam o estado de saúde da criança. Atualmente, o SINASC está implantado em todos os estados brasileiros, e as informações são divulgadas pela página da Internet do De- partamento de Informática do SUS (DATASUS; http://www.datasus.gov.br), estando disponíveis por município de 1994 a 2005. Embora seja per- ceptível que a cobertura do SINASC esteja cres- cendo e que a qualidade da informação venha melhorando desde a sua implantação, sabe-se que a cobertura do sistema ainda não é comple- ta, tendo sido estimada em 89% no ano de 2004 (DATASUS. http://tabnet.datasus.gov.br/cgi/ idb2006/f10.htm, acessado em 16/Ago/2007). A análise dos dados de nascidos vivos mostra ainda deficiências na cobertura do SINASC em alguns municípios do país, embora de menor magni- Live Birth; Low Birth Weight Infant; Information Systems Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 BAIXO PESO AO NASCER NO BRASIL 2565 tude que as apresentadas pelo Sistema de Infor- mações sobre Mortalidade (SIM) do Ministério da Saúde 1. Resultados Na Tabela 1, os indicadores são apresentados se- gundo região e categoria populacional. Analisan- do-se a variável relativa ao grau de escolaridade das mães, percebem-se as grandes desigualdades sócio-espaciais: os percentuais mais elevados de baixa escolaridade encontram-se nas regiões Norte e Nordeste, e dentro das regiões, nos muni- cípios com menos de 50 mil habitantes. Chama a atenção que na Região Nordeste 61,8% das mães têm Ensino Fundamental incompleto e, entre as que residem nos municípios de menor porte, o percentual alcança 72%. No presente estudo, analisam-se as desigual- dades da proporção de baixo peso ao nascer, no Brasil, em 2005. As desigualdades são examina- das à luz de alguns recortes como o geográfico, tamanho da população do município, e escola- ridade da mãe. Examina-se, ainda, a influência da atenção pré-natal em amenizar as desigual- dades. Com relação à assistência pré-natal, os indi- cadores obedecem a um gradiente sociogeográ- fico similar. No que se refere ao percentual de mães com 7 ou mais consultas, os municípios com 50 mil habitantes ou mais de todas as regi- ões são os que apresentam os maiores valores. Esse indicador varia de 33,8% na Região Norte a 69,9% na Região Sudeste. Já o percentual de mães sem atendimento pré-natal chega a atingir 8% entre os municípios com menos de 50 mil ha- bitantes da Região Norte. É importante enfati- zar, porém, que nas regiões mais desenvolvidas do país as proporções de nenhuma consulta são ligeiramente menores entre as mães residentes nos municípios de pequeno tamanho popula- cional. Abstract ao nascer > 6.000g), idade gestacional (< 32 se- manas, 32 a 36 semanas, ≥ 37 semanas), tipo de gravidez (não-múltipla, múltipla), número de consultas de pré-natal (nenhuma consulta, 1 a 6 consultas, 7 ou mais consultas), tipo de parto (vaginal, cesáreo) e grau de escolaridade da mãe (Ensino Fundamental incompleto: < 8 anos de estudo; Ensino Fundamental completo: ≥ 8 anos de estudo). A forma de categorização das variá- veis obedeceu à disponibilidade das informações do SINASC. Um expressivo número de estudos, tanto da literatura nacional como na estrangeira, tem mostrado que filhos de mães socialmente des- favorecidas têm um maior risco de baixo peso ao nascer 2,3,4. A análise dos dados do SINASC permite verificar a ocorrência do baixo peso ao nascer no Brasil, bem como estabelecer as desi- gualdades socioespaciais no território nacional. Com base nessas variáveis, foram construí- dos os indicadores: percentual de baixo peso ao nascer (peso < 2.500g), percentual de prematuri- dade (< 37 semanas de gestação), percentual de mães sem consulta de pré-natal, percentual de mães com 7 ou mais consultas de pré-natal, per- centual de partos cesáreos, percentual de mães com Ensino Fundamental incompleto. O peso do bebê ao nascimento é fortemente associado ao risco de morrer no primeiro ano de vida 5 e em grau menor, com problemas de de- senvolvimento na infância, além da maior pro- babilidade de várias doenças na vida adulta 6. Controlando-se ou não por idade gestacional, a proporção de baixo peso ao nascer é também o indicador mais comumente utilizado para ava- liar os cuidados no pré-natal 7. Paradoxalmente, apesar da associação da ocorrência de baixo peso ao nascer com o menor nível sócio-econômico, as análises em subgru- pos populacionais específicos têm mostrado cor- relações em sentido contrário. Estudos desen- volvidos nos Estados Unidos mostraram que as proporções de baixo peso ao nascer são menores em populações de menor nível sócio-econômico, como entre os mexicanos residentes naquele pa- ís 3,8,9. De maneira similar, estudo no Brasil, com- parando dados de nascidos vivos notificados em São Luís (Maranhão) e Ribeirão Preto (São Paulo) 10 revelou que as regiões mais desenvolvidas do Brasil detêm maiores taxas de baixo peso ao nas- cer do que as regiões menos desenvolvidas. Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Material e métodos As informações usadas neste estudo foram pro- venientes do SINASC para todos os municípios brasileiros em 2005, último ano com informa- ções disponíveis. A análise foi realizada por Gran- de Região e porte populacional (municípios com menos de 50 mil habitantes e municípios com 50 mil habitantes ou mais). Os dados da Tabela 1 mostram uma inversão do gradiente sócio-espacial para a ocorrência de baixo peso ao nascer: os municípios com po- pulação de 50 mil habitantes ou mais apresen- tam, sem exceção, percentuais maiores do que os municípios com menos de 50 mil habitantes, para todas as macrorregiões geográficas. Merece As informações da mãe e da criança foram analisadas utilizando-se as seguintes variáveis: peso ao nascer (eliminando-se da análise nas- cidos vivos com peso ao nascer < 500g ou peso Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Andrade CLT et al. 2566 Tabela 1 Indicadores selecionados construídos com base em informações do Sistema de Informação sobre Nascidos Vivos (SINASC) segundo Grande Região e categoria populacional. Brasil, 2005. Indicadores selecionados construídos com base em informações do Sistema de Informação sobre Nascidos Vivos (SINASC) segundo Grande Região e categoria populacional. Brasil, 2005. Material e métodos Região/Categoria Mães com Mães sem Mães com Baixo peso Prematuridade Parto populacional Ensino Fundamental consulta 7 ou mais consultas ao nascer (%) cesáreo (habitantes) incompleto (%) pré-natal (%) de pré-natal (%) (%) (%) Norte < 50.000 70,6 8,0 22,0 5,9 4,8 25,2 ≥ 50.000 51,7 5,0 33,8 7,3 5,4 37,2 Total 59,2 6,2 29,1 6,8 5,2 32,4 Nordeste < 50.000 72,3 4,0 30,4 6,5 5,1 24,7 ≥ 50.000 51,6 3,4 41,5 8,3 6,2 39,3 Total 61,8 3,7 36,0 7,4 5,7 32,1 Sudeste < 50.000 51,3 1,2 60,8 8,2 6,3 48,7 ≥ 50.000 34,4 1,5 69,9 9,2 7,8 52,6 Total 38,1 1,4 67,9 9,0 7,5 51,7 Sul < 50.000 51,8 0,8 66,8 7,8 6,4 49,1 ≥ 50.000 37,6 1,5 69,3 8,9 8,0 50,3 Total 43,2 1,2 68,3 8,4 7,3 49,8 Centro-oeste < 50.000 53,5 1,4 58,7 6,4 5,3 47,3 ≥ 50.000 38,4 1,9 60,4 8,1 7,4 50,5 Total 43,7 1,7 59,8 7,5 6,7 49,4 Brasil < 50.000 62,6 3,1 44,2 7,1 5,5 35,7 ≥ 50.000 40,8 2,3 58,8 8,7 7,2 47,5 Total 48,5 2,6 53,6 8,1 6,6 43,3 notar que entre os nascidos vivos com menos de 32 semanas de gestação os percentuais de baixo peso ao nascer são, invariavelmente, maiores do que 95% e entre os nascidos a termo, menores do que 5%. Contrariamente, há uma grande varia- ção nos percentuais de baixo peso ao nascer en- tre os nascidos vivos com idade gestacional entre 32 e 36 semanas, de 34,7% na Região Norte, nos municípios pequenos, a 59,2% na Nordeste, nos municípios grandes. Analisando-se o indicador por categoria populacional, chamam a atenção também as diferenças intra-regionais na propor- ção de baixo peso ao nascer na categoria de idade gestacional 32-36 semanas, em que são obser- vados para todas as macrorregiões geográficas percentuais mais elevados nos municípios com 50 mil habitantes ou mais. destacar que as regiões Sudeste e Sul são as que possuem percentuais mais elevados de baixo pe- so ao nascer, atingindo valores de 9% entre os municípios de maior tamanho populacional. No que diz respeito à análise dos dados de idade gestacional por região e categoria popu- lacional, a freqüência relativa de prematuridade apresenta comportamento semelhante ao per- centual de baixo peso ao nascer (Tabela 1). O percentual mais elevado de nascidos vivos pre- maturos é observado nos municípios com 50 mil habitantes ou mais da Região Sul (8%). Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Material e métodos O indicador referente ao tipo de parto, apre- sentado também na Tabela 1, revela que o per- centual de parto cesáreo é maior nos municípios com 50 mil habitantes ou mais em todas as regi- ões, variando de 37,2% na Região Norte a 52,6% na Sudeste. Na Tabela 3, apresenta-se o percentual de bai- xo peso ao nascer por grau de escolaridade da mãe, por categoria populacional e região, entre os nascidos vivos com 37 ou mais semanas de gesta- O percentual de baixo peso ao nascer por ida- de gestacional segundo categoria populacional e região é apresentado na Tabela 2. É interessante Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 BAIXO PESO AO NASCER NO BRASIL 2567 Tabela 2 Percentual de baixo peso ao nascer por idade gestacional segundo Grande Região e categoria populacional. Brasil, 2005. Região/Idade gestacional Baixo peso ao nascer (%) Total (semanas) < 50.000 habitantes 50.000 habitantes Norte < 32 97,8 96,1 96,7 32-36 34,7 55,1 47,4 37 ou mais 4,1 4,2 4,2 Total 5,9 7,3 6,8 Nordeste < 32 97,9 97,8 97,9 32-36 45,6 59,2 53,2 37 ou mais 4,0 4,4 4,2 Total 6,5 8,2 7,3 Sudeste < 32 98,0 98,0 98,0 32-36 56,7 59,0 58,6 37 ou mais 4,5 4,4 4,4 Total 8,2 9,1 8,9 Sul < 32 98,8 98,7 98,7 32-36 56,0 59,0 58,0 37 ou mais 4,1 4,0 4,0 Total 7,8 8,8 8,4 Centro-oeste < 32 98,4 98,0 98,1 32-36 51,5 51,7 51,7 37 ou mais 3,4 4,0 3,8 Total 6,3 8,0 7,4 Brasil < 32 98,1 97,9 98,0 32-36 49,7 58,2 55,6 37 ou mais 4,1 4,3 4,2 Total 7,0 8,6 8,0 escolaridade da mãe. A tendência de decrésci- mo da proporção de baixo peso ao nascer com o aumento do número de consultas é consta- tada em todas as regiões e nas duas categorias de nível de instrução. Na Região Sul, entre as mães sem atendimento pré-natal e com Ensino Fundamental incompleto, o percentual de baixo peso ao nascer atinge 9,2%, mesmo em crianças nascidas a termo. escolaridade da mãe. A tendência de decrésci- mo da proporção de baixo peso ao nascer com o aumento do número de consultas é consta- tada em todas as regiões e nas duas categorias de nível de instrução. Material e métodos Na Região Sul, entre as mães sem atendimento pré-natal e com Ensino Fundamental incompleto, o percentual de baixo peso ao nascer atinge 9,2%, mesmo em crianças nascidas a termo. ção e gravidez não-múltipla. São constatadas as desigualdades do baixo peso ao nascer por grau de instrução da mãe, com percentuais de baixo peso sempre menores entre as mães de maior grau de escolaridade, qualquer que seja o estrato composto por região e categoria populacional. Analisando-se o percentual de baixo peso ao nascer por tipo de parto segundo categoria po- pulacional e Grande Região, entre nascidos vivos com 37 ou mais semanas de gestação e gravidez não-múltipla, observa-se que o percentual de baixo peso ao nascer é sempre menor entre as mães que realizaram partos por via cesárea do que entre aquelas com parto vaginal (Tabela 4). Discussão A análise realizada neste estudo, sobre as in- formações contidas no SINASC, teve o objetivo de avaliar as desigualdades sócio-espaciais do baixo peso ao nascer no Brasil. Observou-se o Na Tabela 5, pode-se analisar o percentual de baixo peso ao nascer por atendimento pré- natal segundo região de residência e grau de Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Andrade CLT et al. 2568 Andrade CLT et al. 2568 Tabela 3 Percentual de baixo peso ao nascer por grau de escolaridade da mãe segundo Grande Região e categoria populacional entre os nascidos vivos de 37 ou mais semanas de gestação e gravidez não-múltipla. Brasil, 2005. Percentual de baixo peso ao nascer por grau de escolaridade da mãe segundo Grande Região e categoria populacional entre os nascidos vivos de 37 ou mais semanas de gestação e gravidez não-múltipla. Brasil, 2005. Percentual de baixo peso ao nascer por grau de escolaridade da mãe segundo Grande Região e categoria populacional entre os nascidos vivos de 37 ou mais semanas de gestação e gravidez não-múltipla. Brasil, 2005. Região/Grau de escolaridade Baixo peso ao nascer (%) Total < 50.000 habitantes 50.000 habitantes Norte Ensino Fundamental incompleto 3,8 4,2 4,0 Ensino Fundamental completo 3,1 3,5 3,4 Total 3,6 3,9 3,8 Nordeste Ensino Fundamental incompleto 3,7 4,4 4,0 Ensino Fundamental completo 3,0 3,5 3,3 Total 3,5 4,0 3,7 Sudeste Ensino Fundamental incompleto 4,6 4,7 4,6 Ensino Fundamental completo 3,5 3,6 3,5 Total 4,0 3,9 4,0 Sul Ensino Fundamental incompleto 4,2 4,3 4,2 Ensino Fundamental completo 3,0 3,2 3,1 Total 3,6 3,6 3,6 Centro-oeste Ensino Fundamental incompleto 3,5 4,0 3,8 Ensino Fundamental completo 2,6 3,3 3,1 Total 3,1 3,6 3,4 Brasil Ensino Fundamental incompleto 3,9 4,4 4,2 Ensino Fundamental completo 3,1 3,5 3,4 Total 3,6 3,9 3,8 dades da Federação 13, ainda há dificuldades em se obter indicadores para o monitoramento da mortalidade perinatal com base nas informações do Ministério da Saúde, devido, principalmente, a falhas no preenchimento das declarações de óbito, não sendo possível, muitas vezes, saber o momento da morte. paradoxo do baixo peso ao nascer com os per- centuais mais baixos de baixo peso ao nascer nas localidades menos favorecidas socialmen- te. Os resultados confirmam achados anteriores encontrados no Brasil, nos Estados Unidos e na Noruega 9,10,11,12. Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Discussão Tanto no que se refere à análi- se por Grande Região como por categoria popu- lacional, foram, invariavelmente, encontrados maiores percentuais de baixo peso ao nascer nas áreas geográficas de maior desenvolvimen- to sócio-econômico. A análise por idade gestacional revelou que existem diferenças substanciais nas proporções de baixo peso ao nascer entre os nascidos vivos com 32 a 36 semanas de gestação, fornecendo evidências da subnotificação de nascidos de 32 a 36 semanas de gestação com baixo peso nas regiões Norte e Nordeste, principalmente nos municípios com menos de 50 mil habitantes. Es- tudo anterior realizado no Brasil, que relacionou o Sistema de Internações Hospitalares (SIH) com o SINASC 14, mostrou, igualmente, maior sub- registro de nascimentos justamente nos municí- pios pequenos, de tal modo que quanto menor o porte populacional, tanto maior a subnotificação de nascidos vivos no SINASC. Uma hipótese explicativa para o paradoxo do baixo peso ao nascer é o aumento de nascimen- tos pré-termo, que antes constituíam natimortos. Nas áreas menos desenvolvidas do país, devido à atenção inadequada e precariedade de acesso à tecnologia perinatal, muitos desses nascidos vi- vos morrem logo após o parto e, freqüentemente, não são registrados ou são classificados indevi- damente como natimortos, reduzindo as propor- ções de prematuridade nestes locais. Conforme evidenciado em estudo realizado em oito Uni- BAIXO PESO AO NASCER NO BRASIL 2569 Tabela 4 Percentual de baixo peso ao nascer por tipo de parto entre os nascidos vivos de 37 ou mais semanas de gestação e gravidez não-múltipla, segundo Grande Região e categoria populacional. Brasil, 2005. Região/Tipo de parto Baixo peso ao nascer (%) Total < 50.000 habitantes 50.000 habitantes Norte Vaginal 3,8 4,1 4,0 Cesáreo 3,0 3,4 3,3 Total 3,6 3,9 3,8 Nordeste Vaginal 3,6 4,4 3,9 Cesáreo 3,1 3,3 3,3 Total 3,5 4,0 3,7 Sudeste Vaginal 4,7 4,3 4,4 Cesáreo 3,3 3,6 3,6 Total 4,0 4,0 4,0 Sul Vaginal 4,2 3,9 4,0 Cesáreo 3,1 3,3 3,2 Total 3,6 3,6 3,6 Centro-oeste Vaginal 3,6 4,0 3,8 Cesáreo 2,4 3,2 3,0 Total 3,1 3,6 3,4 Brasil Vaginal 3,9 4,2 4,1 Cesáreo 3,1 3,5 3,4 Total 3,6 3,9 3,8 Percentual de baixo peso ao nascer por tipo de parto entre os nascidos vivos de 37 ou mais semanas de gestação e gravidez não-múltipla, segundo Grande Região e categoria populacional. Brasil, 2005. As falhas de registro de nascimentos prematu- ros foram também verificadas em outros países. Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Discussão Em estudo realizado na Suíça, os autores con- cluíram que um significante número de crianças com baixo peso ao nascer, cuja morte é próxima ao momento do parto, não é oficialmente regis- trado 15. Análise da adequação dos dados prove- nientes dos certificados de nascimentos do Esta- do de New Jersey, Estados Unidos, foi realizada mediante a comparação com dados obtidos em um programa estadual de pré-natal, entre 1989 e 1992, de acordo com as características sócio-eco- nômicas da mãe 16. Os resultados indicaram que os piores percentuais de concordância do baixo peso ao nascer e da prematuridade ocorreram entre as mães de pior nível sócio-econômico sem proficiência na língua inglesa. dos vivos prematuros. Embora a mortalidade infantil nos Estados Unidos tenha mostrado re- levante decréscimo nas últimas décadas, o per- centual de baixo peso ao nascer tem mostrado nítido acréscimo: de 6,7% em 1984, passou a 8,1% em 2004, acompanhando a tendência de crescimento da prematuridade. Os resultados de Barros et al. 18, que comparam indicado- res de duas coortes em Pelotas (1982 e 1993), Rio Grande do Sul, indicam que o mesmo vem ocorrendo no Brasil. Em outro trabalho realizado no Brasil, com- parando os dados de Ribeiro Preto (Região Su- deste) e São Luís (Região Nordeste), foi obser- vado o mesmo paradoxo para o baixo peso ao nascer, encontrando-se taxa significativamente maior na cidade mais rica. Fatores como deter- minações incorretas da idade gestacional, sub- registro de nascidos vivos, bem como falhas no registro de nascidos vivos como nascidos mor- tos nos municípios menos desenvolvidos são considerados pelos autores como responsáveis, Segundo as estatísticas vitais da população norte-americana, no ano de 2004 17 a propor- ção de prematuridade aumentou de 9,4% em 1981 para 10,6% em 1990 e atingiu 12,5% em 2004, refletindo a maior sobrevida dos nasci- Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Andrade CLT et al. 2570 Andrade CLT et al. Tabela 5 Percentual de baixo peso ao nascer por número de consultas de pré-natal e grau de escolaridade da mãe, segundo Grande Região entre os nascidos vivos de 37 ou mais semanas de gestação e gravidez não-múltipla. Brasil, 2005. Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Discussão Região/Número de Baixo peso ao nascer (%) consultas de pré-natal Ensino Fundamental Ensino Fundamental Total incompleto completo Norte Nenhuma 5,6 5,6 5,6 1-6 4,0 3,6 3,9 7 ou mais 3,4 3,1 3,2 Total 4,0 3,4 3,8 Nordeste Nenhuma 6,3 6,0 6,3 1-6 4,2 3,7 4,0 7 ou mais 3,2 2,7 3,0 Total 4,0 3,3 3,7 Sudeste Nenhuma 9,0 7,4 8,6 1-6 5,4 4,6 5,0 7 ou mais 3,9 3,2 3,4 Total 4,6 3,5 4,0 Sul Nenhuma 9,2 7,8 8,8 1-6 5,0 4,2 4,6 7 ou mais 3,6 2,8 3,1 Total 4,2 3,1 3,6 Centro-oeste Nenhuma 6,6 5,9 6,4 1-6 4,4 3,8 4,1 7 ou mais 3,1 2,7 2,9 Total 3,8 3,1 3,4 Brasil Nenhuma 6,7 6,5 6,7 1-6 4,5 4,0 4,3 7 ou mais 3,6 3,0 3,2 Total 4,2 3,4 3,8 Percentual de baixo peso ao nascer por número de consultas de pré-natal e grau de escolaridade da mãe, segundo Grande Região entre os nascidos vivos de 37 ou mais semanas de gestação e gravidez não-múltipla. Brasil, 2005. percentual de mulheres que têm trabalho remu- nerado é significativamente maior nas cidades de grande porte populacional. pelo menos parcialmente, pelas taxas maiores de baixo peso ao nascer nos municípios de me- lhor nível sócio-econômico 10. Em estudo prospectivo de coorte de nascidos vivos em 1982, 1993 e 2004, na cidade de Pelotas, mostrou-se que a prevalência de prematuridade cresceu de 6,3% em 1982 para 16,2% em 2004, correspondendo a uma redução de 47 gramas na média do peso ao nascer, apesar da melho- ria das condições de vida materna 21. Em ver- tente explicativa complementar, Barros et al. 21 consideram o papel da excessiva medicalização, incluindo indução do parto, maior proporção de partos cesáreos e ultra-sonografias impreci- sas ou mal interpretadas. Outras possíveis explicações para os resulta- dos paradoxais do baixo peso ao nascer recaem no comportamento diferenciado da mulher nas áreas urbanas, tais como a freqüência maior do hábito de fumar na gestação, que é um fator, reconhecidamente, associado ao baixo peso ao nascer 6. O estilo de vida, incluindo o estresse no trabalho, pode ser outro fator associado à maior ocorrência de prematuridade, e, conseqüente- mente, à maior freqüência de baixo peso ao nas- cer 19. Dados da Pesquisa Mundial da Saúde 20, realizada no Brasil em 2003, revelaram que o Cad. Discussão Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 BAIXO PESO AO NASCER NO BRASIL 2571 No Município do Rio de Janeiro, análise se- gundo a esfera administrativa do estabelecimen- to onde se realizou o parto (público ou privado) mostrou a influência da medicalização excessiva, além da enorme proporção de partos cesarianos ocorridos em estabelecimentos não-públicos (87%) 22. Dados do SINASC, no mesmo municí- pio, em 2005, mostram que a proporção de be- bês de baixo peso foi maior entre as mães que tiveram parto cesáreo (DATASUS. http://tabnet. datasus.gov.br/cgi/tabcgi.exe?sinasc/cnv/nvrj. def, acessado em 22/Ago/2007). os percentuais de baixo peso ao nascer são me- nores entre as mães que realizaram 7 ou mais consultas de pré-natal, número de consultas considerado adequado pelo Ministério da Saú- de. Conforme discutido por Koblinsky et al. 26, o impacto do pré-natal poderia ser mais inten- so, dependendo da capacidade dos serviços em identificar as gestantes de alto risco, prevenir ou tratar complicações, e referir para atenção obs- tétrica especializada, quando necessário. O papel da atenção básica como elemento- chave para a promoção da eqüidade tem sido enfatizado, principalmente nas sociedades com grandes disparidades sociais 27, podendo-se in- ferir que o atendimento pré-natal abrangente e adequado poderia ter maior impacto na redu- ção dos resultados adversos da gestação. No caso particular deste estudo, o desempenho favorável do pré-natal indica que muito ainda pode ser fei- to no âmbito do setor saúde para diminuição das desigualdades do baixo peso ao nascer por grau de escolaridade no Brasil. É preciso enfatizar, ainda, a necessidade de melhora da cobertura e da qualidade das informações sobre nascidos vi- vos, sobretudo nas regiões menos desenvolvidas, para possibilitar a análise das informações com fidedignidade. Corroborando os achados de vários estudos que mostram as disparidades do baixo peso ao nascer por nível sócio-econômico da mãe 23, os dados do SINASC, em âmbito nacional, mostram que as proporções de nascidos vivos a termo de gestação não-múltipla com peso inferior a 2.500g são maiores tanto menor o grau de escolaridade da mãe, única variável representativa do nível só- cio-econômico disponível no SINASC. Um outro achado importante deste estudo foi a relevância do atendimento pré-natal no sentido de amenizar as desigualdades do baixo peso ao nascer. Assim como em trabalhos ante- riores realizados em outros países da América Latina 24,25, os dados brasileiros mostram que Nascimento Vivo; Recém-Nascido de Baixo Peso; Siste- mas de Informação Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Referências 14. Drumond EF, Machado CJ, França E. Underreport- ing of live births: measurement procedures using the Hospital Information System. Rev Saúde Públi- ca 2008; 42:55-63. 1. Andrade CLT, Szwarcwald CL. Desigualdades só- cio-espaciais da adequação das informações de nascimentos e óbitos do Ministério da Saúde, Brasil, 2000-2002. Cad Saúde Pública 2007; 23: 1207-16. 15. Muller M, Drack G, Schindler C, Bucher HU. Live and stillborn very low birthweight infants in Swit- zerland: comparison between hospital based birth registers and the national birth register. Swiss Med Wkly 2005; 135:433-9. 2. Gama SG, Szwarcwald CL, Leal MC, Theme-Filha MM. The pregnancy during adolescence as a risk factor for low birth weight, Brazil. Rev Saúde Públi- ca 2001; 35:74-80. 16. Reichman N, Schwartz-Soicher O. Accuracy of birth certificate data by risk factors and outcomes: analysis of data from New Jersey. Am J Obstet Gy- necol 2007; 197:32.e1-8. 3. Parker JD, Schoendorf KC, Kiely JL. Associations between measures of socioeconomic status and low birth weight, small for gestational age, and premature delivery in the United States. Ann Epi- demiol 1994; 4:271-8. 17. Hoyert DL, Mathews TJ, Menacker F, Strobino DM, Guyer B. Annual summary of vital statistics: 2004. Pediatrics 2006; 117:168-83. 4. Menezes AMB, Barros FC, Victora CG, Alves C, Rocha C, Albernaz E, et al. Mortalidade perinatal em duas coortes de base populacional no Sul do Brasil: tendências e diferenças. Cad Saúde Pública 1996; 12 Suppl 1:S33-41. 18. Barros FC, Victora CG, Vaughan JP, Tomasi E, Horta BL, Cesar JA, et al. The epidemiological transition in maternal and child health in a Brazilian city, 1982-93: a comparison of two population-based cohorts. Paediatr Perinat Epidemiol 2001; 15:4-11. 5. Luginaah IN, Lee KS, Abernathy TJ, Sheehan D, Webster G. Trends and variations in perinatal mortality and low birthweight: the contribution of socio-economic factors. Can J Public Health 1999; 90:377-81. 19. Gabbe SG, Turner LP. Reproductive hazards of the American lifestyle: work during pregnancy. Am J Obstet Gynecol 1997; 176:826-32. 20. Leal MC, Gama SGN, Frias P, Szwarcwald CL. Healthy lifestyles and access to periodic health ex- ams among Brazilian women. Cad Saúde Pública 2005; 21 Suppl:S78-88. 6. Wilcox AJ. On the importance – and the unim- portance – of birthweight. Int J Epidemiol 2001; 30:1233-41. 7. Fiscella K. Does prenatal care improve birth out- comes? A critical review. Obstet Gynecol 1995; 85:468-79. 21. Cad. Saúde Pública, Rio de Janeiro, 24(11):2564-2572, nov, 2008 Colaboradores C. L. T. Andrade foi responsável pela elaboração do texto e dos resultados. C. L. Szwarcwald participou na con- cepção do artigo e na elaboração do texto. E. A. Castilho participou na discussão dos resultados. Embora seja notório que a cobertura do Sistema de Informação sobre Nascidos Vivos (SINASC) esteja crescendo e que a qualidade da informação venha melhorando, desde a sua implantação, sabe-se que a enumeração de nascidos vivos ainda não é comple- ta no Brasil. Neste trabalho, objetiva-se analisar as desigualdades da proporção do baixo peso ao nascer no Brasil, em 2005, segundo alguns aspectos como o geográfico, o tamanho da população do município e a escolaridade da mãe. Analisou-se, igualmente, a in- fluência da atenção pré-natal. Considerando a totali- dade dos nascidos vivos, evidenciou-se o paradoxo do baixo peso ao nascer, ou seja, foram encontrados per- centuais mais elevados nas áreas de maior desenvol- vimento sócio-econômico. Os resultados paradoxais são explicados, principalmente, pela menor sobrevida e registro inadequado dos prematuros nos municípios mais pobres. Levando-se em conta os nascidos vivos a termo de gestação não-múltipla, foram encontradas desigualdades por grau de escolaridade da mãe. 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Recebido em 06/Dez/2007 Versão final reapresentada em 16/Abr/2008 Aprovado em 18/Abr/2008 Recebido em 06/Dez/2007 Versão final reapresentada em 16/Abr/2008 Aprovado em 18/Abr/2008
https://openalex.org/W2575815406	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-016-3440-5	English	null	Single genome retrieval of context-dependent variability in mutation rates for human germline	BMC genomics	2,017	cc-by	17,293	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 DOI 10.1186/s12864-016-3440-5 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 DOI 10.1186/s12864-016-3440-5 Correspondence: as952@cam.ac.uk; sb10031@cam.ac.uk 1Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, UK Full list of author information is available at the end of the article Abstract Background: Accurate knowledge of the core components of substitution rates is of vital importance to understand genome evolution and dynamics. By performing a single-genome and direct analysis of 39,894 retrotransposon remnants, we reveal sequence context-dependent germline nucleotide substitution rates for the human genome. Results: The rates are characterised through rate constants in a time-domain, and are made available through a dedicated program (Trek) and a stand-alone database. Due to the nature of the method design and the imposed stringency criteria, we expect our rate constants to be good estimates for the rates of spontaneous mutations. Benefiting from such data, we study the short-range nucleotide (up to 7-mer) organisation and the germline basal substitution propensity (BSP) profile of the human genome; characterise novel, CpG-independent, substitution prone and resistant motifs; confirm a decreased tendency of moieties with low BSP to undergo somatic mutations in a number of cancer types; and, produce a Trek-based estimate of the overall mutation rate in human. Background: Accurate knowledge of the core components of substitution rates is of vital importance to understand genome evolution and dynamics. By performing a single-genome and direct analysis of 39,894 retrotransposon remnants, we reveal sequence context-dependent germline nucleotide substitution rates for the human genome. Results: The rates are characterised through rate constants in a time-domain, and are made available through a Background: Accurate knowledge of the core components of substitution rates is of vital importance to understand genome evolution and dynamics. By performing a single-genome and direct analysis of 39,894 retrotransposon remnants, we reveal sequence context-dependent germline nucleotide substitution rates for the human genome. Results: The rates are characterised through rate constants in a time-domain, and are made available through a dedicated program (Trek) and a stand-alone database. Due to the nature of the method design and the imposed stringency criteria, we expect our rate constants to be good estimates for the rates of spontaneous mutations. Benefiting from such data, we study the short-range nucleotide (up to 7-mer) organisation and the germline basal substitution propensity (BSP) profile of the human genome; characterise novel, CpG-independent, substitution prone and resistant motifs; confirm a decreased tendency of moieties with low BSP to undergo somatic mutations in a number of cancer types; and, produce a Trek-based estimate of the overall mutation rate in human. Abstract Conclusions: The extended set of rate constants we report may enrich our resources and help advance our understanding of genome dynamics and evolution, with possible implications for the role of spontaneous mutations in the emergence of pathological genotypes and neutral evolution of proteomes. Keywords: Nucleotide substitutions, Spontaneous mutations, Germline, Context-dependence, Genome composition, Somatic mutations, Cancer break down of the rate into a number of general compo- nents. For a given genomic position and i→j nucleotide conversion, the substitution rate, as expressed by the rate constant ri,j, can be roughly presented as a single-base average value rsb i,j and fluctuations contributed by short- range context (δrsr i,j), CpG-associated (δrCpG i,j ), long-range (δrlr i,j), gene/functional (δrgene i,j ), and specific (δrspec i,j ) effects (1): Single genome retrieval of context-dependent variability in mutation rates for human germline Aleksandr B. Sahakyan1 and Shankar Balasubramanian1,2,3* Revealing the core single-nucleotide substitution rates Revealing the core single-nucleotide substitution rates g g The repetitive occurrence of mobile DNA elements in different regions within the same genome [34] provides the opportunity to obtain the rcore i,j (2) rate constants that account for the δrsr i,j immediate effects of neighbouring nucleotides. After the initial inactivation at different time epochs [36–39], individual remnants of many transpo- son subfamilies within a genome have been subjected to largely the same overall mutagenic and repair conditions as the rest of the genome [40], hence can also serve as markers of rcore i,j neutral substitution rates applicable to genomic sites that share the immediate sequence-context. For the purpose of this study, we have used the homi- noid lineage of the L1 (long interspersed nuclear element 1, LINE-1) retrotransposons, spanning 3.1 to 20.4 myr (million years) of age [37]. The constituent subfamilies of the lineage are L1PA5, L1PA4, L1PA3, L1PA2 and the most recent L1Hs. Their respective age and the number of insertions in the human genome are presented in Table S1 in Additional file 1. The choice was made through the following reasoning. The L1 elements have a long (∼6 k nt) sequence without extended repeats like in the LTR (long terminal repeat) elements [34]. This enables their robust mapping on a chosen template and provides essen- tial local sequence variability around different nucleotide positions within L1 elements. There are distinct L1 sub- families that were active at different time epochs, with detailed molecular clock analyses available [36–39] to reveal and, importantly, validate the age of each subfam- ily. They are well-represented and, unlike other classes of transposable elements, are uniformly scattered across mostly the intergenic regions of the human genome [34, 41, 42], and are less prone to recombination and con- text bias [43, 44]. The young L1 subfamilies have most of their remnants coming from the genomic regions with G+C content close to the genomic average value (Fig. 1, see also [41]). Unlike SINEs (small interspersed nuclear elements) and LTRs, LINE sites show a very low level of RNA polymerase enrichment, as a marker of transcrip- tional association, in normal tissues [45]. Background The stability, organisation and dynamics of genomes are key factors that influence the molecular evolution of life [1]. Genomic single-nucleotide mutations, with their sub- sequent fixation in a population, occur an order of magni- tude more frequently than common insertions/deletions [2, 3], hence are major contributors in defining the genome evolution. An understanding of the descriptors that govern single-nucleotide mutations and substitutions is thus essential to comprehend genome dynamics and its link to the underlying molecular processes. ri,j = rsb i,j + δrsr i,j + δrCpG i,j + δrlr i,j + δrgene i,j + δrspec i,j (1) (1) For the sake of lucidity in pointing to different contri- butions on substitution rates, which may encompass both mutation- and fixation-based effects, let us introduce a The rsb i,j term can be estimated through genomic aver- ages for the individual i→j substitutions, and has been explored for the genomes of human [4, 5] and other species [6–8]. By investigating the aggregation patterns in substitution frequencies, it was shown that the ri,j vari- ation is subjected to two distinct, short-range (< 10 nt) and long-range (> 1000 nt), effects [9, 10]. In the equation Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 2 of 17 above, the short-range effect is captured through the δrsr i,j term and mainly describes the totality of the intrinsic properties and sequence-dependent interactions of DNA with overall mutagenic and reparation processes in a given organism [11]. The better-studied substitution patterns at a CpG context [12–14] are separated in the δrCpG i,j term, since besides having a specific short-range dyad con- text, the CpG mutations underlying the δrCpG i,j substitution term also depend on a number of regional factors that alter the epigenetic targeting of the CpG sites [10, 15–18]. Many relatively recent studies provide essential insights into the δrlr i,j variation caused by the regional effects that depend on a long-range (megabase) sequence con- text through general mechanisms, such as recombination and GC-biased gene conversion [1, 19–21], transcription- coupled biased genome repair [22] and instability [23], chromatin-organisation- [24] and replication-associated mutational bias [25] and inhomogeneous repair [26], dif- ferential DNA mismatch repair [27], non-allelic gene con- version [28], and male mutation bias [29]. Background The term δrgene i,j captures the change in substitution rates in genes and other functional elements under elevated selection bias and may reflect observations such as the increased neu- tral substitution rates in exons [30, 31] and the possible reduction of mutation rates in the X-chromosome [32]. δrspec i,j holds the highly specific increase or decrease in substitution rates governed by a strong selection or tar- geted underlying hyper- and hypomutations [33] present, for example, in the genes of immune system, and may additionally include other effects not captured in prior terms. random DNA sequence, whose key features are in better agreement with the short-range oligomeric organisation of the human genome. We next calculate the basal substi- tution propensity profile of the human genome, evaluating the core predisposition to single-nucleotide substitutions. We outline the decreased frequency of the sequence motifs that are stable in germline among the sites linked to somatic cancer mutations. Revealing the core single-nucleotide substitution rates 2), we expect the absence of the δrgene i,j contribution, the elim- ination of δrlr i,j at the averaging stage (Fig. 2b) and the removal of the δrCpG i,j and δrspec i,j effects through our robust- ness checks embedded within the Trek procedure (see Fig. 2c, d and Methods). Therefore, our method provides the rcore i,j =rsb i,j + δrsr i,j core variation (Fig. 3) of the substi- tution rates at around the rsb i,j genomic average values for each i→j base substitution. If the above is correct and Trek indeed results in rcore i,j values, further averaging of the core rsb i,j +δrsr i,j rates (median values shown in Fig. 3) should give us the single-base rsb i,j genomic average substitution rates, cancelling out the remaining δrsr i,j contribution. In fact, the comparisons of our Trek-derived rsb i,j with two published datasets that reflect on the genomic average rsb i,j rates [4, 20] show an excellent correlation (Figure S3 in Additional file 1, Pearson’s R > 0.99) confirming the absence of any biased averaging and unusual substitution rates in the time-accumulated substitutions at the L1 sites that pass the Trek procedure. The genome simulation, described later in this work, provides an additional valida- tion for our rate constants. The rcore i,j values (Eqs. 1 and 2) for all possible i→j neutral substitutions inferred for each of the eligible individual L1 positions are thus assumed to be common for any other sites in the human genome that share the short-range sequence context. Fig. 1 Long-range G+C context of the young L1 insertion sites in the human genome. a and b The distribution of the G+C contents for all the w-sized (1000-nt in a and 10,000-nt in b) bins in the human genome (orange lines) is shown, as compared to the same distribution but using only the bins centred at the midpoints of all the remnants of young (L1Hs, L1PA2, L1PA3, L1PA4, L1PA5) L1 elements (green lines) Fig. 1 Long-range G+C context of the young L1 insertion sites in the human genome. Revealing the core single-nucleotide substitution rates The selected most-recent subfamilies are sufficiently young [37] a) to enable an unambiguous identification of the genomic coordinates of the borders for the remnants; b) to assume that each position in those elements would be unlikely Herein, we propose a methodology to obtain the core components (2, capturing only the short-range sequence caused variation) of the neutral single-nucleotide substi- tution rates via the direct analysis of 39,894 L1 mobile DNA remnants [34] in the same, human, genome (a single-genome approach). rcore i,j = rsb i,j + δrsr i,j (2) (2) Our transposon exposed k-mer rate (Trek) method provides the rcore i,j rate constants at single-nucleotide res- olution in L1, where we demonstrate sufficient sequence variability to cover a wide-range of sequence contexts. We use this coverage to determine the core rate constants for all possible nucleotide substitutions (3 per position) at each of the 3.2 billion positions in the human genome. The Trek aims at revealing the rcore i,j variation in a rela- tively model-free manner and at a level beyond accounting for only the two immediate neighbouring nucleotides [35]. We make our dataset of the time-dependent rate con- stants for individual substitutions publicly available. To exemplify the usage of Trek data, we demonstrate that the rcore i,j values can generate a sequence starting from a Page 3 of 17 Page 3 of 17 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Fig. 1 Long-range G+C context of the young L1 insertion sites in the human genome. a and b The distribution of the G+C contents for all the w-sized (1000-nt in a and 10,000-nt in b) bins in the human genome (orange lines) is shown, as compared to the same distribution but using only the bins centred at the midpoints of all the remnants of young (L1Hs, L1PA2, L1PA3, L1PA4, L1PA5) L1 elements (green lines) were thus employed to infer the corresponding rcore i,j val- ues from the analysis of all the young L1 remnants in the human genome. We recorded the data in the Trek database that contains a set of well-defined rcore i,j constants (see below for the extent of sequence context coverage in the Trek database) capturing the influence of the unique arrangement of neighbouring nucleotides at those posi- tions. Owing to the nature of the selected L1 elements, as discussed above, and the Trek procedure design (Fig. Revealing the core single-nucleotide substitution rates a and b The distribution of the G+C contents for all the w-sized (1000-nt in a and 10,000-nt in b) bins in the human genome (orange lines) is shown, as compared to the same distribution but using only the bins centred at the midpoints of all the remnants of young (L1Hs, L1PA2, L1PA3, L1PA4, L1PA5) L1 elements (green lines) to undergo repeated substitutions over the studied period of their existence as remnants in the human genome (see Methods); c) to attribute a time-invariance to the rates during the analysed period of mutation accumula- tion [20, 46, 47]. Finally, many matching positions in our studied five L1 representatives share the same consensus bases, hence, such positions are not polymorphic due to adaptive pressure and can serve as internal references for inferring the rcore i,j rates. The influence range of neighbour nucleotides The comparison of these fractions coming from individual base types across different time periods enables a linear model fitting, through which we can reveal the rates for the substitutions into the b2, b3 and b4 bases from the consensus (b1) state of the given position (d). The steps (c) and (d) are repeated for all the positions in the reference sequence, producing single-nucleotide resolution core substitution rate constants with sequence-context dependency as sampled in the reference sequence of the mobile element. To assure the high quality and neutrality of the retrieved rates, we accounted for the sites in the reference sequence that had at least 700 mapped occurrences in each time group (b), with the same wild-type variant being always the prevalent one (more than 80%) in each subfamily (c) and producing a Pearson’s correlation coefficient of at least 0.7 in the time-evolution plots (d) Fig. 2 Single genome determination of the context-dependent substitution rate constants. a–d The Trek approach is applicable to a genome containing multiple remnants of retrotransposon subfamilies silenced at different time epochs (a). We can consider those subfamilies as substitution counters that had different resetting ages (b). The full consensus sequence of the most recent subfamily is taken as a reference (a). The remnants are then grouped by their age and fully mapped onto the reference sequence (b). For each position i in the reference sequence, the fractions of the four bases in all the time groups are calculated (c). The comparison of these fractions coming from individual base types across different time periods enables a linear model fitting, through which we can reveal the rates for the substitutions into the b2, b3 and b4 bases from the consensus (b1) state of the given position (d). The steps (c) and (d) are repeated for all the positions in the reference sequence, producing single-nucleotide resolution core substitution rate constants with sequence-context dependency as sampled in the reference sequence of the mobile element. The influence range of neighbour nucleotides g g To apply the rcore i,j constants to the human genome, we first established the optimal length of a DNA sequence (k-mer, where k is the length of the sequence) capturing most of the influences that modulate substitution rates of the base at the centre. For this, we evaluated the power of the knowledge of the neighbouring arrangement of nucleotides in predicting the rcore i,j constants for each of the twelve i→j substitution types, where i and j are the four DNA bases. We built test predictors for individual substi- tution types via a tree-based gradient boosting machine (GBM, machine learning technique) [48, 49], while using varying lengths of sequences centred at the positions where the rate constants were to be predicted (see Methods). The aim of the machine learning procedure was to establish the optimal sequence length to min- imise the error in the predicted rate constants (Figures ,j The Trek methodology of obtaining rcore i,j rates, along with the considerations for filtering out the possible selec- tion and non-neutral substitution sites, is presented in Fig. 2 with further details in Methods, Figures S1 and S2 in Additional file 1. The acquired data on the full set of position-specific substitution rates are presented in Fig. 3, to highlight the revealed variation per substitution type, along with the fully averaged values for the rate constants. A total of 661 positions, at the 3’ side of the L1 ele- ments, passed our robustness checks (see Methods) and Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 4 of 17 Fig. 2 Single genome determination of the context-dependent substitution rate constants. a–d The Trek approach is applicable to a genome containing multiple remnants of retrotransposon subfamilies silenced at different time epochs (a). We can consider those subfamilies as substitution counters that had different resetting ages (b). The full consensus sequence of the most recent subfamily is taken as a reference (a). The remnants are then grouped by their age and fully mapped onto the reference sequence (b). For each position i in the reference sequence, the fractions of the four bases in all the time groups are calculated (c). The influence range of neighbour nucleotides To assure the high quality and neutrality of the retrieved rates, we accounted for the sites in the reference sequence that had at least 700 mapped occurrences in each time group (b), with the same wild-type variant being always the prevalent one (more than 80%) in each subfamily (c) and producing a Pearson’s correlation coefficient of at least 0.7 in the time-evolution plots (d) the k-mer to five, hence accounting for two upstream and two downstream bases, we cover 404 unique sequences out of possible 1024 (45). Further reduction of the size to three, allows having data for 56 unique triads out of 64 (leaving out only the CpG containing triads, see below). For the single-base case (1-mers), where we average out all short-range neighbour effects and longer-range sequence variability, we obtain data for all the four bases and 4 × 3 possible substitutions as shown in Fig. 3 (the median val- ues on the top of the figure). The coverage of the longer k- mers is, however, increased nearly twice when we account for the strand-symmetry, as described in Methods. Please note, that for each unique k-mer we obtained three rcore i,j constants via the described analysis of a large pool of L1 remnants from different genomic loci. S4 and S5 in Additional file 1). In agreement with prior evidence [9, 10, 44, 50], but now obtained for each indi- vidual i→j substitution type from Trek data, the optimal window was found to be 5-7-nt (both 5- and 7-nt result- ing in comparable results for many substitution types) and was subsequently used as guidance for the direct map- ping of the Trek rate constants from the L1 sequence onto any given human nuclear DNA sequence for the rcore i,j assignment. S4 and S5 in Additional file 1). In agreement with prior evidence [9, 10, 44, 50], but now obtained for each indi- vidual i→j substitution type from Trek data, the optimal window was found to be 5-7-nt (both 5- and 7-nt result- ing in comparable results for many substitution types) and was subsequently used as guidance for the direct map- ping of the Trek rate constants from the L1 sequence onto any given human nuclear DNA sequence for the rcore i,j assignment. Mapping the Trek rcore i,j data on any DNA sequence ,j The upper 7-nt size window for determining the single- nucleotide substitution rate constants at the central base accounts for three upstream and three downstream bases relative to each nucleotide position. Our neutral substi- tution positions that pass the Trek criteria capture 636 unique 7-mers out of the possible 16,384 (47). Therefore, for many loci in the human genome we need to use a smaller window (< 7-mer) as a match criterion to assign to one of the Trek rate constant sets. By trimming the size of With the above considerations, we created a program (Trek mapper, Methods, Note S1 in Additional file 1) to produce rcore i,j core substitution rate constants for any sequence, accounting for the context information within up to the 7-mer window and pulling the matching core Page 5 of 17 Page 5 of 17 Page 5 of 17 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Fig. 3 Transposon exposed (Trek) rcore i,j substitution rate constants of the human genome. The boxplots are shown for each i→j substitution type inferred from the hominoid L1 remnants spread across the human genome. Each point comes from a specific position in the L1 element, reflecting the substitution rate constant averaged across multiple occurrences of that specific position with the same sequence-context in multiple regions of the human genome. The complementary i→j pairs are plotted in adjacency. The median values of the overall substitution rates (rsb i,j ) in byr−1 (billion years) unit, averaged across the varying sequence-context within the L1 elements, are shown on the top of the CpG sites in the L1 remnants passed our robust- ness checks (Methods), due to the targeted, epigenetic- (δrCpG i,j ) or APOBEC-affected single-nucleotide substitu- tions there [12–14]. The latter effects were likely to be non-uniform with time (active targeting, δrspec i,j , while in the viable epoch for each L1 subfamily, at both DNA and RNA levels) and may had been present in order to silence the active retrotransposons. Our current data are for the human nuclear genome. However, the general approach for obtaining rcore i,j con- stants is applicable to any organism where the genome contains a set of well-characterised and related young mobile elements silenced at different time epochs and without notable genomic context bias. rcore i,j rates and the oligomeric composition of the human genome Fig. 3 Transposon exposed (Trek) rcore i,j substitution rate constants of the human genome. The boxplots are shown for each i→j substitution type inferred from the hominoid L1 remnants spread across the human genome. Each point comes from a specific position in the L1 element, reflecting the substitution rate constant averaged across multiple occurrences of that specific position with the same sequence-context in multiple regions of the human genome. The complementary i→j pairs are plotted in adjacency. The median values of the overall substitution rates (rsb i,j ) in byr−1 (billion years) unit, averaged across the varying sequence-context within the L1 elements, are shown on the top Fig. 3 Transposon exposed (Trek) rcore i,j substitution rate constants of the human genome. The boxplots are shown for each i→j substitution type inferred from the hominoid L1 remnants spread across the human genome. Each point comes from a specific position in the L1 element, reflecting the substitution rate constant averaged across multiple occurrences of that specific position with the same sequence-context in multiple regions of the human genome. The complementary i→j pairs are plotted in adjacency. The median values of the overall substitution rates (rsb i,j ) in byr−1 (billion years) unit, averaged across the varying sequence-context within the L1 elements, are shown on the top The full set of sequence-dependent human rcore i,j substi- tution rates (all three constants per position) enabled us to perform a sophisticated in silico evolution of a random DNA sequence, guided solely by our rcore i,j val- ues. We started from a random sequence of 5 million (mln) nt with a G+C content of 60% (substantially greater than the 40.45% G+C content for the human genome). We performed random nucleotide substitutions weighted by Trek-inferred probabilities (Methods, Figure S7 in Additional file 1), where, after each cycle, the substitution rate constants were updated for the sequence positions that were either mutated or fell within the influence zone of the performed substitutions. The simulation was con- tinued until the overall G+C content of the simulated sequence became constant (see Fig. 4a–c). data from the Trek database. Should a representative match be absent with the full 7-nt long sequence, the window around the given position in a query sequence is shortened into the longest variant possible (out of the 5-nt, 3-nt or 1-nt lengths) with a full match in the Trek database (Methods, Figure S6 in Additional file 1). rcore i,j rates and the oligomeric composition of the human genome In this way, for all the possible 16,384 7-mers, our Trek database reports 49,152 rate constants (3×16,384), of which 3168 (6.4%) account for the 7-mer context, 23,232 (47.3%) account for the nested 5-mer context, 17,120 (34.8%) for 3-mer and only 5632 (11.5%, CpG containing sequences) constants do not account for any context effect on the central base (since we eliminate those by design, due to the δrCpG i,j contribution). Our full dataset reports and makes publicly available (Additional file 2), the time- dependent rcore i,j rates for all individual i→j substitutions accounting for the context effects beyond the 64 triads [35]. If we consider only the unique values in the Trek database, we report 2078 unique rate constants (taking into account different extent of averaging, where multi- ple entries are present for the different context ranges), of which 1208 (58.1%), 782 (37.6%), 85 (4.1%) and 3 (0.1%) entries account for 7-, 5-, 3- and 1-mer contexts respec- tively. The 1-mer averaged data were used for only the k-mers that contain either C or G bases of a CpG dyad at the centre, to assign the overall substitution rate con- stants by the Trek mapper. This was done since none data from the Trek database. Should a representative match be absent with the full 7-nt long sequence, the window around the given position in a query sequence is shortened into the longest variant possible (out of the 5-nt, 3-nt or 1-nt lengths) with a full match in the Trek database (Methods, Figure S6 in Additional file 1). In this way, for all the possible 16,384 7-mers, our Trek database reports 49,152 rate constants (3×16,384), of which 3168 (6.4%) account for the 7-mer context, 23,232 (47.3%) account for the nested 5-mer context, 17,120 (34.8%) for 3-mer and only 5632 (11.5%, CpG containing sequences) constants do not account for any context effect on the central base (since we eliminate those by design, due to the δrCpG i,j contribution). Our full dataset reports and makes publicly available (Additional file 2), the time- dependent rcore i,j rates for all individual i→j substitutions accounting for the context effects beyond the 64 triads [35]. rcore i,j rates and the oligomeric composition of the human genome If we consider only the unique values in the Trek database, we report 2078 unique rate constants (taking into account different extent of averaging, where multi- ple entries are present for the different context ranges), of which 1208 (58.1%), 782 (37.6%), 85 (4.1%) and 3 (0.1%) entries account for 7-, 5-, 3- and 1-mer contexts respec- tively. The 1-mer averaged data were used for only the k-mers that contain either C or G bases of a CpG dyad at the centre, to assign the overall substitution rate con- stants by the Trek mapper. This was done since none The simulation converged to generate a sequence with the A, T, G and C compositions of 30.91, 30.90, 19.06 and 19.13% respectively. Note, that these values are close to the A, T, G and C compositions of the repeat-masked human genome of 29.75, 29.79, 20.24 and 20.22% respec- tively (Methods), being slightly AT rich. Furthermore, the simulated sequence captures the contents of different individual oligomers (k-mers) in the human genome. The data for all the possible 16 dyads, 64 triads, 1,024 pentads and 16,384 heptads are presented in Fig. 4d–g and show a significant (see the correlation coefficients on the plots) correlation between the compositional landscapes of the Trek-simulated sequence and the actual human genome. Regardless of the starting composition of the initial DNA sequences, our simulations always equilibrated to a state with similar oligomer (up to 7-mer) content. The k-mer contents shown in Fig. 4d–g for the actual human genome were calculated from the repeat-masked version of the RefSeq human genome, where all the identified repeat ele- ments, including the L1, were disregarded. This assured the removal of a potential bias due to the presence of L1 elements (Methods), used to infer the rate constants, in Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 6 of 17 Fig. 4 Comparison of the in silico evolved sequence and the actual human genome. a The 5-mln-nt starting sequence is randomly generated with 60% G+C content. (b and c) The sequence is then neutrally evolved using rcore i,j only, until the base-compositional equilibrium is established (c). This was reached after about 20 mln substitutions (or an average of 4 substitutions per site (b), where x-axis shows the number of substitutions divided by the simulated sequence length). The equilibration converges faster when we start from a sequence with lower G+C content. rcore i,j rates and the oligomeric composition of the human genome d–g The plots showing the correlation of the k-mer contents in the equilibrated genome with the corresponding content in the real human genome. The lengths of the k-mers along with the correlation coefficients are shown on the bottom right corners of the plots. Two correlation coefficients are shown with the exclusion and the inclusion (the value in the bracket) of CpG containing oligomers (red points in the plots). The dashed lines depict the diagonals for the ideal match of the k-mer contents Fig. 4 Comparison of the in silico evolved sequence and the actual human genome. a The 5-mln-nt starting sequence is randomly generated with 60% G+C content. (b and c) The sequence is then neutrally evolved using rcore i,j only, until the base-compositional equilibrium is established (c). This was reached after about 20 mln substitutions (or an average of 4 substitutions per site (b), where x-axis shows the number of substitutions divided by the simulated sequence length). The equilibration converges faster when we start from a sequence with lower G+C content. d–g The plots showing the correlation of the k-mer contents in the equilibrated genome with the corresponding content in the real human genome. The lengths of the k-mers along with the correlation coefficients are shown on the bottom right corners of the plots. Two correlation coefficients are shown with the exclusion and the inclusion (the value in the bracket) of CpG containing oligomers (red points in the plots). The dashed lines depict the diagonals for the ideal match of the k-mer contents the human genome. As rcore i,j constants are free of the δrCpG i,j contribution (see above), the simulated genome produced higher alterations in representing the k-mer contents that have CpGs (red points in Fig. 4d–g). These alterations visually demonstrate the role of δrCpG i,j in the background compositional landscape of the human genome. The cor- relations in Fig. 4 are from simulations where the rate constants were symmetrised according to the inherent strand-symmetry in double-helical DNA (see Methods). The results without such equalisation are still significant, though producing slightly worse correlation coefficients (Figure S8 in Additional file 1). long sequence is equal to the product of the occurrence probabilities of their constituent bases. For instance, the probability of observing the AGT triad is the pAGT = pApGpT product, where the individual pi probabilities are the base contents expressed in fractions. rcore i,j rates and the oligomeric composition of the human genome The comparison of the k-mer fractions obtained in this way with the human genome data (Fig. 5) shows a substantially reduced corre- lation (for the genomic 7-mer content, Pearson’s R = 0.59 compared to 0.74 using Trek rates). The discrepancies are minimal while accounting for only the dyad and therefore singleton contents, however, while using context-invariant singleton substitution rates in the simplistic in silico sim- ulations descried above, we observe slight but system- atic underestimation of the overall G+C content in the sequence at compositional equilibrium (Fig. 6). Although excluded via the specificities of the Trek methodology (Fig. 2), to completely rule out the presence of any circu- larity in the reproduction of the human genome higher k-mer (up to 7 nt) content through the Trek rate constants, we also demonstrated the overall poor agreement between the k-mer contents of L1 elements used to infer the rate constants and the human nuclear genome (Figure S9 in Additional file 1). To confirm that the observed correlations for differ- ent k-mer contents (Fig. 4d–g) present an improvement due to our sequence-context-dependent rates, rather than being a side effect, by a pure chance, in a sequence where the simulation makes only the single-base compo- sition converge to that of the real human genome (such as in a sequence generated using an ideal 4 × 4 single- nucleotide substitution rate matrix), we calculated the expected distribution of different k-mers in a genome with fully random base arrangement but with the exact human A, T, G and C overall base composition. In the complete absence of any sequence-context effects, the probability of the occurrence (fraction) of any k-mer in a sufficiently The described simulations therefore support the attri- bution of a contributory role that the rcore i,j variability plays Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 7 of 17 Fig. 5 Oligomeric composition of the ideal neighbour-invariant sequence. a–d The oligomeric content of the human genome (x-axis) is compared to the content expected by chance (y-axis) in a sequence that has the exact single-base composition as the human genome, but has substitution rates that are purely context independent. This corresponds to a hypothetic genome simulation with perfectly correct rsb i,j single-base rate constants, but without any δrsr i,j sequence-context dependency present. rcore i,j rates and the oligomeric composition of the human genome The lengths of the k-mers, along with the Pearson’s correlation coefficients without and with (the values in the brackets) the CpG containing oligomer data (red points) are shown on the bottom right corners of the plots. The correlation coefficients are notably smaller compared to the in silico sequence equilibrated based on the full set of context-dependent Trek rcore i j Fig. 5 Oligomeric composition of the ideal neighbour-invariant sequence. a–d The oligomeric content of the human genome (x-axis) is compared to the content expected by chance (y-axis) in a sequence that has the exact single-base composition as the human genome, but has substitution rates that are purely context independent. This corresponds to a hypothetic genome simulation with perfectly correct rsb i,j single-base rate constants, but without any δrsr i,j sequence-context dependency present. The lengths of the k-mers, along with the Pearson’s correlation coefficients without and with (the values in the brackets) the CpG containing oligomer data (red points) are shown on the bottom right corners of the plots. The correlation coefficients are notably smaller compared to the in silico sequence, equilibrated based on the full set of context-dependent Trek rcore i,j constants. The dashed lines depict the diagonals for the ideal match of the k-mer contents Basal substitution propensity profile and cancer-linked somatic mutations A recent study, which correlated the cancer and cell divi- sion frequencies, suggested that cancers, with their multi- etiologic nature, are linked to random mutation events upon cell division/DNA replication [56], in addition to expressing unique type-dependent mutational signatures [57]. To this end, genomic sites with higher intrinsic BSP (lower stability) may potentially exhibit a higher preva- lence of cancer-related genome alterations, as compared to sites of lower intrinsic BSP (higher stability), should the germline and cancer-linked somatic mutations share com- mon mechanisms [54]. Although the sequence context signatures of cancer mutations and their variation across different cancer types is out of the scope of the present work and is covered in detail elsewhere [57–62], here we examined the simple relationship between our calculated germline BSP values and the observed cancer-associated somatic mutations accessed via the annotated COSMIC database of somatic mutations in cancer [63] (Methods). Since the Trek data are for the core neutral substitu- tions, we restricted the analysis to the non-coding and non-polymorphic (not identified as SNP) point mutations (6 mln) in cancer. By mapping these sites to the human genome and retrieving the sequence-context information Fig. 6 G+C content equilibration of random sequences with 60 and 30% initial G+C contents. Here, the in silico genome is equilibrated by using either the Trek rcore i,j constants with up-to 7-mer context dependence (green lines), or only the singleton rates from Trek without any context dependence (red line, shown for only the 30% G+C content start). The simulation accounting for the context dependence results in 38.19 and 38.18% of G+C content (compare to 40.45% for the repeat-masked human genome), starting from random sequences with 60% and 30% G+C contents respectively. The simulation with only the singleton rates converged at lower 37.36% value for the G+C content, where we expected the least disagreement between the “most-ideal” singleton vs. 7-mer description of the substitution rates (see Fig. 5a). Please note, that in the hypothetic case of the most ideal singleton substitution rate constants, the discrepancy is more pronounced while analysing the genomic contents of the higher k-mers, as presented in Fig. 5 Fig. 7 Sequence-context dependence of the rcore i,N basal substitution propensity (BSP) constants. Basal substitution propensity profile of the human genome, substitution prone and resistant motifs Here, the in silico genome is equilibrated by using either the Trek rcore i,j constants with up-to 7-mer context dependence (green lines), or only the singleton rates from Trek without any context dependence (red line, shown for only the 30% G+C content start). The simulation accounting for the context dependence results in 38.19 and 38.18% of G+C content (compare to 40.45% for the repeat-masked human genome), starting from random sequences with 60% and 30% G+C contents respectively. The simulation with only the singleton rates converged at lower 37.36% value for the G+C content, where we expected the least disagreement between the “most-ideal” singleton vs. 7-mer description of the substitution rates (see Fig. 5a). Please note, that in the hypothetic case of the most ideal singleton substitution rate constants, the discrepancy is more pronounced while analysing the genomic contents of the higher k-mers, as presented in Fig. 5 stability of C in the wntCnwn context, and analogously, G in the nwnGanw context. Those bases become prone to substitutions, independently from the well-studied CpG context, in the nmrCarn and nytGykn motifs for the C and G bases respectively. Furthermore, A and T bases become prone to more frequent substitutions in the ncwAtnn and analogous ngwTann motifs (Fig. 7). Basal substitution propensity profile and cancer-linked somatic mutations a–h Sequence logos [55] are shown for all the unique 7-mer sequences grouped by the central base type (columns) and the category of the BSP range the sequences fall in (rows, BSP range is shown in byr−1 rate constants). The y-axes in the individual sequence logos show the information content in bits. The x-axes outline the neighbouring base positions relative to the central base. For each sequence, the BSP of the central base (i) depicts the sum of the core rate constants for the substitutions to the three other (non-i) bases, rcore i,N = rcore i,b2 + rcore i,b3 + rcore i,b4 . As can be seen from the plots, the bases A and T are highly mutable when the neighbouring positions are enriched in the same, A and T, bases (compare the logos a and d with e and h). The adjacent enrichment in A increases the BSP of C (b), and decreases the BSP of G (g) bases. Conversely, the adjacent enrichment in T increases the BSP of G (c) and decreases that of C (f) bases. Note, that our data are for rcore i,N , thus independent from the methylation-driven increased mutation rates in CpG dyads [12–14] Fig. 7 Sequence-context dependence of the rcore i,N basal substitution propensity (BSP) constants. a–h Sequence logos [55] are shown for all the unique 7-mer sequences grouped by the central base type (columns) and the category of the BSP range the sequences fall in (rows, BSP range is shown in byr−1 rate constants). The y-axes in the individual sequence logos show the information content in bits. The x-axes outline the neighbouring base positions relative to the central base. For each sequence, the BSP of the central base (i) depicts the sum of the core rate constants for the substitutions to the three other (non-i) bases, rcore i,N = rcore i,b2 + rcore i,b3 + rcore i,b4 . As can be seen from the plots, the bases A and T are highly mutable when the neighbouring positions are enriched in the same, A and T, bases (compare the logos a and d with e and h). The adjacent enrichment in A increases the BSP of C (b), and decreases the BSP of G (g) bases. Conversely, the adjacent enrichment in T increases the BSP of G (c) and decreases that of C (f) bases. Basal substitution propensity profile of the human genome, substitution prone and resistant motifs in shaping the compositional landscape of the human nuclear genome [51]. Importantly, our study demonstrates that the non-specific core substitution rates are capa- ble of producing apparent selection or depletion patterns in higher k-mers, beyond dyads, in the human genome. To this end, the 7-mer content from our in silico equili- brated sequences, obtained solely based on the set of rcore i,j constants, can serve as a background standard to reveal specific selection [52] for or against different sequence motifs in the human genome. Trek mapper provides the full set of rcore i,j constants for each position in the whole human genome. Such data enables us to calculate the germline context-dependent basal substitution propensity (BSP) by taking the sum of the individual rate constants for the three possible sub- stitutions at each base position, thus producing the core rcore i,N constant for the substitution of a given base i by any other base N. Figure S10 in Additional file 1 shows the BSP profiles calculated for the individual chromo- somes (red) as compared with the whole genome profile (green), where most of the chromosomes exhibit the same overall distribution as the whole genome. Further group- ing and analysis [55] of the unique sequences found in regions of different BSP for the whole human genome reveals motifs with varying substitution propensities of the bases at the centre of 7-mers (Fig. 7). In particular, adhering to the standard nucleobase notation in small letters (a=A, c=C, g=G, k={G, T}, m={A, C}, n={A, C, G, T}, r={A, G}, t=T, w={A, T}, y={C, T}), the compar- ative examinations of the sequences reveal the overall Similar link between the context-dependent mutation pressure and the nucleotide composition has recently been shown for the triad counts in the bacterial genome of Mesoplasma forum [53] and in the human mitochondrial genome [54]. Interestingly, the latter study employed the somatic mutation propensities found by analysing can- cer genomes to reproduce the triad count of the human mitochondrial genome, as an evidence of the link between the somatic and germline mutation rates in human mito- chondria. That link is further visible in the cancer anal- ysis presented here (vide infra) for the human nuclear genome. Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 8 of 17 Fig. 6 G+C content equilibration of random sequences with 60 and 30% initial G+C contents. Basal substitution propensity profile and cancer-linked somatic mutations substitutions accumulate in a genome [5, 51, 67], (also see Box 1 in [68]), and, we have applied stringent cri- teria for filtering out L1 positions with a potential to be non-neutral, our Trek rcore i,j substitution rate con- stants may serve as contributory estimates for germline mutation rates (especially for the relative mutation rates across individual base conversions). The Trek method complements previous studies on the germline muta- tion rates [5], and retrieves rate constants (per site, in time domain, byr−1) in a single genome manner, where all the detected substitutions in L1 remnants (their neu- tral positions only) retrieved from a single genome have accumulated while evolving in multiple (39,894) copies within a lineage of a single organism. This may have a potential to minimise fixation biases and further eliminate biases attributed to the change (mutations) in background mutagenic and reparatory machineries, from an individ- ual to individual, when comparing data from multiple genomes. Taking into account the singleton composi- tion of the unmasked human genome (Additional file 2) and the rsb i,j constants for the individual transitions and transversions brought in Fig. 3, as estimated through Trek method, we can provide a new estimate for the overall germline mutation rate for the human genome to be 1.176 mutations per site per billion year for the used reference human genome. This makes 2.35 × 10−8 mutations per site per generation, assuming 20 years of average gen- eration span [2]. Interestingly, the value is slightly lower than the ∼2.5 × 10−8 estimate [2, 5, 67, 69, 70] based on phylogenetic-based approaches, which can be attributed to the more aggressive elimination of biases built in the Trek design (see above). However, the estimate is still greater than the more recent 1.2 × 10−8 to 1.45 × 10−8 evaluations from pedigree-based studies [71–74], known to be free from background recombination influence but highly dependent on the paternal age [75]. Less so for the relative values of the rates for individual base-conversions, we expect the absolute values in Trek to be dependent on the absolute timing of the used five subfamilies of L1 ele- ments. Although the latter reliance may be the cause of the relatedness of the Trek estimate to the phylogeny-based ones, our estimate is still a useful addition to the ongoing debate on calibrating the average germline mutation rates [76–78]. Basal substitution propensity profile and cancer-linked somatic mutations Note, that our data are for rcore i,N , thus independent from the methylation-driven increased mutation rates in CpG dyads [12–14] Fig. 7 Sequence-context dependence of the rcore i,N basal substitution propensity (BSP) constants. a–h Sequence logos [55] are shown for all the unique 7-mer sequences grouped by the central base type (columns) and the category of the BSP range the sequences fall in (rows, BSP range is shown in byr−1 rate constants). The y-axes in the individual sequence logos show the information content in bits. The x-axes outline the neighbouring base positions relative to the central base. For each sequence, the BSP of the central base (i) depicts the sum of the core rate constants for the substitutions to the three other (non-i) bases, rcore i,N = rcore i,b2 + rcore i,b3 + rcore i,b4 . As can be seen from the plots, the bases A and T are highly mutable when the neighbouring positions are enriched in the same, A and T, bases (compare the logos a and d with e and h). The adjacent enrichment in A increases the BSP of C (b), and decreases the BSP of G (g) bases. Conversely, the adjacent enrichment in T increases the BSP of G (c) and decreases that of C (f) bases. Note, that our data are for rcore i,N , thus independent from the methylation-driven increased mutation rates in CpG dyads [12–14] Page 9 of 17 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 substitutions accumulate in a genome [5, 51, 67], (also see Box 1 in [68]), and, we have applied stringent cri- teria for filtering out L1 positions with a potential to be non-neutral, our Trek rcore i,j substitution rate con- stants may serve as contributory estimates for germline mutation rates (especially for the relative mutation rates across individual base conversions). The Trek method complements previous studies on the germline muta- tion rates [5], and retrieves rate constants (per site, in time domain, byr−1) in a single genome manner, where all the detected substitutions in L1 remnants (their neu- tral positions only) retrieved from a single genome have accumulated while evolving in multiple (39,894) copies within a lineage of a single organism. Basal substitution propensity profile and cancer-linked somatic mutations g Overall, the results show that the intrinsic BSP of differ- ent sites in DNA may contribute to their absence/presence in pathological genotypes. In particular, we observe that 7-mers with low germline BSPs of the central base are rel- atively depleted in cancer-linked somatic mutation data (Figure S12 in Additional file 1 and Fig. 8). They present a cancer enrichment ratio that is smaller than 1, whereas for the unstable 7-mers, the enrichment ratio, on aver- age, tends to 1, meaning that their presence in cancer is overall comparable to the one in the whole genome. Focusing on the average trend (red lines in Fig. 8) that does not expand to the k-mers with > 1 cancer enrich- ment ratio, we cannot comment about the behaviour of the sites of higher mutability in cancer by the present work. Those could potentially be better reflected in the cancer mutational signature analyses done by others [57, 60, 61]. Our results, however, further outline the potential role of the general imbalance in repair machin- ery in determining the accumulation of somatic mutations in cancer, where the mutations are originally caused by errors in replication that possibly emerge via mechanisms more or less common in somatic and germline cells [54]. Conclusions We have employed a single-genome approach (Trek) that reveals the core (rcore i,j =rsb i,j + δrsr i,j) component of the spon- taneous single-nucleotide substitution rates and basal substitution propensity constants (rcore i,N ) for the human nuclear genome (Figs. 2 and 3). Although the mobile DNA elements have been used before [20, 40, 46, 79] for estimating averaged substitution rates, the increased quality of the human reference sequence and the detailed The relation between the Trek rcore i,j and germline mutation rates Basal substitution propensity profile and cancer-linked somatic mutations This may have a potential to minimise fixation biases and further eliminate biases attributed to the change (mutations) in background mutagenic and reparatory machineries, from an individ- ual to individual, when comparing data from multiple genomes. Taking into account the singleton composi- tion of the unmasked human genome (Additional file 2) and the rsb i,j constants for the individual transitions and transversions brought in Fig. 3, as estimated through Trek method, we can provide a new estimate for the overall germline mutation rate for the human genome to be 1.176 mutations per site per billion year for the used reference human genome. This makes 2.35 × 10−8 mutations per site per generation, assuming 20 years of average gen- eration span [2]. Interestingly, the value is slightly lower than the ∼2.5 × 10−8 estimate [2, 5, 67, 69, 70] based on phylogenetic-based approaches, which can be attributed to the more aggressive elimination of biases built in the Trek design (see above). However, the estimate is still greater than the more recent 1.2 × 10−8 to 1.45 × 10−8 evaluations from pedigree-based studies [71–74], known to be free from background recombination influence but highly dependent on the paternal age [75]. Less so for the relative values of the rates for individual base-conversions, we expect the absolute values in Trek to be dependent on the absolute timing of the used five subfamilies of L1 ele- ments. Although the latter reliance may be the cause of the relatedness of the Trek estimate to the phylogeny-based ones, our estimate is still a useful addition to the ongoing debate on calibrating the average germline mutation rates [76–78]. (7-nt long sequences centred at the mutation points), we processed the data with Trek mapper and obtained the BSP (rcore i,N ) profile for the non-coding sites detected in human cancer. The outcome in Figure S11 in Additional file 1, overlapped with the whole-genome BSP profile, shows that stable sites in the human genome, assigned by the Trek mapper to have rcore i,N below 1.13 byr−1, are signif- icantly less likely to undergo somatic mutations in cancer. Like many other disease-causing mutation sites [64], most of the sites that are highly enriched in cancer (Figure S12a in Additional file 1) are CpGs [65], which, even without accounting for the methylation driven increase [12–14] of the mutation rates, show high basal mutability [66]. Basal substitution propensity profile and cancer-linked somatic mutations However, Figure S12b, c in Additional file 1 demonstrates the discussed trend in the 7-mer cancer enrichment ratio (Methods) vs. BSP dependence even when all the CpG sites are removed from the analysis. Furthermore, while investigating the same relationship in different varieties of cancer (as classified based on the primary tissue and primary cancer types, see Methods) we can see that the trend is mostly in place for the 11 cancer types where we have enough data on non-coding somatic mutations (Fig. 8), with the only exception being the oesophageal carcinoma (Fig. 8e, l). The latter deviation might stem from the greater role of carcinogen driven mechanisms of somatic mutations in a tissue (oesophageal) more exposed to external carcinogens. (7-nt long sequences centred at the mutation points), we processed the data with Trek mapper and obtained the BSP (rcore i,N ) profile for the non-coding sites detected in human cancer. The outcome in Figure S11 in Additional file 1, overlapped with the whole-genome BSP profile, shows that stable sites in the human genome, assigned by the Trek mapper to have rcore i,N below 1.13 byr−1, are signif- icantly less likely to undergo somatic mutations in cancer. Like many other disease-causing mutation sites [64], most of the sites that are highly enriched in cancer (Figure S12a in Additional file 1) are CpGs [65], which, even without accounting for the methylation driven increase [12–14] of the mutation rates, show high basal mutability [66]. However, Figure S12b, c in Additional file 1 demonstrates the discussed trend in the 7-mer cancer enrichment ratio (Methods) vs. BSP dependence even when all the CpG sites are removed from the analysis. Furthermore, while investigating the same relationship in different varieties of cancer (as classified based on the primary tissue and primary cancer types, see Methods) we can see that the trend is mostly in place for the 11 cancer types where we have enough data on non-coding somatic mutations (Fig. 8), with the only exception being the oesophageal carcinoma (Fig. 8e, l). The latter deviation might stem from the greater role of carcinogen driven mechanisms of somatic mutations in a tissue (oesophageal) more exposed to external carcinogens. The relation between the Trek rcore i,j and germline mutation rates Since the rates of point mutations can be indirectly estimated by determining the rates at which neutral Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 10 of 17 Fig. 8 Enrichment of 7-mers with varying BSPs in somatic mutation sites linked to different cancer types. a–k Each point in the plots corresponds to a unique 7-mer sequence. All the 7-mers that had either C or G of a CpG dyad at the centre were excluded from the plots representing the zoomed [0, 3] byr−1 range of BSPs. The plots (a–k) show data from 11 cancer types, with their primary tissue and primary cancer types indicated at the top-right corner of each plot. The red lines in (a–k) represent the Lowess [87] smoothing fits, outlining the decrease of the cancer enrichment ratio with the decrease in BSP. The numbers at the top-left corners of the plots show the slopes of the linear fits (not shown) for the data points below the 1.3 byr−1 (vertical line), where the depletion of stable k-mers is the most pronounced. The linear model slopes coming from all 11 cancer types are shown in (l) for comparing the extent of the cancer enrichment ratio vs. BSP dependence across the analysed cancer types Fig. 8 Enrichment of 7-mers with varying BSPs in somatic mutation sites linked to different cancer types. a–k Each point in the plots corresponds to a unique 7-mer sequence. All the 7-mers that had either C or G of a CpG dyad at the centre were excluded from the plots representing the zoomed [0, 3] byr−1 range of BSPs. The plots (a–k) show data from 11 cancer types, with their primary tissue and primary cancer types indicated at the top-right corner of each plot. The red lines in (a–k) represent the Lowess [87] smoothing fits, outlining the decrease of the cancer enrichment ratio with the decrease in BSP. The numbers at the top-left corners of the plots show the slopes of the linear fits (not shown) for the data points below the 1.3 byr−1 (vertical line), where the depletion of stable k-mers is the most pronounced. The linear model slopes coming from all 11 cancer types are shown in (l) for comparing the extent of the cancer enrichment ratio vs. The relation between the Trek rcore i,j and germline mutation rates BSP dependence across the analysed cancer types subfamily divergence studies for the L1 elements [36–39] done during the past decade enabled the construction of a specific direct method for the single-genome retrieval of the core rcore i,j rate constants at a single-nucleotide resolution, while also accounting for the comprehensive short-range context effects beyond the previous estimates for the +1/-1 base effects [35]. The retrieval of our rcore i,j data in a single-genome manner adds additional value, since it ensures the absence of potential bias present a) in the comparison of the genomes of different species due to the differences in the molecular machinery (presence of mutations in the respective genes) that influence the overall mutation rates, and b) in the SNP-counting based methods that rely on sites where the visible polymorphism may contain significant selection bias. compared to the analogous contents from the calculations based on the singleton substitution rates. The calculated basal substitution propensities revealed motifs that are prone or resistant to substitutions (Fig. 7), and confirmed the presence of a link between core substitution rates in the germline and the somatic mutations in cancer, outlin- ing possible commonalities in the mechanisms of muta- tions at both levels (Fig. 8). The discussed parallel between the Trek rcore i,j substitution rates and the germline muta- tions makes the observed link with the somatic mutations, inferred from a cancer database, be more logical. We have become aware of a recently published paral- lel study [80], where the authors revealed the context- dependent mutation probabilities by analysing the vari- ation reflected in the multiple genomes of the 1000 Genomes Project [81, 82]. By arriving to the CAAT muta- ble motif (Fig. 7a) and outlining the importance of the heptameric context (Figure S5 in Additional file 1) in explaining the mutational patterns, the work provided Our context-dependent rate constants were then used to drive the equilibration of a random DNA sequence (Fig. 4). The resulting DNA had k-mer (up to 7) contents in better agreement with those of the human genome, as Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 11 of 17 means to additionally validate part of our results by com- pletely different means. Appealingly, the Trek method retrieves rate constants (in time domain, byr−1) in a single genome manner (see above). The relation between the Trek rcore i,j and germline mutation rates Besides the potential minimisation of fixation biases, this may provide an opportunity to detect some of the biases attributed to the change (because of mutations) in background muta- genic and repair machineries, from individual to individ- ual, when comparing two or more genomes. In particular, Fig. 9 demonstrates that neutral substitution probabili- ties may be elevated in one population as compared to the other. The difference was detectable owing to the multi-genomic analyses within each population done in [80]. Trek may thus serve as a single-genome independent and complementary method to assess inter-individual and inter-population variation of the substitution rates, and retrieve the context-dependent rates for species with only single sequenced genome available. Fig. 9 Agreement between the 7-mer context-dependent Trek rate constants (in byr−1), determined in a single-genome manner, and the context-dependent substitution probabilities from the 1000 Genomes Project. a–c The correlation plots are shown for the substitution The extended set of core substitution rate constants and the associated program we report may enrich our resources and potentially help advance our understanding of genome dynamics, with possible implications for the role of random substitutions in the emergence of patho- logical genotypes and the neutral evolution of proteomes. Methods The human reference genome sequence was taken from the Ensembl database (www.ensembl.org), and was of the version hg19/GRCh37. The positions and span of the retrotransposons were taken from the output of the RepeatMasker [83] processing, accessed through the UCSC genome database (www.genome.ucsc.edu). The repeat annotations were those corresponding to the ver- sion of the used human RefSeq genome. The R [84] programming language (www.r-project.org) was used for all the consecutive analyses. Most of the computations were performed on the available Linux workstation and computing cluster facilities hosted at the Department of Chemistry, University of Cambridge, and the Cancer Research UK Cambridge Institute. The specific details on the employed methods are brought below, with the general flow and the reasoning behind the approach pre- sented in Results and discussion. Fig. 9 Agreement between the 7-mer context-dependent Trek rate constants (in byr−1), determined in a single-genome manner, and the context-dependent substitution probabilities from the 1000 Genomes Project. a–c The correlation plots are shown for the substitution probabilities inferred [80] from three different (African, European and Asian) populations [81]. Only 7-mers with data present in both works were used for the comparisons. Overall, the plots highlight the good agreement between the two methods, and show the variation in substitution probabilities across different populations. This variation may be caused by genetic differences in those populations affecting genes/proteins involved in the background mutagenic and repair machineries, thus altering the spontaneous mutation rates Fig. 9 Agreement between the 7-mer context-dependent Trek rate constants (in byr−1), determined in a single-genome manner, and the context-dependent substitution probabilities from the 1000 Genomes Project. a–c The correlation plots are shown for the substitution probabilities inferred [80] from three different (African, European and Asian) populations [81]. Only 7-mers with data present in both works were used for the comparisons. Overall, the plots highlight the good agreement between the two methods, and show the variation in substitution probabilities across different populations. This variation may be caused by genetic differences in those populations affecting genes/proteins involved in the background mutagenic and repair machineries, thus altering the spontaneous mutation rates Revealing the core substitution rate constants Therefore, by assuming a threshold of 80%, we allow up to 15 times the variation of the rates from the average esti- mate, which is a safe range [4] for the direct estimation of the single-nucleotide substitution rates and their core variation. Having the substitution fraction data, from five different ages and for three (b1→b2, b1→b3, b1→b4) possible sub- stitutions at the position i, allowed the fitting of a linear model via the least squares methodology for the fraction- versus-time dependence for each substitution separately (Fig. 2d). If the data, hence the fitted line, were of high quality, the slope was expected to represent the rcore i,j sub- stitution rate constant. We applied the final robustness filtering at this stage, by making sure that the rates were calculated for only the cases where the time correlation of the substitution fractions in Fig. 2d had greater than 0.7 Pearson’s correlation coefficient (3rd stringency cri- terion, Figure S1 in Additional file 1). This ensured that the retrieved fractions of the substitutions comprised of only the time-accumulated substitutions, rather than of targeted substitutions during the active life-span of the L1 elements, before their silencing. The acceptability of taking 700 as the minimum num- ber of L1 remnants mapped onto a given ith position of the L1 reference sequence, while retrieving substitution rate constants, was checked via the following test. We divided the L1 sequence pool into two and retrieved the substitution rates twice, in each case using the L1 rem- nants from the halved pool only. This resulted in two sets of position-specific rate constants, however, with the dataset division resulting in a less number of positions that passed the 700 threshold and the other filtering cri- teria described below. The high correlation between the two sets (Pearson’s R > 0.9), demonstrated the validity of 700 as a threshold. The high correlation held when decreasing the threshold to 500, decaying after. We have, however, adhered to 700 to ensure the quality of the final values. Please note, however, that the correlation coefficients in most of such time correlations that passed the whole Trek procedure were substantially higher (the observed Pearson’s correlation coefficients were centred at 0.92 with 0.07 standard deviation). Furthermore, the resulting slope (rate constant) estimates had significantly high t- values, averaged at 6.2, showing that the standard error in estimates was, on average, 6.2 times smaller than the esti- mated value. Revealing the core substitution rate constants Project. a c The correlation plots are shown for the substitution probabilities inferred [80] from three different (African, European and Asian) populations [81]. Only 7-mers with data present in both works were used for the comparisons. Overall, the plots highlight the good agreement between the two methods, and show the variation in substitution probabilities across different populations. This variation may be caused by genetic differences in those populations affecting genes/proteins involved in the background mutagenic and repair machineries, thus altering the spontaneous mutation rates All the remnant sequences of the selected L1 subfamilies (Fig. 1, Table S1 in Additional file 1) were first aligned onto the 6064 nucleotide (nt) reference sequence. As the refer- ence, we took the consensus sequence of the human L1Hs retrotransposon (Fig. 2a, b, Figures S1 and S2 in Addi- tional file 1). The alignment was done in a pairwise man- ner with high end-gap penalties (“overlap” mode) that, while allowing insertions and deletions, did not severely break the queried sequences for false mappings with a Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 12 of 17 Page 12 of 17 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 12 of 17 better global alignment score. R [84] with the Biostrings library for alignment was used. After the alignment, all the relevant substitution fractions were collected for each position in the five L1 subfamilies reporting on a specific time epoch (Fig. 2b, c). For example, if the position i in the reference sequence was G (b1), the substitution rate con- stants were calculated for the G→A (b1→b2) transition and G→C (b1→b3), G→T (b1→b4) transversions. First, the base fractions were calculated for five time-reporting L1 subfamilies; i.e. to get the fraction of substitutions accumulated in ∼20.4 myr (age of L1PA5 [37]), all bases in L1PA5 remnants that were precisely mapped on the ith position of the reference sequence were counted, and the fractions of G, A, C and T bases retrieved (Fig. 2c). Here, we applied one of the robustness checks (1st stringency criterion) and made sure that the fractions were estimated if at least 700 mapped bases were present for the ith posi- tion in each time-reporting subfamily (Fig. 2b, Figure S1 in Additional file 1). Revealing the core substitution rate constants Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 13 of 17 of the second substitution to another, k ̸= j, base happen- ing at the same position to be (rδt)2, which is the product of individual substitution probabilities assuming that the rate constant does not change from our average estimate r across those two substitution types. We can permit this for the sake of the back-of-the-envelope estimation of the order of the effect expected from the repeated substitu- tions hitting the same site within 20.4 myr period. To this end, the δf app j apparent change in i→j substitution fraction that we would observe by neglecting the addi- tional j→k substitution, would underestimate the more realistic δfj and be equal to δf app j =rδt-(rδt)2, as we would not count the j bases that emerged but became addition- ally substituted by k. Hence, the corresponding apparent rate constant that neglects second substitution would also underestimate the actual value r, and can be expressed as rapp=δf app j /δt=(rδt-(rδt)2)/δt=r(1-rδt). This means that the underestimation of the actual rate constant would be by [ r-r(1-rδt)] ×100/r=100 × rδt %. Putting 0.64 byr−1 for r and the 0.0204 byr for δt, we can expect only 1.3% contribution to rate constants from the repeated second substitution at the same position within 20.4 myr. Since some of the other non-j bases could revert to js and bal- ance the underestimation of the δfj fraction, the error could be even smaller. This shows that repeated substi- tutions could be neglected in our 20.4 myr time-scale. Furthermore, the validity of the rcore i,j rate constants (Fig. 3) was further checked through the independent analyses reflected in Figure S3 in Additional file 1 and Fig. 9. was of the bi (1) or any other base type (0). For instance, if we want to develop a model accounting for only a single upstream (pos = −1) and a single downstream (pos = +1) nucleotides, hence predicting the substitution rates for different 3-mers, where the central base is the one that mutates, then we would produce 8 pos/bi features for the GBM fitting. There, 4 binary features (-1/A, -1/C, -1/G and -1/T) would describe whether the upstream -1 posi- tion is of base type A, C, G or T, and 4 binary features (pos/bi) would describe the same for the downstream +1 position. Revealing the core substitution rate constants The individual t-values are presented in the Additional file 2 along with the rate constants. The procedure was done for all the 6064 positions in the L1 reference sequence, except the positions 5856– 5895 and 6018–6064, close to the 3’-end (Figure S2 in Additional file 1) that engulf low-complexity G-rich and A-rich sequences correspondingly, prone to alignment errors. We also aimed at calculating such substitution rates for only the positions where the substitutions are neutral and not specifically selected for or against (2nd stringency cri- terion). In other words, the position should not be a poly- morphic or a subfamily speciation-defining nucleotide. We filtered out such cases by ensuring that any eligi- ble ith position had the same nucleotide of the reference sequence as its most prevalent variant with a minimum of 80% occurrence in all subfamilies (Fig. 2c, Figure S1 in Additional file 1). One of the reasons for the usage of only the young L1 subfamilies (spanning 20.4 myr age) was to minimise the potential error in rate constant determination in the Trek procedure caused by repeated substitutions hitting the same position during the considered period of the sub- stitution accumulation. The effect is indeed negligible for 20.4 myr span, as we can estimate using the above men- tioned 0.64 byr−1 value [4] for the average i→j substitu- tion rate constant, r. Since the rate constant is sufficiently small to induce only a small δfj change in substituted base fraction during the δt = 0.0204 byr (20.4 myr) time period (see above), we can equate the δfj change in the fraction of the base j (at the given position that had the original base i identity in a large population of homologous sequences) to the p ≈δfj ≈rδt substitution probability within δt period. We can thus make a crude estimation for the probability The 80% threshold was taken from the following consid- eration. The average crude single-nucleotide substitution rate is noted to be 12.85 × 10−9 substitutions per site per generation [4]. Assuming an average generation length of 20 years [2], the substitution rate constant in a time domain can be crudely approximated as 0.64 byr−1. In the course of 20.4 myr (the age of L1PA5), this should result in only a 1.31% substituted base fraction at a given site, caused by the average spontaneous substitution rates. Revealing the core substitution rate constants We built the models using 3-, 5-, 7-, 9-, and 11- mers, thus accounting for one, two, three, four and five upstream and the same number of downstream neigh- bour bases (Figure S4 in Additional file 1). The absence of the coupling in the binary features, unlike in the case where, for instance, one employs only two binary features per 4 states, enabled us to also investigate the predictive significance of each nucleobase identity at a given neigh- bouring position, which was useful in deciding against the construction of more complex machine learning mod- els (see below) using additional features with higher level of abstraction for the sequence information (overall base content, sequence-derivative properties). The GBM mod- els were then fitted by systematically trying different per- mutations of the tuning values [49] for the number of trees (50-7500), interaction depth (1-10), shrinkage (0.001, 0.01 and 0.1), the number of minimum observations per node (1-28) and the bag fraction (0.25-0.65). The optimal combinations of the tuning parameters were found per substitution type and sequence length, via a 16-fold cross validation repeated 7 times. The found best parameters are accessible in the Additional file 2, and the predic- tive performances of the best models, from the repeated cross validation studies, are presented in Figure S5 in Additional file 1. To make a predictor of rcore i,j based on a sequence only, we, however, found it much better to use direct averaged values coming from the proposed Trek methodology for each k-mer, rather than the GBM mod- els, as the Trek values are already well averaged across multiple occurrences of the same sequence in different loci of the human genome (Figs. 1, 2 and 3, Figure S3 in Additional file 1). Furthermore, the overall poor perfor- mance of the decision-tree-based GBM models implies that the influence of the immediate context is highly non-additive and non-Bayesian, which is expected tak- ing into account the nature of the core context-dependent mutations. The latter reflect the intrinsic short-range sequence properties, interactions and recognition with the overall mutagenic and repair machinery present in a given organism. There, the whole sequence at a cer- tain small (< 10 nt) scale [9] is what likely defines the interaction [11], and it is hard to represent such effects through even smaller-scale constituents linked with each other at a certain dimension The direct model-free Mapping the Trek substitution data We developed an open source Trek mapper program. For each i position in a query sequence (Figure S6 in Addi- tional file 1), the program looks at the bases i −3 to i + 3. If the exact 7-mer, with the associated rate con- stant values, is not available in the Trek database, the program reduces the size of the sequence to 5, by con- sidering i −2 to i + 2 positions, or, if necessary, to 3- or 1-mers, until an exact match is found in the database. This would mean that some reported substitution rates would come from the actual triad data. In any case, about half will come from pentads and some from heptads, account- ing for more precise sequence-context information (see Results and discussion for the numbers). A few will orig- inate from the fully averaged single-base (1-mer) Trek rates. For each unique sequence in the discussed 7-, 5-, 3- and 1-mers, if the k-mer appears more than once in the reference L1 sequence, of course with different neigh- bours at the positions out of the k-mer range, we average the Trek values by taking the median. For instance, the rG,A substitution rate constant in the 3-mer AGT rep- resents the rate averaged across all the appearances of AGT in the L1 reference sequence, which would nor- mally be with varying other neighbour bases, out of the 3-mer range. The rG,A in AGT will therefore represent the average rate constant across all the representatives of the significant range, the NNAGTNN 7-mers, present in L1, where N can be any of the four bases. In the same way, the substitution rate constants for the single bases (1-mers) can be considered as fully averaged across all the possible neighbour effects in NNNGNNN sequences. Our algo- rithm therefore makes the most of transposon exposed substitution rate data of the human genome, returning the best possible values inferable from our Trek database and, where uncertainty is present, returning the best averaged values for a shorter context range. Furthermore, we have enabled the usage of symmetrized Trek parameterisation, assuming an overall strand-invariance of the substitution rates. Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 14 of 17 k-mers were equalised. For example, the G→A substitu- tion rate constant (r1) in the 3-mer AGC was set equal to the complementary C→T rate constant (r2) in the reverse complementary GCT. The data equalisation was done in the following way: if both r1 and r2 were of the same quality accounting for the whole sequence-context infor- mation in both 3-mer variants, then both values were set to (r1 + r2)/2; however, if one of the rate constants was determined with a better quality, since the full 3- mer data for the other case was missing and the 1-mer average was used as a replacement, then the rate con- stant of the better quality variant was assigned to both r1 and r2. Accounting for the strand symmetry improves the results of the validation studies, further refining the substitution rate constant values and increasing the cover- age of longer k-mers in the Trek database. The described open source program, along with the associated data can be accessed through the http://trek.atgcdynamics.org web page. Future improvements in data and the program, through extending the types of mobile DNA in the Trek procedure, will be reflected on the same web site. The Trek mapper server application was written in R, using the Shiny library and server backend (http://shiny.rstudio. com). The stand-alone program, supporting both graph- ical and programmatic (terminal) interaction and multi- processor computing, can be obtained from the same web page, to be used for larger projects and genomes. approach used in our Trek mapper methodology (see below) thus seems preferable in mapping the rcore i,j rate constants throughout the human genome. To this end, the GBM models here had a sole purpose of identifying the optimal range of influence for accounting the neigh- bouring nucleotides. The optimal range was found to be captured, on average, by a 5-7-nt long window (Figures S4 and S5 in Additional file 1) which is in an excellent agreement with the prior < 10 nt estimate [9–11, 44, 50]. We thus used the maximum 7-nt length to strat- ify the Trek data for the further model-free mapping on any provided sequence, including the whole human genome. Finding the influence range of neighbour nucleotides Finding the influence range of neighbour nucleotides We have used gradient boosting machines (GBM) [48, 49] to elucidate the effective range for the core sequence- context effects. This was achieved by developing test mod- els to evaluate the predictive strength of only the neigh- bouring bases in defining the core substitution rate of the central base. Tree-based gradient boosting machines (GBM) are a class of machine learning methodologies that produce strong regression or classification models by creating an ensemble of weaker models. The technique consecutively adds weaker models in the forms of decision trees that aim to reduce the residuals of the predictions [48, 49]. It was used as implemented in the gbm library for R. For each i→j substitution type, all the found Trek data were taken without the possible outliers, which were filtered by allowing only the usage of the values that were within the 1.65 × standard deviation range (keeps ∼90% data if normally distributed) of the constants in a given substitution category. The sequences were then processed to produce pos/bi decoupled features that were associated with the relative adjacent positions (pos, - for upstream and + for downstream positions) and their possible four bi base types. Those features took values 0 or 1, depend- ing on whether the base at an associated relative position Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 Page 14 of 17 Page 14 of 17 Funding g This research was supported by the Herchel Smith Fund and Cancer Research UK (C9681/A18618). S.B. is a Wellcome Trust Senior Investigator (099232/Z/12/Z). The cancer enrichment score (Figure S12 in Additional file 1) for a given k-mer sequence was calculated by taking the ratio of the occurrence fractions, calculated in (numer- ator) only the cancer-linked sites (where the linked base is the central one in the k-mer) and (denominator) in the whole repeat-masked human genome (Additional file 2). We next repeated the above analysis by examining non- coding somatic mutations from different types of cancer (Fig. 8), as guided by the primary tissue (primT) and primary cancer (primC) types recorded in the COSMIC database. To stratify data, we examined the primT:primC pair as a cancer type identifier for each mutation. Abbreviations bln: Billion; BSP: Basal substitution propensity; byr: Billion years; COSMIC: Catalogue of somatic mutations in cancer; GBM: Gradient boosting machines; L1: Long interspersed nuclear element 1; LINE: Long interspersed nuclear element; LTR: Long terminal repeat; mln: Million; myr: Million years; nt: Nucleotide; SINE: Short interspersed nuclear element; SNP: Single-nucleotide polymorphism Mapping the Trek substitution data We thus analysed the results from the top 11 identifiers with the most number of recorded somatic mutations. For the comparison of the simulated sequence at equi- librium with the real human genome (RefSeq), we calcu- lated the fractions of different oligomers (k-mers) in both sequences (Additional file 2). The k-mer contents of the human genome were calculated by sliding a window of size k (from 1 to 7) and counting the occurrence of each 4k unique sequence. We used a direct calculation of the lexicological index [85] of a string to increase the compu- tational efficiency of the k-mer counting. Although, data from the masked human genome were used in the k-mer analyses to rule out any bias from the presence of the same L1 elements in the object of application of Trek data, the comparison of the masked and unmasked genomes showed only negligible differences in both single base and short k-mer contents. If, however, we consider only the L1 elements, the k-mer content in L1 is markedly differ- ent from the rest of the genome (Figure S9 in Additional file 1), additionally signifying the absence of circularity in our procedure and results. Basal substitution propensity at cancer-linked sites The basal substitution propensities were defined and retrieved for the human genome as presented in the Results and discussion (Fig. 7, Figure S10 in Additional file 1). We took all the non-coding somatic point mutation data associated with cancer from the COSMIC database [63] (http://cancer.sanger.ac.uk/cosmic, NCV dataset accessed in February, 2015). Since our Trek rate constants are for the spontaneous core substitutions, we only con- sidered the sites that were also not declared as known SNPs (the status was present in the NCV dataset). This was to ensure that we excluded sites where an active poly- morphism is potentially encouraged by natural selection. About 3.7% data from the remaining set of cancer-linked somatic mutations were duplicates, with no differences found in genomic location and mutation types. We removed those, keeping only the single first-encountered copies of such entries (Figure S11 in Additional file 1). The resulting data contained 5,984,711 mutation entries. Availability of data and materials The preprint of this work was first deposited in BioRxiv in August 2015 [86]. The Trek rcore i,j database and the mapper are housed at http://trek.atgcdynamics.org. The source code can be accessed through http://github.com/aleksahak/Trek. Mapping the Trek substitution data In the latter case, the complementary rate con- stants of the central bases in two reverse complementary Equilibration of a random DNA sequence with Trek rates A 5 million (mln) nt sized random sequence (in silico “genome”) was created with the initial A, T, G and C base contents set to 20, 20, 30 and 30% correspondingly, hence with 60% genomic G+C content. The length was selected to cope with the finite computational and time resources, though operating on lengthier sequences will not change the outcome of the calculations, since the cap- tured sequence-context effects are within 7-nt window. We first calculated the probabilities of all the possible sub- stitution in this random sequence, which basically meant the assignment of three substitution rate constants per position in the genome, describing the conversion into the three bases other than the base already present in the respective position. This was done using the Trek mapper described above. Next, at each step, we sampled 5000 substitution weighted by the calculated 3 × 5 mln rate constants. We then identified those 5000 positions and the corresponding substitution types that were sam- pled to happen (Figure S7 in Additional file 1), performed those substitutions, and, updated the probability values via the Trek mapper. Repeated multiple times, the process evolved the sequence ruled by the core spontaneous sub- stitution rate constants that are sensitive to the changes in the sequence composition at the immediate vicinity in the genome (Figs. 4, 5 and 6, Figure S8 in Additional file 1). Page 15 of 17 Page 15 of 17 Page 15 of 17 Sahakyan and Balasubramanian BMC Genomics (2017) 18:81 The same simulation was also done by using the substitu- tion rates averaged to singletons only, thus with no context dependence (Fig. 6). The data contained 29 unique primT:primC pairs, of which 11 had a substantial number of records (2,422,060 non-coding mutations for liver:carcinoma, 1,287,384 for pancreas:carcinoma, 851,028 for ovary:other, 411,076 for kidney:other, 331,520 for oesophagus:carcinoma, 286,800 for haematopoetic and lymphoid tissue:lymphoid neoplasm, 92,276 for breast:carcinoma, 79,342 for cen- tral nervous system (CNS):primitive neuroectodermal tumour/medulloblastoma, 69,721 for prostate:carcinoma, 62,714 for pancreas:carcinoid endocrine/tumour, 38,835 for lung carcinoma). This list was followed by 19,004 records with non-specified primary tissue and cancer types, and substantially low number of records for the rest (at or below ∼10 k records). Acknowledgements We thank Prof. Michael Lynch and Dr. Chris Lowe for their helpful comments and suggestions. Authors’ contributions ABS and SB designed the study, performed the research, interpreted the results and wrote the paper. Both authors read and approved the final manuscript. Additional files Additional file 1: Supplementary note, table and figures. Additional supporting note, table and figures referenced in the text (Note S1, Table S1, Figures S1-S12), as well as the detailed description of the Additional file 2 content. The file is in the PDF format. (PDF 3307 kb) Additional file 2: Supplementary numerical data. The raw data on the substitution rate constants in the reference L1 sequence (data 1), the resulting Trek database processed with (data 2) and without (data 3) the strand-symmetry considerations, the k-mer content for the masked and unmasked human genomes (data 4), the full set of 7-mer sequences with the respective cancer enrichment scores and basal substitution propensity values (data 5), and the GBM parameters that were minimising the error of the tree-based test models (data 6). The file is in the plain TXT format. (TXT 3266 kb) Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. References 29. Ellegren H. Characteristics, causes and evolutionary consequences of male-biased mutation. Proc Roy Soc. B. 2007;274(1606):1–10. 1. Lynch M. The Origins of Genome Architecture. Sunderland, 1. Lynch M. The Origins of Genome Architecture. 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Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, Xie M, Zhang Q, McMichael JF, Wyczalkowski MA, Leiserson MDM, Miller CA, Welch JS, Walter MJ, Wendl MC, Ley TJ, Wilson RK, Raphael BJ, Ding L. Mutational landscape and significance across 12 major cancer types. Nature. 2013;502(7471):333–9. 81. 1000 Genomes Project Consortium:. A map of human genome variation from population-scale sequencing. Nature. 2010;467(7319):1061–1073. 82. 1000 Genomes Project Consortium etal. A global reference for human genetic variation. Nature. 2015;526(7571):68–74. genetic variation. Nature. 2015;526(7571):68–74. 83. Smit AFA, Hubley R, Green P. RepeatMasker Open-4.0. 2015. http://www. repeatmasker.org. 60. Alexandrov LB, Nik-Zainal S, Wedge DC, Campbell PJ, Stratton MR. Deciphering signatures of mutational processes operative in human cancer. Cell Rep. 2013;3(1):246–59. 84. References Rate of de novo mutations and the importance of father’s age to disease risk. Nature. 2012;488(7412):471–5. Submit your next manuscript to BioMed Central and we will help you at every step: 72. Fu Q, Li H, Moorjani P, Jay F, Slepchenko SM, Bondarev AA, Johnson PLF, Aximu-Petri A, Prüfer K, de Filippo C, Meyer M, Zwyns N, Salazar-García DC, Kuzmin YV, Keates SG, Kosintsev PA, Razhev DI, Richards MP, Peristov NV, Lachmann M, Douka K, Higham TFG, Slatkin M, Hublin JJ, Reich D, Kelso J, Viola TB, Pääbo S. Genome sequence of a 45,000-year-old modern human from western Siberia. Nature. 2014;514(7523):445–9. • 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 p y y p • We accept pre-submission inquiries 73. Rahbari R, Wuster A, Lindsay SJ, Hardwick RJ, Alexandrov LB, Al Turki S, Dominiczak A, Morris A, Porteous D, Smith B, Stratton MR, UK10K Consortium, Hurles ME. Timing, rates and spectra of human germline mutation. Nat Genet. 2016;48(2):126–33. 74. Narasimhan VM, Rahbari R, Scally A, Wuster A, Mason D, Xue Y, Wright J, Trembath RC, Maher ER, van Heel DA, Auton A, Hurles ME, Tyler-Smith C, Durbin R. A direct multi-generational estimate of the human mutation
https://openalex.org/W4361833440	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_Caveolin-1_Increases_Aerobic_Glycolysis_in_Colorectal_Cancers_by_Stimulating_HMGA1-Mediated_i_GLUT3_i_Transcription/22393104/1/files/39838683.pdf	English	null	Supplementary Figure 1 from Caveolin-1 Increases Aerobic Glycolysis in Colorectal Cancers by Stimulating HMGA1-Mediated <i>GLUT3</i> Transcription	null	2,023	cc-by	788	Supplementary Fig. S1 A 1/GAPDH) 3.0 2.5 Normal Tumor N > 2T T > 2N V1 mRNA level (CAV1 2.0 1.5 1.0 0.5 CAV 0 P3 P5 P9 P11 P15 P17 P18 P22 P25 P28 P31 P8 P19 P27 P36 P46 P53 60 Low CAV1 High CAV1 Low CAV1 High CAV1 B ge of specimes (%) 60 50 40 30 20 15 10 9 * * 10 18 4 * * Percenta 20 10 0 3 3 5 I II III I II III (N= 33 23 44) 4 7 2 WDMDPD WDMDPD (N= 32 56 12) E - + - + - + - + - + - + 5-AzadC CAV1 er of cell (x 105) SW480 10 8 6 4 pcDNA3.1 WT-CAV1 * CAV1 mbe 2 Supplementary Fig. S1 A 1/GAPDH) 3.0 2.5 Normal Tumor N > 2T T > 2N V1 mRNA level (CAV1 2.0 1.5 1.0 0.5 CAV 0 P3 P5 P9 P11 P15 P17 P18 P22 P25 P28 P31 P8 P19 P27 P36 P46 P53 60 Low CAV1 High CAV1 Low CAV1 High CAV1 B ge of specimes (%) 60 50 40 30 20 15 10 9 * * 10 18 4 * * Percenta 20 10 0 3 3 5 I II III I II III (N= 33 23 44) 4 7 2 WDMDPD WDMDPD (N= 32 56 12) E - + - + - + - + - + - + 5-AzadC CAV1 er of cell (x 105) SW480 10 8 6 4 pcDNA3.1 WT-CAV1 * CAV1 mbe 2 Supplementary Fig. Supplementary Fig. S1 A 1/GAPDH) 3.0 2.5 Normal Tumor N > 2T T > 2N V1 mRNA level (CAV1 2.0 1.5 1.0 0.5 CAV 0 P3 P5 P9 P11 P15 P17 P18 P22 P25 P28 P31 P8 P19 P27 P36 P46 P53 60 Low CAV1 High CAV1 Low CAV1 High CAV1 B ge of specimes (%) 60 50 40 30 20 15 10 9 * * 10 18 4 * * Percenta 20 10 0 3 3 5 I II III I II III (N= 33 23 44) 4 7 2 WDMDPD WDMDPD (N= 32 56 12) E - + - + - + - + - + - + 5-AzadC CAV1 er of cell (x 105) SW480 10 8 6 4 pcDNA3.1 WT-CAV1 * CAV1 mbe 2 S1 A 1/GAPDH) 3.0 2.5 Normal Tumor N > 2T T > 2N V1 mRNA level (CAV1 2.0 1.5 1.0 0.5 CAV 0 P3 P5 P9 P11 P15 P17 P18 P22 P25 P28 P31 P8 P19 P27 P36 P46 P53 A V1 mRNA le 1.0 0.5 CAV 0 P3 P5 P9 P11 P15 P17 P18 P22 P25 P28 P31 P8 P19 P27 P36 P46 P53 60 Low CAV1 High CAV1 Low CAV1 High CAV1 B ge of specimes (%) 60 50 40 30 20 15 10 9 * * 10 18 4 * * Percenta 20 10 0 3 3 5 I II III I II III (N= 33 23 44) 4 7 2 WDMDPD WDMDPD (N= 32 56 12) C E - + - + - + - + - + - + 5-AzadC CAV1 er of cell (x 105) SW480 10 8 6 4 pcDNA3.1 WT-CAV1 * D 893 +1 CAV1 GAPDH Numbe LoVo Days after transfection 2 0 0 1 2 3 4 5 l (x 105) 20 15 si-Control si-CAV1 -893 37 35 33 32 31 29 28 27 24 20 19 16 12 11 9 6 54 3 2 1 Exon 1 +1 Number of cel Days after transfection 10 5 0 0 1 2 3 4 5 * 60 Low CAV1 High CAV1 Low CAV1 High CAV1 B ge of specimes (%) 60 50 40 30 20 15 10 9 * * 10 18 4 * * Percenta 20 10 0 3 3 5 I II III I II III (N= 33 23 44) 4 7 2 WDMDPD WDMDPD (N= 32 56 12) B B C E - + - + - + - + - + - + 5-AzadC CAV1 D 893 +1 CAV1 GAPDH -893 37 35 33 32 31 29 28 27 24 20 19 16 12 11 9 6 54 3 2 1 Exon 1 +1 er of cell (x 105) SW480 10 8 6 4 pcDNA3.1 WT-CAV1 * Numbe LoVo Days after transfection 2 0 0 1 2 3 4 5 l (x 105) 20 15 si-Control si-CAV1 Number of cel Days after transfection 10 5 0 0 1 2 3 4 5 * er of cell (x 105) SW480 10 8 6 4 pcDNA3.1 WT-CAV1 * Numbe Days after transfection 2 0 0 1 2 3 4 5 C E D Exon 1 Days after transfection 1 2 3 4
https://openalex.org/W4387095992	https://github.com/elifesciences/enhanced-preprints-data/raw/master/data/90597/v1/90597-v1.pdf	English	null	Hippocampome.org v2.0: a knowledge base enabling data-driven spiking neural network simulations of rodent hippocampal circuits	eLife	2,024	cc-by	21,382	Diek W. Wheeler, Jeffrey D. Kopsick, Nate Sutton, Carolina Tecuatl, Alexander O. Komendantov, Kasturi Nadella, Giorgio A. Ascoli Center for Neural Informatics, Structures, & Plasticity; Krasnow Institute for Advanced Study; George Mason University, Fairfax, VA, USA • Bioengineering Department and Center for Neural Informatics, Structures, & Plasticity; College of Engineering and Computing; George Mason University, Fairfax, VA, USA • Interdisciplinary Program in Neuroscience; College of Science; George Mason University, Fairfax, VA, USA https://en.wikipedia.org/wiki/Open_access https://en.wikipedia.org/wiki/Open_access https://creativecommons.org/licenses/by/4.0/ Abstract Hippocampome.org (https://hippocampome.org/) is a mature open-access knowledge base of the rodent hippocampal formation focusing on neuron types and their properties. Hippocampome.org (https://hippocampome.org/) v1.0 established a foundational classification system identifying 122 hippocampal neuron types based on their axonal and dendritic morphologies, main neurotransmitter, membrane biophysics, and molecular expression. Releases v1.1 through v1.12 furthered the aggregation of literature-mined data, including among others neuron counts, spiking patterns, synaptic physiology, in vivo firing phases, and connection probabilities. Those additional properties increased the online information content of this public resource over 100-fold, enabling numerous independent discoveries by the scientific community. Hippocampome.org (https://hippocampome.org/) v2.0, introduced here, incorporates over 50 new neuron types and extends the functionality to build real-scale, biologically detailed, data-driven computational simulations. In all cases, the freely downloadable model parameters are directly linked to the specific peer-reviewed empirical evidence from which they were derived. Possible research applications include quantitative, multiscale analyses of circuit connectivity and spiking neural network simulations of activity dynamics. These advances can help generate precise, experimentally testable hypotheses and shed light on the neural mechanisms underlying associative memory and spatial navigation. Hippocampome.org (https://hippocampome.org/) is a mature open-access knowledge base o the rodent hippocampal formation focusing on neuron types and their properties. Reviewed Preprint Reviewed Preprint Reviewed Preprint Published from the original preprint after peer review and assessment by eLife. About eLife's process Reviewed preprint posted September 27, 2023 (this version) Sent for peer review July 13, 2023 Posted to bioRxiv June 29, 2023 Published from the original preprint after peer review and assessment by eLife. Published from the original preprint after peer review and assessment by eLife. Neuroscience Neuroscience Neuroscience Introduction Neuroscience knowledge continues to increase every year (Eke et al., 2022 ; Yeung et al., 2017 ), making it challenging for researchers to keep abreast of mounting data and evolving information even in their own domain of expertise. Large-scale endeavors, such as the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative (Insel et al., 2013 ) and the European Union’s Human Brain Project (Amunts et al., 2016 ), are contributing to this tremendous growth along with the ‘long tail’ of independent labs and individual scientists (Ferguson et al., 2014 ). A key organizing principle for neuroscience knowledge is the seminal notion of neuron types (Petilla Interneuron Nomenclature Group et al., 2008; Zeng and Sanes, 2017 ), which constitute the conceptual ‘parts list’ of functional circuits. The National Institutes of Health launched the BICCN (see abbreviations in Materials and Methods) to help establish a comprehensive reference of the cell type diversity in the human, mouse, and non-human primate brains (BRAIN Initiative Cell Census Network, 2021). This multi-institution collaboration is already producing innovative results (Muñoz-Castañeda et al., 2021 ) and actionable community resources (BICCN Data Ecosystem Collaboration, 2022 ). Hippocampome.org (https://hippocampome.org/) is an open-access knowledge base of the rodent hippocampal circuit (dentate gyrus, CA3, CA2, CA1, subiculum, and entorhinal cortex) at the mesoscopic level of neuron types (Wheeler et al., 2015 ). This resource has proven popular and effective thanks to the adoption of a simple yet powerful classification system for defining neuron types. Specifically, a key property for the identification of neuron types in Hippocampome.org (https://hippocampome.org/) is the location of axons and dendrites across the subregions and layers of the hippocampal formation. This approach can be broadly extended to classify neurons in other brain regions and neural systems (Ascoli and Wheeler, 2016 ). Focusing on axonal and dendritic distributions provides two considerable advantages. First, these features mediate neuronal connectivity, thus immediately revealing the underlying blueprint of network circuitry (Rees et al., 2017 ). Second, they are widely used in the neuroscience community as a reliable and concrete anchoring signature to correlate electrophysiological and transcriptomic profiles (DeFelipe et al., 2013 ). Therefore, starting from the foundational morphology-based identification of 122 neuron types in the first release (version 1.0 or v1.0), Hippocampome.org (https://hippocampome.org/) progressively amassed an increasing amount of complementary data, such as firing patterns, molecular expression, cell counts, synaptic communication, in vivo oscillations, and connection probabilities. eLife assessment The authors have updated a helpful resource for the neuroscience community regarding hippocampal cell types and their interactions, called the "hippocampome." The updates expand content, tools, and potential applications, and therefore the significance is important. The strong presentation makes the paper compelling. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 1 of 47 Characterizing properties of hippocampal neuron types Characterizing properties of hippocampal neuron types Hippocampome.org (https://hippocampome.org/) v1.0 (Wheeler et al., 2015 ) established the morphological encoding of axonal and dendritic locations and the main neurotransmitter (glutamate or GABA) as the primary determinants of neuron types in the rodent hippocampal formation. For example, a DG Basket cell (with name capitalized to indicate a formally defined neuron type) is a GABAergic cell with axon contained in the granular layer and dendrites spanning all DG layers (Figures 1A1-4 ). In this framework, two neurons releasing the same neurotransmitter belong to different types if the axon or dendrites of only one of them invades any of the 26 layers across 6 subregions of the hippocampal formation (hippocampome.org/morphology). In other words, neurons of the same type share the same potential inputs, outputs, and excitatory vs. inhibitory function. These properties were initially supplemented with additional empirical evidence for molecular expression of major protein biomarkers (Figure 1A5 ; hippocampome.org/markers) and membrane biophysics (Figure 1A6- 7 ; hippocampome.org/electrophysiology). Many neuronal properties and functionalities were progressively added in 12 subsequent releases (Table 1 ). The numerical sequencing of these Hippocampome.org (https://hippocampome.org/) versions depended on the order of peer-review and publication of the corresponding scientific reports. Here we will describe them instead in logical groupings. The first two updates enhanced the user functionality of the knowledge base. Specifically, v1.1 integrated a web-based interactive thesaurus mapping of synonyms and definitions (Hamilton et al., 2017a ; hippocampome.org/find-term) to help disambiguate the many terminological inconsistencies in the neuroscience literature (Shepherd et al., 2019 ; Yuste et al., 2020 ). Release v1.2 introduced the capability to browse, search, and analyze the potential connectivity between neuron types (Rees et al., 2016 ; Hippocampome.org (https://hippocampome.org/) /connectivity) as derived from the compiled overlapping locations of all the presynaptic axons and postsynaptic dendrites. Transcriptomic information was greatly expanded in both v1.3 (Hamilton et al., 2017b ), which incorporated in situ hybridization data from the Allen Brain Atlas (Lein et al., 2007 ), and v1.5 (White et al., 2020 ), which leveraged relational inferences interlinking the region-specific expression of two or more genes. The quantifications of firing pattern phenotypes, such as rapid adapting spiking, transient stuttering, and persistent slow-wave bursting, in v1.6 (Komendantov et al., 2019 ; hippocampome.org/firing_patterns) were fitted by dynamical systems modeling (Izhikevich, 2003 ) in v1.7 (Venkadesh et al., 2019 ; hippocampome.org/Izhikevich). Introduction In all cases, the public repository provided direct links to the specific peer-reviewed empirical evidence supporting the added knowledge. Having established a web-based integrated storehouse of hippocampal information, Hippocampome.org (https://hippocampome.org/) also expanded its scope by including data- driven computational models of neuronal excitability and synaptic signaling, as well as ties to community resources such as NeuroMorpho.Org (Akram et al., 2018 ), SenseLab ModelDB (McDougal et al., 2017 ), CARLsim (Niedermeier et al., 2022 ), and the Allen Brain Atlas (Jones et al., 2009 ). Altogether, these extensions resulted in the emergence of a complete framework for launching real-scale, biologically detailed computer simulations of the hippocampal formation. The present report thus marks a new phase in the life cycle of this community resource. Hippocampome.org (https://hippocampome.org/) v2.0, introduced here, also incorporates over 50 newly identified neuron types, updating the classification to the most recent literature findings. The following “Description of resource” section begins with a concise, referenced overview of the neural properties collated from Hippocampome.org (https://hippocampome.org/) v1.0 through release v1.12. We then briefly describe the new neuron types and data currently being added in Hippocampome.org (https://hippocampome.org/) v2.0. Next is an abridged summary of the usage and recognition of this online portal in biomedical research. This is followed by an explanation of Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 2 of 47 the latest capabilities to search, filter, and download the complete set of computational parameters enabling quantitative connectomic analyses and spiking neural network simulations. The section concludes with an outlook of possible research applications allowed by the expansion of this scientific resource. the latest capabilities to search, filter, and download the complete set of computational parameters enabling quantitative connectomic analyses and spiking neural network simulations. The section concludes with an outlook of possible research applications allowed by the expansion of this scientific resource. Description of resource Characterizing properties of hippocampal neuron types Hippocampome.org (https://hippocampome.org/) v1.0 (Wheeler et al., 2015 ) established the morphological encoding of axonal and dendritic locations and the main neurotransmitter (glutamate or GABA) as the primary determinants of neuron types in the rodent hippocampal formation. For example, a DG Basket cell (with name capitalized to indicate a formally defined neuron type) is a GABAergic cell with axon contained in the granular layer and dendrites spanning all DG layers (Figures 1A1-4 ). In this framework, two neurons releasing the same neurotransmitter belong to different types if the axon or dendrites of only one of them invades any of the 26 layers across 6 subregions of the hippocampal formation (hippocampome.org/morphology). In other words, neurons of the same type share the same potential inputs, outputs, and excitatory vs. inhibitory function. These properties were initially supplemented with additional empirical evidence for molecular expression of major protein biomarkers (Figure 1A5 ; hippocampome.org/markers) and membrane biophysics (Figure 1A6- 7 ; hippocampome.org/electrophysiology). Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Characterizing properties of hippocampal neuron types B1) Morphology of a DG (i)2210 Basket GRALDEN (NeuroMorpho.Org cell NMO_146159: Vaden et al., 2020 ) with red axons in SG and blue dendrites in SMo and SMi. B2) Schematic interpretation of the reconstruction (same symbols as in A2). B3-4) Hippocampome.org (https://hippocampome.org/) representation and numerical coding of the morphology (same symbols as in A3-4). B5) Biomarker expression. B6) Firing pattern phenotype (TSTUT.SLN; adapted from Figure S4 in Markwardt et al., 2011 ). B7) Membrane biophysics values recorded at room temperature (from Figure 4D in Vaden et al., 2020 ), and at 22 °C (from Figure S4 in Markwardt et al., 2011 ); emboldened values were extracted from the firing pattern trace in B6. Abbreviations are defined in Materials and Methods. Characterizing properties of hippocampal neuron types Although the above properties were largely measured from slice preparations, v1.9 made available measurements from in vivo recordings (Sanchez-Aguilera et al., 2021 ; hippocampome.org/in-vivo). Release v1.10 provided a compendium of cognitive functions linked to specific hippocampal neurons (Sutton and Ascoli, 2021 ; hippocampome.org/cognome), while the v1.11 neuron type census estimated the population counts for each neuron type (Attili et al., 2022 ; hippocampome.org/census). Last but not least are a set of properties pertaining not to individual neuron types but to synaptic connections between a pair of pre- and post-synaptic neuron types. In particular, v1.8 calculated the synaptic probabilities and the numbers of contacts per connected pair (Tecuatl et al., 2021b ; hippocampome.org/syn_probabilities), and v1.4 data mined synaptic physiology (Moradi and Ascoli, 2020 ; hippocampome.org/synaptome), with conductance, time constant, and short-term Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 3 of 47 Figure 1. Defining neuron types in Hippocampome.org (https://hippocampome.org/) . A) Properties of a Hippocampome.org (https://hippocampome.org/) v1.0 neuron type. A1) Morphology of a DG (i)2232 Basket cell (NeuroMorpho.Org cell NMO_34300: Hosp et al., 2014 ) with axons (red) in SG and dendrites (blue) in all four layers. A2) Schematic interpretation of the morphological tracing, where the circle represents the location of the soma in SG, the red triangle the location of the axons in SG, and the blue rectangles the locations of the dendrites in all four layers. A3) Hippocampome.org (https:// hippocampome.org/) representation of the morphology, where a blue square with a vertical line (\|) indicates dendritic presence in SMo and SMi and H, a purple square with a cross (+) indicates both axonal and dendritic presence in SG, and a black dot (•) indicates the soma location in SG. A4) Hippocampome.org (https://hippocampome.org/) numerical coding of the reconstructed neuron, where 2 indicates the presence of dendrites (in SMo, SMi, and H); and 3 indicates the presence of both axons and dendrites (in SG). A5) Biomarker expressions, where a green triangle indicates positive expression, and a blue triangle indicates negative expression. A6) Firing pattern phenotype (NASP; adapted from Figure 1B 1 in Savanthrapadian et al., 2014 ). A7) Membrane biophysics values (from Figure 3C and Table 1 in Lübke et al., 1998 ) recorded at 35-37 °C. B) Properties for a Hippocampome.org (https://hippocampome.org/) v2.0 neuron type. Figure 1. Defining neuron types in Hippocampome.org (https://hippocampome.org/) . A) Properties of a Hippocampome.org (https://hippocampome.org/) v1.0 neuron type. A1) Morphology of a DG (i)2232 Basket cell (NeuroMorpho.Org cell NMO_34300: Hosp et al., 2014 ) with axons (red) in SG and dendrites (blue) in all four layers. A2) Schematic interpretation of the morphological tracing, where the circle represents the location of the soma in SG, the red triangle the location of the axons in SG, and the blue rectangles the locations of the dendrites in all four layers. A3) Hippocampome.org (https:// hippocampome.org/) representation of the morphology, where a blue square with a vertical line (\|) indicates dendritic presence in SMo and SMi and H, a purple square with a cross (+) indicates both axonal and dendritic presence in SG, and a black dot (•) indicates the soma location in SG. A4) Hippocampome.org (https://hippocampome.org/) numerical coding of the reconstructed neuron, where 2 indicates the presence of dendrites (in SMo, SMi, and H); and 3 indicates the presence of both axons and dendrites (in SG). A5) Biomarker expressions, where a green triangle indicates positive expression, and a blue triangle indicates negative expression. A6) Firing pattern phenotype (NASP; adapted from Figure 1B 1 in Savanthrapadian et al., 2014 ). A7) Membrane biophysics values (from Figure 3C and Table 1 in Lübke et al., 1998 ) recorded at 35-37 °C. B) Properties for a Hippocampome.org (https://hippocampome.org/) v2.0 neuron type. B1) Morphology of a DG (i)2210 Basket GRALDEN (NeuroMorpho.Org cell NMO_146159: Vaden et al., 2020 ) with red axons in SG and blue dendrites in SMo and SMi. B2) Schematic interpretation of the reconstruction (same symbols as in A2). B3-4) Hippocampome.org (https://hippocampome.org/) representation and numerical coding of the morphology (same symbols as in A3-4). B5) Biomarker expression. B6) Firing pattern phenotype (TSTUT.SLN; adapted from Figure S4 in Markwardt et al., 2011 ). B7) Membrane biophysics values recorded at room temperature (from Figure 4D in Vaden et al., 2020 ), and at 22 °C (from Figure S4 in Markwardt et al., 2011 ); emboldened values were extracted from the firing pattern trace in B6. Abbreviations are defined in Materials and Methods. Defining neuron types in Hippocampome.org (https://hippocampome.org/) . A) Properties of a Hippocampome.org (https://hippocampome.org/) v1.0 neuron type. A1) Morphology of a DG (i)2232 Basket cell (NeuroMorpho.Org cell NMO_34300: Hosp et al., 2014 ) with axons (red) in SG and dendrites (blue) in all four layers. Figure 1. A2) Schematic interpretation of the morphological tracing, where the circle represents the location of the soma in SG, the red triangle the location of the axons in SG, and the blue rectangles the locations of the dendrites in all four layers. A3) Hippocampome.org (https:// hippocampome.org/) representation of the morphology, where a blue square with a vertical line (\|) indicates dendritic presence in SMo and SMi and H, a purple square with a cross (+) indicates both axonal and dendritic presence in SG, and a black dot (•) indicates the soma location in SG. A4) Hippocampome.org (https://hippocampome.org/) numerical coding of the reconstructed neuron, where 2 indicates the presence of dendrites (in SMo, SMi, and H); and 3 indicates the presence of both axons and dendrites (in SG). A5) Biomarker expressions, where a green triangle indicates positive expression, and a blue triangle indicates negative expression. A6) Firing pattern phenotype (NASP; adapted from Figure 1B 1 in Savanthrapadian et al., 2014 ). A7) Membrane biophysics values (from Figure 3C and Table 1 in Lübke et al., 1998 ) recorded at 35-37 °C. B) Properties for a Hippocampome.org (https://hippocampome.org/) v2.0 neuron type. B1) Morphology of a DG (i)2210 Basket GRALDEN (NeuroMorpho.Org cell NMO_146159: Vaden et al., 2020 ) with red axons in SG and blue dendrites in SMo and SMi. B2) Schematic interpretation of the reconstruction (same symbols as in A2). B3-4) Hippocampome.org (https://hippocampome.org/) representation and numerical coding of the morphology (same symbols as in A3-4). B5) Biomarker expression. B6) Firing pattern phenotype (TSTUT.SLN; adapted from Figure S4 in Markwardt et al., 2011 ). B7) Membrane biophysics values recorded at room temperature (from Figure 4D in Vaden et al., 2020 ), and at 22 °C (from Figure S4 in Markwardt et al., 2011 ); emboldened values were extracted from the firing pattern trace in B6. Abbreviations are defined in Materials and Methods. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 4 of 47 4 of 47 Table 1. Expanding the catalog of neuron types and properties from Hippocampome.org (https://hippocampome.org/) v1.x to v2.0 The Hippocampome.org (https://hippocampome.org/) framework to classify neuron types and collate their properties allows agile content updates as new data are continuously reported in the peer-reviewed literature. For example, the description of a parvalbumin-positive DG GABAergic interneuron with axon contained in the granular layer and dendrites invading the molecular layer but not the hilus (Vaden et al., 2020 ) supported the definition of a new neuron type (Figures 1B1-5 ), referred to in Hippocampome.org (https://hippocampome.org/) v2.0 as DG Basket GRALDEN. Moreover, such an identification made it possible to unequivocally ascribe to this neuron type previously reported electrophysiological characteristics (Figures 1B6-7 ; Markwardt et al., 2011 ). Comprehensive literature mining following the same process expanded the Hippocampome.org (https://hippocampome.org/) v2.0 catalog with 51 new neuron types1 across 5 of the 6 subregions of the hippocampal formation (Figure 2 ), including axonal-dendritic morphological patterns (Figure 2A ), molecular expression (Figure 2B ), and membrane biophysics (Figure 2C ). Besides identifying new neuron types, the Hippocampome.org (https://hippocampome.org/) classification system also allows the ongoing accumulation of new properties onto existing neuron types as well as the reconciliation of fragmented descriptions from scientific publications (Figure 3 ). For instance, converging evidence indicates that EC Layer III Pyramidal cells have axonal projections in all layers of CA1 (Deller et al., 1996 ; Takács et al., 2012 ), not just in stratum lacunosum-moleculare (SLM) as originally reported (Steward, 1976 ). Hippocampome.org (https:// hippocampome.org/) v2.0 captures both the new extracted knowledge and the corresponding experimental evidence (Figure 3A ). The annotation of neuron type-specific firing phases relative to in vivo oscillations in v1.9 highlighted a clear distinction between Superficial and Deep CA1 Pyramidal cells (Sanchez-Aguilera et al., 2021 ). The present release enriches that description with accompanying novel molecular markers (Figure 3B1 ), membrane biophysics values (Figure 3B2 ), and differential connectivity with other subregions and neuron types (Figure 3B3 ). Similarly, numerous additional firing patterns (Figure 3C ) have been datamined for existing neuron types, such as adapting spiking in CA3 Basket cells and non-adapting spiking in CA3 Bistratified cells (Fidzinski et al., 2015) or transient stuttering in CA1 Radiatum Giant cells (Kirson and Yaari, 2000 ). Notably, this includes a novel phenotype, TSTUT.PSTUT in CA1 Interneuron Specific O-targeting QuadD cells (Chamberland et al., 2010 ). With this report, we also release new differential connection probabilities to various CA1 neuron type targets from traditional CA3 Pyramidal cells vs. Table 1. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 5 of 47 Added knowledge and functioning in Hippocampome.org (https://hippocampome.org/) releases v1.1-12. and functioning in Hippocampome.org (https://hippocampome.org/) releases v1.1-12. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 5 of 47 5 of 47 plasticity values normalized by age, temperature, species, sex, and recording method in v1.12 (Moradi et al., 2022 ; hippocampome.org/synapse), leveraging machine learning and a phenomenological model (Tsodyks et al., 1998 ). Expanding the catalog of neuron types and properties from Hippocampome.org (https://hippocampome.org/) v1.x to v2.0 CA3c Pyramidal cells (Figure 3D ) and from DG Granule cells to mossy fiber CA3 targets (Table 2 ). Quantifying the content and impact of Quantifying the content and impact of Hippocampome.org (https://hippocampome.org/) Hippocampome.org (https://hippocampome.org/) Over the course of subsequent releases, we have measured Hippocampome.org (https:// hippocampome.org/) content using two metrics. The number of pieces of knowledge (PoK) tallies the distinct units of structured information, such as the statements that DG Granule cell axons invade the hilus or that CA1 Basket cells express parvalbumin. The pieces of evidence (PoE) are specific excerpts of peer reviewed publications (portion of text, figure, or table) or database entries (e.g., from the Allen Brain Atlas) always linked to each PoK. Both PoK and PoE continued to grow with successive releases of Hippocampome.org (https://hippocampome.org/) (Figure 4A ). Notably, the largest increases in PoK and PoE were related to synaptic properties (Moradi and Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 6 of 47 Figure 2. New neuron types added to Hippocampome.org (https://hippocampome.org/) v2.0. A) Morphology encodings of the 56 new neuron types. (Left) Increase in number of neuron types for each subregion. B) Biomarker expressions of the neuron types. C) Membrane biophysics values for the neuron types. Abbreviations are defined in Materials and Methods. Figure 2. New neuron types added to Hippocampome.org (https://hippocampome.org/) v2.0. A) Morphology encodings of the 56 new neuron types. (Left) Increase in number of neuron types for each subregion. B) Biomarker expressions of the neuron types. C) Membrane biophysics values for the neuron types. Abbreviations are defined in Materials and Methods. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 7 of 47 7 of 47 Figure 3. Extensions to the neuronal properties of Hippocampome.org (https://hippocampome.org/) v1.x. A1) Additions to the axonal projections (circled in green) for two v1.0 neuron types. A2) Evidence for the axonal extensions in strata oriens, pyramidale, and radiatum from A1 are circled in green (adapted from Figure 2b in Deller et al., 1996 ). Scale bar: 75 μm. B1) Biomarker expressions for the two CA1 Pyramidal sub-types added in Hippocampome.org (https://hippocampome.org/) v1.9 (Sanchez-Aguilera et al., 2021 ). B2) Membrane biophysics values for the two sub-types. B3) CA2 projects preferentially to the deep sublayer of CA1 (Kohara et al., 2014 ). More perisomatic PV+ GABAergic boutons are found at CA1 Deep Pyramidal cells (Valero et al., 2015 ). CA1 Superficial Pyramidal cells form more frequent connections to PV+ CA1 Basket cells, and PV+ CA1 Basket cells form significantly more perisomatic axon terminals on CA1 Deep Pyramidal cells (Lee et al., 2014 ). C1) Additions to the firing pattern phenotypes of v1.0 neuron types. C2a) Example of ASP. in a CA1 Oriens-Bistratified cell (adapted from Figure 4B in Craig and McBain, 2015 ). C2b) Example of NASP in a CA3 Basket CCK+ cell (adapted from Figure 3A in Szabadics and Soltesz, 2009 ). C2c) Example of TSTUT. in a CA1 Radiatum Giant cell (adapted from Figure 2Bb in Kirson and Yaari, 2000 ). C2d) Example of TSTUT.PSTUT in a CA1 Interneuron Specific O-targeting QuadD (adapted from Figure 2D in Chamberland et al., 2010 ). C2e) Example of TSTUT.SLN in a DG MOLAX cell (adapted from Figure S2c in Lee et al., 2016 ). neuron types. Abbreviations are defined in Materials and Methods. Figure 3. Extensions to the neuronal properties of Hippocampome.org (https://hippocampome.org/) v1.x. A1) Additions to the axonal projections (circled in green) for two v1.0 neuron types. A2) Evidence for the axonal extensions in strata oriens, pyramidale, and radiatum from A1 are circled in green (adapted from Figure 2b in Deller et al., 1996 ). Scale bar: 75 μm. B1) Biomarker expressions for the two CA1 Pyramidal sub-types added in Hippocampome.org (https://hippocampome.org/) v1.9 (Sanchez-Aguilera et al., 2021 ). B2) Membrane biophysics values for the two sub-types. B3) CA2 projects preferentially to the deep sublayer of CA1 (Kohara et al., 2014 ). More perisomatic PV+ GABAergic boutons are found at CA1 Deep Pyramidal cells (Valero et al., 2015 ). CA1 Superficial Pyramidal cells form more frequent connections to PV+ CA1 Basket cells, and PV+ CA1 Basket cells form significantly more perisomatic axon terminals on CA1 Deep Pyramidal cells (Lee et al., 2014 ). C1) Additions to the firing pattern phenotypes of v1.0 neuron types. C2a) Example of ASP. in a CA1 Oriens-Bistratified cell (adapted from Figure 4B in Craig and McBain, 2015 ). C2b) Example of NASP in a CA3 Basket CCK+ cell (adapted from Figure 3A in Szabadics and Soltesz, 2009 ). C2c) Example of TSTUT. in a CA1 Radiatum Giant cell (adapted from Figure 2Bb in Kirson and Yaari, 2000 ). C2d) Example of TSTUT.PSTUT in a CA1 Interneuron Specific O-targeting QuadD (adapted from Figure 2D in Chamberland et al., 2010 ). C2e) Example of TSTUT.SLN in a DG MOLAX cell (adapted from Figure S2c in Lee et al., 2016 ). neuron types. Abbreviations are defined in Materials and Methods. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 8 of 47 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Probabilities of connection and number of contacts per connected pair from DG Granule cell to mossy fibers targets in CA3. Table 2. Probabilities of connection and number of contacts per connected pair from DG Granule cell to mossy fibers targets in CA3. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 9 of 47 9 of 47 Ascoli, 2020 ; Tecuatl et al., 2021b ; Moradi et al., 2022 ). Specifically, the data underlying synaptic physiology and connection probabilities were supported by over 23,000 PoE and yielded a remarkable 500,000 PoK thanks to the normalized collection of signaling and short-term plasticity modeling parameters for multiple combinations of experimental conditions. To assess community usage of Hippocampome.org (https://hippocampome.org/) , we tracked the number of citations of the original publication (Wheeler et al., 2015 ) and of the subsequent versions (Figure 4B ), separating simple references from actual employment of information extracted from Hippocampome.org (https://hippocampome.org/) for secondary analyses (Table 3 ). At the time of this writing, year 2021 proved to be the most prolific citation-wise; however, more than a third of the releases (v1.8-12) appeared after 2021 and most PoK were added in 2022, so usage could potentially accelerate further in coming years. An early application of Hippocampome.org (https://hippocampome.org/) -sourced data used subthreshold biophysical measures, such as input resistance and membrane time constant, for multicompartmental models of signal integration and extracellular field generation (Gulyas et al., 2016). That study concluded that somatic and proximal dendritic intracellular recordings in pyramidal cells and calretinin- positive interneurons, in particular, do not capture a sizable portion of the synaptic inputs. As a recent usage example, another lab employed Hippocampome.org (https://hippocampome.org/) as the primary information resource for neuron types in DG, CA3, and CA1 (Schumm et al., 2022 ). They discovered that mild traumatic brain injury, in the form of alterations in spike-timing- dependent plasticity, may affect the broadband power in CA3 and CA1 and the phase coherence between CA3 and CA1. From experimental data to biologically realistic computational models Several key neural properties collated into Hippocampome.org (https://hippocampome.org/) have gradually transformed the site from an organized repository of hippocampal knowledge to a computational framework for launching real-scale neural network simulations. Specifically, building a data-driven circuit model of a neural system (such as the hippocampal formation or portion thereof) requires four essential quantities besides the full list of neuron types (Bahmer et al., 2023 ; DePasquale et al., 2023 ): (i) the number of neurons in each type; (ii) the input-output response function for each neuron type; (iii) the connection probability for each pair of interacting neuron types; and (iv) the unitary synaptic signals for each pair of connected neuron types (Figure 5 ). Of those quantities, (i) and (ii) are neuron type properties, while (iii) and (iv) are properties of directional connections, defined as a distinct pair of a presynaptic and a postsynaptic neuron type. Moreover, (i) and (iii) are structural features, while (ii) and (iv) are electrophysiological ones. Hippocampome.org (https://hippocampome.org/) v2.0 provides estimates of the number of neurons in each neuron type (i) for both rats and mice (Figure 5A ). These values were derived in a two-step process (Attili et al., 2020 ): first, literature mining extracted suitable quantitative relations such as the cellular density in a given layer (Attili et al., 2019 ), the total count of neurons expressing a certain gene, or the fraction of sampled cells with a particular morphology; second, numerical optimization of the corresponding equations yielded a complete census for all neuron types. Hippocampome.org (https://hippocampome.org/) represents the neuronal input- output response function (ii) in the form of single- and multi-compartment Izhikevich models (Figure 5B ) fitted by evolutionary algorithms to accurately reproduce the observed firing behavior of each neuron type (Venkadesh et al., 2018 ). The connection probability (iii) from one neuron type to another (Figure 5C ) was computed from measurements of the appropriate axonal and dendritic lengths in each invaded subregion and layer (hippocampome.org/A- D_lengths). Additionally, users can also access the presynaptic and postsynaptic path distances from the respective somata (hippocampome.org/soma_distances) and the number of contacts per connected neuron pairs (hippocampome.org/num_contacts). As for the synaptic communication Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 10 of 47 . Hippocampome.org (https://hippocampome.org/) data, knowledge, citations, and usage since v1.0. A) Increase in pieces of knowl nce (red) with Hippocampome.org (https://hippocampome.org/) version number. From experimental data to biologically realistic computational models B) Number of citations, in which the publication d (blue and gray portions), and usage cases, in which the citing work makes use of the information contained within the Hippocam Figure 4. Trends in Hippocampome.org (https://hippocampome.org/) data, knowledge, citations, and usage since v1.0. A) Increase in pieces of knowledge (blue) and evidence (red) with Hippocampome.org (https://hippocampome.org/) version number. B) Number of citations, in which the publication is simply referenced (blue and gray portions), and usage cases, in which the citing work makes use of the information contained within the Hippocampome.org (https://hippocampome.org/) -related work (orange and yellow portions), by year. Figure 4. Trends in Hippocampome.org (https://hippocampome.org/) data, knowledge, citations, and usage since v1.0. A) Increase in pieces of knowledge (blue) and evidence (red) with Hippocampome.org (https://hippocampome.org/) version number. B) Number of citations, in which the publication is simply referenced (blue and gray portions), and usage cases, in which the citing work makes use of the information contained within the Hippocampome.org (https://hippocampome.org/) -related work (orange and yellow portions), by year. Trends in Hippocampome.org (https://hippocampome.org/) data, knowledge, citations, and usage since v1.0. A) Increase in pieces of knowledge (blue) and evidence (red) with Hippocampome.org (https://hippocampome.org/) version number. B) Number of citations, in which the publication is simply referenced (blue and gray portions), and usage cases, in which the citing work makes use of the information contained within the Hippocampome.org (https://hippocampome.org/) -related work (orange and yellow portions), by year. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 11 of 47 11 of 47 Table 3. Examples of independent studies utilizing unique neuronal properties from Hippocampome.org (https://hippocampome.org/) v1.0. Examples of independent studies utilizing unique neuronal properties from Hippocampome.org (https://hippocampome.org/) v1.0. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 12 of 47 Figure 5. Transitional knowledge enabling Hippocampome.org (https://hippocampome.org/) to support spiking neural network simulations. Center: General diagram of the hippocampal formation and the number of cell types in Hippocampome v1.0. A) Neuron type census. Top: General pipeline for obtaining cell counts for specific collections of neurons from the peer reviewed literature. Left: Neuron count proportions for the different subregions of the hippocampal formation. Insert: Normalized neuron counts for the inhibitory vs. excitatory balance by subregion. Right: Neuron counts for five identified CA2 neuron types (green schematic: excitatory, red schematic: inhibitory). B) Neuron dynamics. Top: General pipeline for obtaining Izhikevich models to reproduce the firing pattern phenotypes from peer reviewed data. Right: Simulated firing pattern from a Sub CA1-Projecting Pyramidal cell in response to a 250-pA current injection pulse lasting 1 s. Izhikevich model parameters are shown in bold and the membrane biophysics properties are shown in regular font. C) Synaptic probabilities. Left: General pipeline for obtaining the connection probabilities, number of contacts, and dendritic and axonal path lengths from 2D reconstructions. Middle: Example of a connectivity diagram of a DG Granule cell and two interneurons across the different parcels of DG. Probabilities of connection (mean ± SD) are shown in black, numbers of contacts in gray, dendritic path lengths in blue, and axonal lengths in red. Right top: Total number of connections within DG by connection type. Right bottom: Breakdown of the total number of connections by parcel and connection type. D) Synaptic physiology. Left: General pipeline for obtaining normalized synaptic parameters from paired recordings with a TPM model. Right top: Digitized synaptic data between two EC LII-III Pyramidal-Tripolar cells. Experimental data are shown in blue, initiation synaptic points in pink, model data in orange, and corrected data in green. Right bottom: Simulated modeling conditions, electrophysiological parameters, and TPM parameters. Abbreviations are defined in Materials and Methods. Figure 5. Transitional knowledge enabling Hippocampome.org (https://hippocampome.org/) to support spiking neural network simulations. Center: General diagram of the hippocampal formation and the number of cell types in Hippocampome v1.0. A) Neuron type census. Top: General pipeline for obtaining cell counts for specific collections of neurons from the peer reviewed literature. Left: Neuron count proportions for the different subregions of the hippocampal formation. Insert: Normalized neuron counts for the inhibitory vs. excitatory balance by subregion. Right: Neuron counts for five identified CA2 neuron types (green schematic: excitatory, red schematic: inhibitory). B) Neuron dynamics. Top: General pipeline for obtaining Izhikevich models to reproduce the firing pattern phenotypes from peer reviewed data. Right: Simulated firing pattern from a Sub CA1-Projecting Pyramidal cell in response to a 250-pA current injection pulse lasting 1 s. Izhikevich model parameters are shown in bold and the membrane biophysics properties are shown in regular font. C) Synaptic probabilities. Left: General pipeline for obtaining the connection probabilities, number of contacts, and dendritic and axonal path lengths from 2D reconstructions. Middle: Example of a connectivity diagram of a DG Granule cell and two interneurons across the different parcels of DG. Probabilities of connection (mean ± SD) are shown in black, numbers of contacts in gray, dendritic path lengths in blue, and axonal lengths in red. Right top: Total number of connections within DG by connection type. Right bottom: Breakdown of the total number of connections by parcel and connection type. D) Synaptic physiology. Left: General pipeline for obtaining normalized synaptic parameters from paired recordings with a TPM model. Right top: Digitized synaptic data between two EC LII-III Pyramidal-Tripolar cells. Experimental data are shown in blue, initiation synaptic points in pink, model data in orange, and corrected data in green. Right bottom: Simulated modeling conditions, electrophysiological parameters, and TPM parameters. Abbreviations are defined in Materials and Methods. Transitional knowledge enabling Hippocampome.org (https://hippocampome.org/) to support spiking neural network simulations. Center: General diagram of the hippocampal formation and the number of cell types in Hippocampome v1.0. A) Neuron type census. Top: General pipeline for obtaining cell counts for specific collections of neurons from the peer reviewed literature. Left: Neuron count proportions for the different subregions of the hippocampal formation. Insert: Normalized neuron counts for the inhibitory vs. excitatory balance by subregion. Right: Neuron counts for five identified CA2 neuron types (green schematic: excitatory, red schematic: inhibitory). B) Neuron dynamics. Figure 5. Those in turn are linked to an identified neuron type based on qualitative features, like calbindin expression or laminar distribution of axons and dendrites. In addition to enabling computational applications as described below, such integration also allows the meta-analysis of correlations between morphological features, molecular profiles, electrophysiological properties, and dynamic circuit functions. At the same time, several components of Hippocampome.org (https://hippocampome.org/) are also synergistically linked to external community resources (Figure 6B ). For example, each neuron page links out to all three-dimensional morphological reconstructions of the same cell type available in NeuroMorpho.Org (Ascoli et al., 2007 ), and selected data from NeuroMorpho.Org were used to compute axonal and dendritic length and connection probabilities. Each neuron page also links out to all computational models (including Hodgkin-Huxley, stochastic diffusion, mean firing rate, etc.) involving the same cell type on ModelDB (McDougal et al., 2017 ), while conversely ModelDB includes the Izhikevich models for all Hippocampome.org (https://hippocampome.org/) neuron types. Moreover, simulation parameters from Hippocampome.org (https://hippocampome .org/) are exportable to the CARLsim simulation environment (Nageswaran et al., 2009 ), enabling fast execution of spiking neural network models optimized for GPUs. Furthermore, Hippocampome.org (https://hippocampome.org/) harnessed data from the Allen Brain Atlas (Lein et al., 2007 ) to infer gene expression for principal neurons and cell densities for use in the neuron type census. To facilitate construction of spiking neural network simulations, Hippocampome.org (https:// hippocampome.org/) v2.0 also includes a new graphical user interface (GUI)2 . With this GUI, users can download sets of simulation parameter values for arbitrarily selected neuron types, a subregion of interest, or the whole hippocampal formation (Figure 7 ). The sets consist of files for the instantiation of CARLsim simulations and a CSV spreadsheet of parameters for use in a different simulation environment of the user’s choice. For the convenience of users interested in simplified circuit models, Hippocampome.org (https://hippocampome.org/) informally ranks the importance of each neuron type from 1 (essential) to 5 (dispensable). For instance, a user may choose to simulate only the canonical, or rank 1, neuron types of the tri-synaptic circuit and entorhinal cortex, consisting of DG granule, CA3 pyramidal, CA1 Pyramidal, and MEC LII Stellate cells. When Hippocampome.org (https://hippocampome.org/) is missing a parameter value due to insufficient experimental evidence, the GUI exports a default value clearly indicating so in the downloadable files. Figure 5. Top: General pipeline for obtaining Izhikevich models to reproduce the firing pattern phenotypes from peer reviewed data. Right: Simulated firing pattern from a Sub CA1-Projecting Pyramidal cell in response to a 250-pA current injection pulse lasting 1 s. Izhikevich model parameters are shown in bold and the membrane biophysics properties are shown in regular font. C) Synaptic probabilities. Left: General pipeline for obtaining the connection probabilities, number of contacts, and dendritic and axonal path lengths from 2D reconstructions. Middle: Example of a connectivity diagram of a DG Granule cell and two interneurons across the different parcels of DG. Probabilities of connection (mean ± SD) are shown in black, numbers of contacts in gray, dendritic path lengths in blue, and axonal lengths in red. Right top: Total number of connections within DG by connection type. Right bottom: Breakdown of the total number of connections by parcel and connection type. D) Synaptic physiology. Left: General pipeline for obtaining normalized synaptic parameters from paired recordings with a TPM model. Right top: Digitized synaptic data between two EC LII-III Pyramidal-Tripolar cells. Experimental data are shown in blue, initiation synaptic points in pink, model data in orange, and corrected data in green. Right bottom: Simulated modeling conditions, electrophysiological parameters, and TPM parameters. Abbreviations are defined in Materials and Methods. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 13 of 47 13 of 47 between neurons (iv), Hippocampome.org (https://hippocampome.org/) adopts the Tsodyks- Pawelzik-Markram formulation, representing unitary signals and short-term plasticity with five constants for each directional pair of interacting neuron types: the synaptic conductance, decay time, recovery time, facilitation time, and the utilization ratio (Tsodyks et al., 1998 ; Moradi et al., 2022 ). Once again, these parameters were fitted from the experimental data (Moradi and Ascoli, 2018 ) employing deep learning to account for (and predict the effects of) numerous experimental variables (Figure 5D ), including species (rat vs. mouse), sex (male vs. female), age (young vs. adult), recording temperature (room vs. body), and clamping configuration (voltage vs. current). The above description underscores the crucial interconnectedness of individually measured neuronal properties forming a cohesive whole in Hippocampome.org (https://hippocampome.org/) (Figure 6 ). In particular, normalized simulation parameters (e.g., the sensitivity of recovery variable in Izhikevich models) are derived from quantitative experimental measurements, such as the spiking adaptation rate (Figure 6a ). Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Figure 5. For missing Izhikevich and synaptic signaling parameters, the default values are those provided by the CARLsim simulation environment. For missing synaptic probabilities, Hippocampome.org (https://hippocampome.org/) precomputes values averaged by connection type, namely excitatory-excitatory (0.0117), excitatory-inhibitory (0.0237), inhibitory-excitatory (0.00684), and inhibitory-inhibitory (0.00423). Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 14 of 47 14 of 47 rg (https://hippocampome.org/) data provenance. A) The internal web of constituent neuron-type properties (thin bute to the instantiation of spiking neural simulations (thick arrows). Properties described qualitatively, such as mor r or molecular biomarker expressions, are in black font. Properties described by quantitative values, such as memb are in red font. Properties with v2.0 updated information, such as connectivity and firing pattern phenotypes, are de .x information, such as Izhikevich modeling parameter values and neuron-type census values, is visualized by black ntribute data to and receive data from Hippocampome.org (https://hippocampome.org/) (the ModelDB logo has be Figure 6. Hippocampome.org (https://hippocampome.org/) data provenance. A) The internal web of constituent neuron-type properties (thin arrows) that ultimately contribute to the instantiation of spiking neural simulations (thick arrows). Properties described qualitatively, such as morphological presence of axons in a layer or molecular biomarker expressions, are in black font. Properties described by quantitative values, such as membrane biophysics and neurite lengths, are in red font. Properties with v2.0 updated information, such as connectivity and firing pattern phenotypes, are depicted by blue hexagons, and v1.x information, such as Izhikevich modeling parameter values and neuron-type census values, is visualized by black circles. B) External resources that contribute data to and receive data from Hippocampome.org (https://hippocampome.org/) (the ModelDB logo has been modified from the original). Hippocampome.org (https://hippocampome.org/) data provenance. A) The internal web of constituent neuron-type properties (thin arrows) that ultimately contribute to the instantiation of spiking neural simulations (thick arrows). Properties described qualitatively, such as morphological presence of axons in a layer or molecular biomarker expressions, are in black font. Properties described by quantitative values, such as membrane biophysics and neurite lengths, are in red font. Properties with v2.0 updated information, such as connectivity and firing pattern phenotypes, are depicted by blue hexagons, and v1.x information, such as Izhikevich modeling parameter values and neuron-type census values, is visualized by black circles. B) External resources that contribute data to and receive data from Hippocampome.org (https://hippocampome.org/) (the ModelDB logo has been modified from the original). Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 15 of 47 15 of 47 Figure 7. CARLsim simulation parameters selection and file generation interface. A) The user chooses which subset of the available neuron types to include in the generated downloadable parameter file. Neuron types can be selected (check boxes and gray highlights) either individually or by groupings, such as by subregion and/or by importance rank. B) Representative user selection. C) Downloadable neuron-level parameters. D) Downloadable connection-level parameters. Potential applications to connectomic analyses and spiking neural networks simulations Hippocampome.org (https://hippocampome.org/) v2.0 enables the multiscale analysis of circuit connectivity (Figure 8 ). At the highest echelon are the connections between hippocampal subregions, which are comprised of the mesoscopic level potential connectivity between individual neuron types (Figure 8A ). Expanding, for example, upon the 147 connections between DG and CA3 neuron types reveals all the connections every individual neuron type forms with the other neuron types within and across the subregions (Figure 8B ). Zooming in onto a single neuron type each from DG and CA3, it is possible to quantify the efferent and afferent connections with other neuron types from throughout the hippocampal formation in terms of synaptic probabilities and number of neuronal partners (Figure 8C ). Diving even deeper into the isolated connection between two neuron types, such as the mossy fiber contacts from DG Granule cells to CA3 Basket cells, expands the connectivity analysis to several physiological factors affecting neuronal communication: the subcellular location of the synaptic contact (e.g., soma in stratum pyramidale and proximal dendrites in stratum lucidum), the transfer function (product of synaptic conductance and decay time constant), the in vivo firing rate of the presynaptic neuron type, and the relationship between input current and resulting output spiking frequency (F-I curve) of the post-synaptic neuron (Figure 8D ). Release of v2.0 makes the original objective of Hippocampome.org (https://hippocampome.org/) , to enable data-driven spiking neural network simulations of rodent hippocampal circuits (Ascoli, 2010), finally achievable. An ongoing line of research in this regard focuses on a real-scale mouse model of CA3, with the eventual goal of investigating the cellular mechanisms of pattern completion. Initial work included excitatory Pyramidal cells and seven main inhibitory interneuron types: Axo-axonic, Basket, Basket CCK+, Bistratified, Ivy, Mossy Fiber-Associated ORDEN, and QuadD-LM (Kopsick et al., 2022 ). Use of Hippocampome.org (https://hippocampome .org/) parameters for cell census, Izhikevich models, synaptic signals, and connection probabilities resulted in robust, realistic, rhythmic resting state activity for all neuron types (Figure 9A ). Another pursuit seeks to replicate the spatial representation in grid cells (Sargolini et al., 2006 ), modeled utilizing the Hippocampome.org (https://hippocampome.org/) MEC LII Stellate cells and supported by various GABAergic interneuron types (Dhillon and Jones, 2000 ). This study aims to reproduce the in vivo firing of these neuron types as a virtual rodent explores an open field (Figure 9B ). Figure 7. CARLsim simulation parameters selection and file generation interface. A) The user chooses which subset of the available neuron types to include in the generated downloadable parameter file. Neuron types can be selected (check boxes and gray highlights) either individually or by groupings, such as by subregion and/or by importance rank. B) Representative user selection. C) Downloadable neuron-level parameters. D) Downloadable connection-level parameters. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 16 of 47 Potential applications to connectomic analyses and spiking neural networks simulations While preliminary, these illustrative examples highlight the potential of Hippocampome.org (https://hippocampome.org/) -enabled data-driven spiking neural network simulations to investigate computational theories of cognitive functions in hippocampal circuits at the level of biologically detailed mechanisms (Sutton and Ascoli, 2021 ). Discussion Hippocampome.org (https://hippocampome.org/) , through its continuous updates and conspicuous usage, has established itself prominently amongst other readily accessible, evidence- based, expert-curated bioscience public resources of note, such as FlyBase for Drosophila molecular biology (The FlyBase Consortium, 1994; dos Santos et al., 2015), WormBase for nematode genomics (Stein et al., 2001 ), the Blue Brain Project for somatosensory cortex (Markram, 2006 ), SynGO for synaptic functions (Koopmans et al., 2019 ), and RegenBase for spinal cord injury biology (Callahan et al., 2016 ). The growth of Hippocampome.org (https://hippocampome.org/) since the initial release of v1.0 (Wheeler et al., 2015 ) has been prodigious. To date, the site has been visited over 136,000 times with over 33,000 unique visits, and the original publication has been cited more than hundred times. Each successive release of Hippocampome.org (https://hippocampome.org/) has added new dimensions of knowledge and/or functionality and has been building toward assembling all the 17 of 47 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Figure 8. Hierarchy of neuronal connectivity in Hippocampome.org (https://hippocampome.org/) . A) Subregional connectivity, where the number of connections between subregions is shown, and the node size is proportional to the number of neuron types in each subregion. B) The reciprocal connectivity between DG and CA3 neuron types consists of 147 connections. The node size is proportional to the census size for each neuron type. C) The full connectivity involving DG Granule and CA3 Basket neuron types consists of 98 connections. The node size is proportional to the census size for each neuron type, and the thicknesses of the connecting arrows are proportional to the synaptic probability. The dashed lines are connections for which the synaptic probability has been approximated based on the means of known values. D) The electrophysiological connection between a DG Granule cell and a CA3 Basket cell. The in vivo firing rate is shown for the presynaptic neuron. The transfer function between the two neuron types is proportional to the synaptic conductance times the single-exponential decay time constant (g ⋅ τd; rat, male, P56, 32°C, current clamp). The frequency-current (F-I) curve of the single-compartment Izhikevich model of a CA3 Basket cell was obtained with 10 pA current steps. Inset: Izhikevich model firing pattern of a CA3 Basket cell simulated with 430 pA of current applied for 500 ms (vertical and horizontal scale bars, respectively). Figure 8. Hierarchy of neuronal connectivity in Hippocampome.org (https://hippocampome.org/) . A) Subregional connectivity, where the number of connections between subregions is shown, and the node size is proportional to the number of neuron types in each subregion. B) The reciprocal connectivity between DG and CA3 neuron types consists of 147 connections. The node size is proportional to the census size for each neuron type. C) The full connectivity involving DG Granule and CA3 Basket neuron types consists of 98 connections. The node size is proportional to the census size for each neuron type, and the thicknesses of the connecting arrows are proportional to the synaptic probability. The dashed lines are connections for which the synaptic probability has been approximated based on the means of known values. D) The electrophysiological connection between a DG Granule cell and a CA3 Basket cell. The in vivo firing rate is shown for the presynaptic neuron. The transfer function between the two neuron types is proportional to the synaptic conductance times the single-exponential decay time constant (g ⋅ τd; rat, male, P56, 32°C, current clamp). The frequency-current (F-I) curve of the single-compartment Izhikevich model of a CA3 Basket cell was obtained with 10 pA current steps. Inset: Izhikevich model firing pattern of a CA3 Basket cell simulated with 430 pA of current applied for 500 ms (vertical and horizontal scale bars, respectively). Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 18 of 47 18 of 47 etwork simulations. A) Full-scale CA3 model. (Aa) Neuron type connectivity schematic. (Ab) Theta (4-12 Hz; top), Gamma (25-100 Hz; arp-Wave Ripple (150-200 Hz; bottom) filtered local field potentials from 175 ms of the simulation. (Ac) Raster plot of 500 Pyramidal cells rons of each type (top), and representative voltage traces for each neuron type (bottom) during the same 175 ms of the simulation in epresentation through grid cell firing. (Ba) Neuron type connectivity schematic. Bb) Simulated animal trajectory (black) with red dots ing of a neuron in those locations. Bc) Raster plot of 50 randomly selected neurons from each type (top), and representative voltage neuron type. Abbreviations are defined in Materials and Methods. Figure 9. Spiking neural network simulations. A) Full-scale CA3 model. (Aa) Neuron type connectivity schematic. (Ab) Theta (4-12 Hz; top), Gamma (25-100 Hz; middle), and Sharp-Wave Ripple (150-200 Hz; bottom) filtered local field potentials from 175 ms of the simulation. (Ac) Raster plot of 500 Pyramidal cells and 50 interneurons of each type (top), and representative voltage traces for each neuron type (bottom) during the same 175 ms of the simulation in (Ab). B) Spatial representation through grid cell firing. (Ba) Neuron type connectivity schematic. Bb) Simulated animal trajectory (black) with red dots indicating the firing of a neuron in those locations. Bc) Raster plot of 50 randomly selected neurons from each type (top), and representative voltage traces for each neuron type. Abbreviations are defined in Materials and Methods. Spiking neural network simulations. A) Full-scale CA3 model. (Aa) Neuron type connectivity schematic. (Ab) Theta (4-12 Hz; top), Gamma (25-100 Hz; middle), and Sharp-Wave Ripple (150-200 Hz; bottom) filtered local field potentials from 175 ms of the simulation. (Ac) Raster plot of 500 Pyramidal cells and 50 interneurons of each type (top), and representative voltage traces for each neuron type (bottom) during the same 175 ms of the simulation in (Ab). B) Spatial representation through grid cell firing. (Ba) Neuron type connectivity schematic. Bb) Simulated animal trajectory (black) with red dots indicating the firing of a neuron in those locations. Bc) Raster plot of 50 randomly selected neurons from each type (top), and representative voltage traces for each neuron type. Abbreviations are defined in Materials and Methods. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 19 of 47 19 of 47 components necessary to produce real-scale computational models of the rodent hippocampal formation. The culmination of all this work is the release of v2.0, which introduces a framework for launching computer simulations directly from the accumulated knowledge. However, achieving simulations does not mark the end point for this project, because Hippocampome.org (https://hippocampome.org/) will continue to aggregate new knowledge as it is published in the peer-reviewed literature. Gradually, the focus of this resource will shift from development to exploitation through the in silico emulation of complex dynamics observed in vivo and in vitro, with the goal of shedding light on the underlying synaptic-level computational mechanisms. Figure 9. The creation of real-scale spiking neural network models of the hippocampal formation and its subregions can foster biologically realistic, data-driven, mesoscopic simulations of cognitive function and dysfunction (Sutton and Ascoli, 2021 ). For instance, simulations with Hippocampome.org (https://hippocampome.org/) ’s real-scale model of the dentate gyrus can build on previous network models of epileptogenesis (Dyhrfjeld-Johnsen et al., 2007 ) by providing further clarity to the roles of all documented neuron types and their corresponding potential connections in seizure initiation and propagation. A real-scale model of CA1 can aim to further the insights into the spatiotemporal dynamics of the circuit during theta oscillations (Bezaire et al., 2016 ; Navas-Olive et al., 2020 ; Romani et al., 2023 ). Furthermore, network models involving multiple subregions can open new vistas on unexplored territories, such as the use of real-scale models of the entorhinal cortex and CA2 to simulate the neuron- and connection-type specific mechanisms of social memory (Lopez-Rojas et al., 2022 ). Moreover, open source sharing of the real-scale models replicating those functions (Gleeson et al., 2017 ) will facilitate cross-talk within the systems neuroscience community to better understand the role of distinct neuron types in hippocampal function. A notable aspect of Hippocampome.org (https://hippocampome.org/) is that all freely downloadable model parameters are directly linked to the specific peer-reviewed empirical evidence from which they were derived. Thus, if users disagree with a specific interpretation, or are not fully convinced by an individual experimental measurement, they maintain control in selecting the information sources. Conversely, researchers can choose to reuse the collated experimental data to constrain different computational models they may prefer, such as adopting the Hodgkin-Huxley formalism instead of Izhikevich dynamics. At the same time, Hippocampome .org (https://hippocampome.org/) is not only a collection of model parameters and corresponding empirical evidence, but it also provides an opportunity to unearth knowledge gaps, as facilitated by an intuitive search functionality (hippocampome.org/find-neuron). Missing data can serve to guide the design of targeted “low hanging fruit” experiments or to generate new hypotheses. Another important element of Hippocampome.org (https://hippocampome.org/) is the careful annotation of the experimental metadata for each piece of evidence, including the species (rat or mouse), sex (male or female), age (young or adult) as well as any and all reported details that could affect the recorded neuronal property. Materials and Methods Hippocampome.org (https://hippocampome .org/) v2.0 vs. the legacy status of v1.12 With the release of v2.0 of Hippocampome.org (https://hippocampome.org/) upon publication of this article, v1.12 of the website will no longer be updated and will transition to legacy status (hippocampome.org/legacy_v1). In this way, users may avail themselves of the full benefits of the new content and functionality of v2.0, while maintaining access to reference content as published through v1.12. In the near term, neuron types new to v2.0 are tagged with an asterisk on the web site to differentiate them from v1.1x types. Hippocampome.org (https://hippocampome .org/) v2.0 vs. the legacy status of v1.12 With the release of v2.0 of Hippocampome.org (https://hippocampome.org/) upon publication of this article, v1.12 of the website will no longer be updated and will transition to legacy status (hippocampome.org/legacy_v1). In this way, users may avail themselves of the full benefits of the new content and functionality of v2.0, while maintaining access to reference content as published through v1.12. In the near term, neuron types new to v2.0 are tagged with an asterisk on the web site to differentiate them from v1.1x types. Figure 9. Examples of these confounding factors abound especially for in vitro electrophysiological data, such as the exact chemical composition of the solution in the electrode and in the bath, slice thickness and orientation, clamping configuration, recording temperature, and animal weight. Because these covariates, when reported by the original investigators, are also stored in the database, it is possible to account for them in subsequent analyses and simulations. Hippocampome.org (https://hippocampome.org/) therefore constitutes a considerably rich one-stop resource to compare and “translate” key parameters, such as the amplitude and duration of a synaptic signal between two specifically identified neuron types, for instance, from 14 day old male rat at 22 °C in voltage clamp to a 56 day old female mouse at 32 °C in current clamp. When fed into spiking neural network simulations, these differential parameter values can foster intuition while attempting to reconcile neuroscience theories and observations. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 20 of 47 20 of 47 Hippocampome.org (https://hippocampome.org/) is yet poised for the onset of an information deluge from current and future big science projects, which will need to be integrated into a complete cohesive picture (de La Prida and Ascoli, 2021 ). Although morphological identification will continue to play a fundamental role in defining neuron types and circuit connectivity, the manner in which knowledge is cross-referenced in this resource will allow its effective linkage to rapidly accumulating molecular and imaging data. The ongoing spatial transcriptomics revolution is already transforming the frontiers of cellular neuroscience, often using the hippocampus as its favorite sandbox (Lein et al., 2017 ; Yao et al., 2021 ; Zeisel et al., 2015 ). Single-cell transcriptomics via scRNAseq can bolster the current morphological information by offering distinct transcription factor codes for existing neuron types and assist in defining new ones (Cembrowski and Spruston, 2019 ; Winnubst et al., 2020 ; Yuste et al., 2020 ). From the functional side, optical imaging via genetically encoded voltage indicators (Knöpfel and Song, 2019 ) will provide in vivo voltage traces for defined neuron types that can greatly enhance the repertoire of firing pattern phenotypes to utilize in simulations (Adam et al., 2019 ). Data-driven computational models can provide a useful conceptual bridge between molecular sequencing and activity imaging by investigating the effects of specific subcellular distributions of voltage- and ligand-gated conductances on neuronal excitability (Migliore et al., 2018 ). Linking neuron types to NeuroMorpho.Org morphological reconstructions Hippocampome.org (https://hippocampome.org/) neuron types are regularly linked to appropriately identified digital reconstructions of neuronal morphology from NeuroMorpho.Org (Ascoli et al., 2007 ). Identification of suitable reconstructions with individual neuron types depends on the correspondence of dendritic and axonal locations across hippocampal subregions and layers, as they appear in the reference publication. Alternatively, direct cell typing by the authors in the reference publication text is accepted as evidence for canonical (principal cell) types, such as CA1 pyramidal cells or DG granule cells. Reconstructions are not linked to a neuron type if the experimental conditions are inconsistent with the inclusion criteria of Hippocampome .org (https://hippocampome.org/) , as in the case of cell cultures or embryonic development. Lack of either axonal or dendritic tracing also disqualifies reconstructions of non-canonical neurons from being linked. Figure 9. With the converging maturation of these young techniques and the advent of others yet on the horizon, Hippocampome .org (https://hippocampome.org/) will be able to integrate multidimensional knowledge on the solid foundation of neuronal classification. Connections from DG Granule cells to CA3 To compute estimates of connection probabilities and numbers of contacts per connected pair for the rat mossy fiber-CA3 circuit, we used previously calculated average convex hull volume (Tecuatl et al., 2021b ) and several measurements from a seminal anatomical study (Acsády et al., Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 21 of 47 21 of 47 1998 ): DG Granule cell axonal length within CA3 (3,236 µm), inter-bouton distances for mossy boutons on Pyramidal cell targets in CA3c (162 µm) and in the rest of CA3 (284 µm), and inter- bouton distances for en-passant and filipodia boutons onto CA3 interneurons (67.4 µm, considering that 48 interneurons can be contacted by a single GC). Given that the mossy fibers innervate mainly CA3 SL, and due to the lack of information regarding the exact proportion of axons innervating CA3 SP, these calculations assume that GCs only innervate SL. The probabilities of connection and numbers of contacts per connected pair (Table 2 ) are estimated as previously described (Tecuatl et al., 2021a ) utilizing the CA3 dendritic lengths reported in Hippocampome .org (https://hippocampome.org/) . hippocampome.org/) spiking neural simulations Hippocampome.org (https://hippocampome.org/) utilizes CARLsim (Nageswaran et al., 2009 ) as its default simulation environment (socsci.uci.edu/∼jkrichma/CARLsim/). CARLsim is a graphics processing unit (GPU)-accelerated library of functions for simulating spiking neural networks based on Izhikevich neuron models (Izhikevich, 2003 ). The current version is CARLsim 6 (Niedermeier et al., 2022 ), and the most up-to-date Hippocampome.org (https://hippocampome.org /) -optimized code base, including features not yet released in the main CARLsim version, can be found at hippocampome.org/CARLsim (Kopsick et al., 2022 ). Connections from CA3 and CA3c Pyramidal cells to CA1 To compute estimates of connection probabilities and numbers of contacts per connected pair for the rat Schaffer collaterals-CA1 circuit, we utilized previously reported values for the distinct axonal innervation patterns (Ropireddy et al., 2011 ; Sik et al., 1993 ; Wittner et al., 2007 ) in CA1 SR and SO from CA3 Pyramidal cells (27.5% of total axonal length: 64% to SR, 15% to SP, 21% to SO) and CA3c Pyramidal cells (64.1% of total axonal length: 94% to SR, 3% to SP, 3% to SO). In addition, we used the average inter-bouton distance reported for the Schaffer collaterals (Li et al., 1994 ) in SR (4.47 µm) and SO (5.8 µm). Total axonal length was measured with L-Measure (Scorcioni et al., 2008 ) from three NeuroMorpho.Org reconstructions for CA3c (NMO_00187, NMO_00191) and CA3b (NMO_00931). We extracted parcel-specific convex hull volumes from Janelia MouseLight (Winnubst et al., 2019 ) Pyramidal cell reconstructions (AA0304, AA0307, AA0420, AA0960, AA0997, AA0999, AA1548) mapped to the 2022 version of the Allen Institute Common Coordinate Framework (CCF). The probabilities of connection and number of contacts per connected pair were estimated as previously described (Tecuatl et al., 2021a ) using CA1 dendritic lengths from Hippocampome.org (https://hippocampome.org/) . We used separate values for inter-bouton distances in CA1 SR for CA3c Pyramidal cells (5.5 µm: Wittner et al., 2007 ) and CA3 Pyramidal cells (3.7 µm: Shepherd et al., 2002; 4.4 µm: Li et al., 1994 ; 4.29 µm: Sik et al., 1993 ; averaged as 4.1 µm). Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Web portal, database, and source code Hippocampome.org (https://hippocampome.org/) runs on current versions of Chrome, Safari, and Edge web browsers, and it is deployed on a CentOS server running Apache. The website runs off of PHP from a MySQL database. The code for Hippocampome.org (https://hippocampome.org/) is available open source at github.com/Hippocampome-Org. This includes all code for displaying the pages of the website, all scripts for importing spreadsheets into the database, code for using evolutionary algorithms to optimize Izhikevich model parameters, code for the graph theory analysis of the potential connectome, code for the implementation of the firing pattern classification algorithm, and code for analyzing network simulations in CARLsim. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 22 of 47 22 of 47 Glossary of abbreviations 5HT-3 serotonin receptor 3 AA EC LII Axo-axonic cell AAC Axo-axonic cell ABA Allen Brain Atlas APampl action potential amplitude APwidth action potential width ASP. adapting spiking Astn2 astrotactin 2 BC Basket cell BC MP EC LII Basket-Multipolar cell BiC Bistratified cell BICCN BRAIN Initiative Cell Census Net C Capacitance 5HT-3 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 ABA APwidth ASP. Capacitance Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 23 of 47 CB CB calbindin CB1 cannabinoid receptor type 1 CCK+ cholecystokinin-positive CR calretinin CSV comma-separated value Dcn decorin DG dentate gyrus E-E excitatory to excitatory E-I excitatory to inhibitory EC entorhinal cortex ENK enkephalin fAHP fast after-hyperpolarizing pote g conductance constant GABAa α1 GABAa α1 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 24 of 47 GABA-a alpha 1 subunit // / / GC granule cell Gpc3 glypican 3 GPUs graphical processing units GRALDEN GRAnule-Like DENdrites Garci grid cell Grp gastrin releasing peptide GUI graphical user interface H hilus HIPROM Hilar Interneuron with PRojections to the Outer Molecular layer Htr2c 5-hydroxytryptamine receptor 2c I-E inhibitory to excitatory I-I inhibitory to inhibitory IS interneuron specific LI-II layers 1-2 h //d i /10 7554/ Lif 90597 1 GC granule cell Gpc3 glypican 3 GPUs graphical processing units GRALDEN GRAnule-Like DENdrites Garci grid cell Grp gastrin releasing peptide GUI graphical user interface H hilus HIPROM Hilar Interneuron with PRojections to the Out Htr2c 5-hydroxytryptamine receptor 2c I-E inhibitory to excitatory I-I inhibitory to inhibitory IS interneuron specific LI-II layers 1-2 GC HIPROM Htr2c 5-hydroxytryptamine receptor 2c I-E inhibitory to excitatory I-I inhibitory to inhibitory Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 25 of 47 layer 2 LIII layer 3 Max FR maximum firing rate MEC medial entorhinal cortex MFA Mossy Fiber-Associated mGluR1a metabotropic glutamate receptor 1 alpha MOCAP cell with MOlecular Commissural-Associational Pathway-related axons and dendrites MOLAX MOlecular Layer Axons MP PC EC LI-II Multipolar Pyramidal cell Mus2R muscarinic type 2 receptor NASP non-adapting spiking Ndst4 N-deacetylase and N-sulfotransferase 4 nNOS neuronal nitric oxide synthase layer 2 Max FR maximum firing rate Max FR maximum firing rate MEC medial entorhinal cortex MFA Mossy Fiber-Associated mGluR1a mGluR1a metabotropic glutamate receptor 1 alpha Ndst4 nNOS neuronal nitric oxide synthase Nov nephroblastoma overexpressed Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 26 of 47 neuropeptide Y Nr3c2 nuclear receptor subfamily 3 group C member 2 Nr4a1 nuclear receptor subfamily 4 group A member 1 ORAX ORiens AXons ORDEN ORiens DENdrites PC pyramidal cell / CA1 Pyramidal cell (Fig. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 ABA 9B ) PL polymorphic layer PoE pieces of evidence PoK pieces of knowledge Prss12 serine protease 12 Prss23 serine protease 23 PV+ parvalbumin-positive QuadD-LM quadrilaminar dendrites – lacunosum-moleculare Rin Nr3c2 nuclear receptor subfamily 3 group C member 2 Nr4a1 nuclear receptor subfamily 4 group A member 1 ORAX ORiens AXons ORDEN ORiens DENdrites PC pyramidal cell / CA1 Pyramidal cell (Fig. 9B ) PL polymorphic layer PoE pieces of evidence PoK pieces of knowledge Prss12 serine protease 12 Prss23 serine protease 23 PV+ parvalbumin-positive QuadD-LM quadrilaminar dendrites – lacunosum-molecular Rin input resistance Nr3c2 nuclear receptor subfamily 3 group C member 2 Nr3c2 Nr4a1 nuclear receptor subfamily 4 group A member 1 Prss23 serine protease 23 PV+ parvalbumin-positive QuadD-LM quadrilaminar dendrites – lacunosum-moleculare Rin input resistance Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 27 of 47 reelin slow after-hyperpolarizing potential slow after-hyperpolarizing potenti SC MEC LII Stellate cell SD standard deviation SG stratum granulosum SL stratum lucidum SLM stratum lacunosum-moleculare SMi inner stratum moleculare SMo outer stratum moleculare SO stratum oriens SOM somatostatin SP stratum pyramidale SR stratum radiatum Sub subiculum subiculum Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 28 of 47 sharp-wave ripple SynGO synaptic gene ontologies TPM TSTUT.PSTUT transient stuttering followed by persistent stuttering TSTUT.SLN silence preceded by transient stuttering holding potential Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 29 of 47 vasoactive intestinal polypeptide vasoactive intestinal polypeptide Vmin post-spike reset potential Vmin post-spike reset potential Vmin post-spike reset potential Vpeak spike cutoff potential Vpeak Vr / Vrest resting membrane potential Vr / Vrest resting membrane potential Vt / Vthresh firing threshold potential Vt / Vthresh Vt / Vthresh firing threshold potential WA BC Wide-arbor Basket cell WA BC WA BC Wide-arbor Basket cell Wfs1 Wfs1 wolframin ER transmembrane glycoprotein Acknowledgements We thank David J. Hamilton, Charise M. White, Christopher L. Rees, Maurizio Bergamino, Keivan Moradi, Siva Venkadesh, Alberto Sanchez-Aguilera, Teresa Jurado-Parras, Manuel Valero, Miriam S. Nokia, Elena Cid, Ivan Fernandez-Lamo, Daniel García-Rincón, Liset M. de la Prida, Sarojini M. Attili, Iqbal Addou, and the many student interns (hippocampome.org/thx) for invaluable help advancing from v1.0 to v2.0. Competing Interests The authors have no competing interests. Contributions DWW contributed to the conceptualization, methodology, software, investigation, data curation, writing – original draft, visualization, supervision, and project administration. JDK contributed to the methodology, software, formal analysis, investigation, data curation, writing – review and editing, and visualization. NS contributed to the methodology, software, investigation, data curation, writing – review and editing, and visualization. CT contributed to the methodology, validation, formal analysis, investigation, data curation, writing – review and editing, visualization, and supervision. AOK contributed to the formal analysis, writing – review and Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 30 of 47 30 of 47 editing, and visualization. KN contributed to the software and writing – review and editing. GAA contributed to the conceptualization, methodology, resources, investigation, data curation, writing – review and editing, visualization, supervision, project administration, and funding acquisition. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Funding information This work was supported in part by grants R01NS39600, RF1MH128693, and U01MH114829 from the National Institutes of Health (NIH), and DE-SC0022998 from the Department of Energy (DOE). The funding sources were not involved in study design, data collection and interpretation, or the decision to submit the work for publication. Diek W. 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Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Yeung AWK, Goto TK, Leung WK (2017) The Changing Landscape of Neuroscience Research, 2006-2015: A Bibliometric Study Frontiers in Neuroscience 11 https://doi.org/10.3389/fnins .2017.00120 Scorcioni R, Polavaram S, Ascoli GA (2008) L-Measure: a web-accessible tool for the analysis, comparison and search of digital reconstructions of neuronal morphologies Nature Protocols 3:866–876 https://doi.org/10.1038/nprot.2008.51 (2018) volving Simple Models of Diverse Intrinsic Dynamics in Hippocampal Neuron Types ontiers in Neuroinformatics 12 https://doi.org/10.3389/fninf.2018.00008 Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 40 of 47 Walker AS, Raliski BK, Nguyen DV, Zhang P, Sanders K, Karbasi K, Miller EW (2021) Imaging Voltage in Complete Neuronal Networks Within Patterned Microislands Reveals Preferential Wiring of Excitatory Hippocampal Neurons Frontiers in Neuroscience 15 https:// doi.org/10.3389/fnins.2021.643868 Wittner L, Henze DA, Záborszky L, Buzsáki G (2007) Three-dimensional reconstruction of the axon arbor of a CA3 pyramidal cell recorded and filled in vivo Brain Structure & Function 212:75–83 https://doi.org/10.1007/s00429‑007‑0148‑y https://doi.org/10.7554/eLife.90597.1 41 of 47 Yuste R, Hawrylycz M, Aalling N, Aguilar-Valles A, Arendt D, Armañanzas R, Ascoli GA, Bielza C, Bokharaie V, Bergmann TB, Bystron I, Capogna M, Chang Y, Clemens A, de Kock CPJ, DeFelipe J, Dos Santos SE, Dunville K, Feldmeyer D, Fiáth R, Fishell GJ, Foggetti A, Gao X, Ghaderi P, Goriounova NA, Güntürkün O, Hagihara K, Hall VJ, Helmstaedter M, Herculano-Houzel S, Hilscher MM, Hirase H, Hjerling-Leffler J, Hodge R, Huang J, Huda R, Khodosevich K, Kiehn O, Koch H, Kuebler ES, Kühnemund M, Larrañaga P, Lelieveldt B, Louth EL, Lui JH, Mansvelder HD, Marin O, Martinez-Trujillo J, Chameh HM, Mohapatra AN, Munguba H, Nedergaard M, Němec P, Ofer N, Pfisterer UG, Pontes S, Redmond W, Rossier J, Sanes JR, Scheuermann RH, Serrano-Saiz E, Staiger JF, Somogyi P, Tamás G, Tolias AS, Tosches MA, García MT, Wozny C, Wuttke TV, Liu Y, Yuan J, Zeng H, Lein E. (2020) A community-based transcriptomics classification and nomenclature of neocortical cell types Nature Neuroscience 23:1456–1468 https://doi.org/10 .1038/s41593‑020‑0685‑8 Zagrean A-M, Georgescu I-A, Iesanu MI, Ionescu R-B, Haret RM, Panaitescu AM, Zagrean L (2022) Oxytocin and vasopressin in the hippocampus Vitamins and Hormones 118:83– 127 https://doi.org/10.1016/bs.vh.2021.11.002 Zeisel A, Mvoz-Manchado AB, Codeluppi S, Lönnerberg P, Manno GL, Juréus A, Marques S, Munguba H, He L, Betsholtz C, Rolny C, Castelo-Branco G, Hjerling-Leffler J, Linnarsson S (2015) Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq Science 347:1138–1142 https://doi.org/10.1126/science.aaa1934 Zeng H, Sanes JR (2017) Neuronal cell-type classification: challenges, opportunities and the path forward Nature Reviews Neuroscience 18:530–546 https://doi.org/10.1038/nrn.2017 Jeffrey D. Kopsick Center for Neural Informatics, Structures, & Plasticity; Krasnow Institute for Advanced Study; George Mason University, Fairfax, VA, USA, Interdisciplinary Program in Neuroscience; College of Science; George Mason University, Fairfax, VA, USA Nate Sutton Center for Neural Informatics, Structures, & Plasticity; Krasnow Institute for Advanced Study; George Mason University, Fairfax, VA, USA, Bioengineering Department and Center for Neural Informatics, Structures, & Plasticity; College of Engineering and Computing; George Mason University, Fairfax, VA, USA Diek W. Wheeler Center for Neural Informatics, Structures, & Plasticity; Krasnow Institute for Advanced Study; George Mason University, Fairfax, VA, USA, Bioengineering Department and Center for Neural Informatics, Structures, & Plasticity; College of Engineering and Computing; George Mason University, Fairfax, VA, USA ORCID iD: 0000‑0001‑8635‑0033 Wittner L, Henze DA, Záborszky L, Buzsáki G (2007) Three-dimensional reconstruction of the axon arbor of a CA3 pyramidal cell recorded and filled in vivo Brain Structure & Function 212:75–83 https://doi.org/10.1007/s00429‑007‑0148‑y Wynne ME, Lane AR, Singleton KS, Zlatic SA, Gokhale A, Werner E, Duong D, Kwong JQ, Crocker AJ, Faundez V (2021) Heterogeneous Expression of Nuclear Encoded Mitochondrial Genes i i i h hibi d i h //d / / Wynne ME, Lane AR, Singleton KS, Zlatic SA, Gokhale A, Werner E, Duong D, Kwong JQ, Crocker AJ, Faundez V (2021) Heterogeneous Expression of Nuclear Encoded Mitochondrial Genes Distinguishes Inhibitory and Excitatory Neurons eNeuro 8 https://doi.org/10.1523/ENEURO 0232‑21 2021 Wynne ME, Lane AR, Singleton KS, Zlatic SA, Gokhale A, Werner E, Duong D, Kwong JQ, Crocker AJ, Faundez V (2021) Heterogeneous Expression of Nuclear Encoded Mitochondrial Genes Distinguishes Inhibitory and Excitatory Neurons eNeuro 8 https://doi.org/10.1523/ENEURO .0232‑21.2021 Distinguishes Inhibitory and Excitatory Neurons eNeuro 8 https://doi.org/10.1523/ENEURO .0232‑21.2021 Yao Z, Van Velthoven CTJ, Nguyen TN, Goldy J, Sedeno-Cortes AE, Baftizadeh F, Bertagnolli D, Casper T, Chiang M, Crichton K, Ding S-L, Fong O, Garren E, Glandon A, Gouwens NW, Gray J, Graybuck LT, Hawrylycz MJ, Hirschstein D, Kroll M, Lathia K, Lee C, Levi B, McMillen D, Mok S, Pham T, Ren Q, Rimorin C, Shapovalova N, Sulc J, Sunkin SM, Tieu M, Torkelson A, Tung H, Ward K, Dee N, Smith KA, Tasic B, Zeng H. (2021) A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation Cell 184:3222–3241 Yao Z, Van Velthoven CTJ, Nguyen TN, Goldy J, Sedeno-Cortes AE, Baftizadeh F, Bertagnolli D, Casper T, Chiang M, Crichton K, Ding S-L, Fong O, Garren E, Glandon A, Gouwens NW, Gray J, Graybuck LT, Hawrylycz MJ, Hirschstein D, Kroll M, Lathia K, Lee C, Levi B, McMillen D, Mok S, Pham T, Ren Q, Rimorin C, Shapovalova N, Sulc J, Sunkin SM, Tieu M, Torkelson A, Tung H, Ward K, Dee N, Smith KA, Tasic B, Zeng H. (2021) A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation Cell 184:3222–3241 Yeung AWK, Goto TK, Leung WK (2017) The Changing Landscape of Neuroscience Research, 2006-2015: A Bibliometric Study Frontiers in Neuroscience 11 https://doi.org/10.3389/fnins .2017.00120 Yeung AWK, Goto TK, Leung WK (2017) The Changing Landscape of Neuroscience Research, 2006-2015: A Bibliometric Study Frontiers in Neuroscience 11 https://doi.org/10.3389/fnins .2017.00120 Diek W. Wheeler et al., 2023 eLife. Alexander O. Komendantov Center for Neural Informatics, Structures, & Plasticity; Krasnow Institute for Advanced Study; George Mason University, Fairfax, VA, USA, Bioengineering Department and Center for Neural Informatics, Structures, & Plasticity; College of Engineering and Computing; George Mason University, Fairfax, VA, USA Editors Editors Reviewing Editor Helen Scharfman Nathan Kline Institute and New York University Langone Medical Center, United States of America Senior Editor Laura Colgin University of Texas at Austin, United States of America Kasturi Nadella Center for Neural Informatics, Structures, & Plasticity; Krasnow Institute for Advanced Study; George Mason University, Fairfax, VA, USA, Bioengineering Department and Center for Neural Informatics, Structures, & Plasticity; College of Engineering and Computing; George Mason University, Fairfax, VA, USA Carolina Tecuatl Center for Neural Informatics, Structures, & Plasticity; Krasnow Institute for Advanced Study; George Mason University, Fairfax, VA, USA Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 42 of 47 42 of 47 Giorgio A. Ascoli Center for Neural Informatics, Structures, & Plasticity; Krasnow Institute for Advanced Study; George Mason University, Fairfax, VA, USA, Interdisciplinary Program in Neuroscience; College of Science; George Mason University, Fairfax, VA, USA, Bioengineering Department and Center for Neural Informatics, Structures, & Plasticity; College of Engineering and Computing; George Mason University, Fairfax, VA, USA Reviewer #2 (Public Review): Summary and strengths: The authors have developed a helpful resource for the community regarding hippocampal cell types and their interactions from many perspectives. There have been many updates to hippocampome v1.0 to v1.12, that are nicely summarized and explained (e.g., Table 1). The content and impact are also presented (Fig. 4). Weaknesses: My main comment is that it is not completely clear and/or it is a bit buried as to what makes this v2.0 (rather than v1.13). The title would seem to encompass it ('... enabling data-driven spiking neural network simulations...), but in the introduction, the authors seem to emphasize "50 newly identified neuron types...". Is it the case that launching network simulations (using CARLsim) was not possible up to v1.12? I don't think so? I think that this research advance is to announce and summarize the various updates and to demonstrate how network simulations can be easily done? If so, this should and could be made more clear so that the reader does not necessarily have to go through all the previous versions to understand what is 'special' or different about v2.0. This could perhaps be achieved by situating their tool and its goals relative to other efforts (e.g., blue brain project) that are mentioned in the Discussion? Reviewer #1 (Public Review): Summary: ummary: he authors made significant updates to Hippacampome.org including 50 new cell types. Strengths: The authors have been thorough in basing their views on peer-reviewed literature. They ha made the data highly accessible and the user has the ability to control what is included. Weaknesses: There are many inconsistencies in the literature regarding cell types and how these are incorporated into hippocampome.org is not clear. Weaknesses: here are many inconsistencies in the literature regarding cell types and how these are corporated into hippocampome.org is not clear. Properties are often a result of modeling and not biological data, and caveats to this approach, and other assumptions are unclear. Properties are often a result of modeling and not biological data, and caveats to this approach, and other assumptions are unclear. Several interneuron subtypes in the dentate gyrus do not appear to be listed, such as neurogliaform cells. Several interneuron subtypes in the dentate gyrus do not appear to be listed, such as neurogliaform cells. The nomenclature HIPROM should be distinguished or made synonymous with HIPP. Same for MOCAP and MOPP/HICAP. The nomenclature HIPROM should be distinguished or made synonymous with HIPP. Same for MOCAP and MOPP/HICAP. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 43 of 47 43 of 47 Dorsal ventral and sex differences are not mentioned. Weaknesses: The figures are complex, containing a heavy information load with many abbreviations. You need some general knowledge of the system in order to grasp the enormous potential of what is provided. Reviewer #3 (Public Review): Summary: Summary: The authors aim to provide a multidisciplinary resource on the structural and physiological organization of the hippocampal system and make the available experimental data available for further theoretical work, providing tools to do so in a very flexible and user-friendly way. Since this is a new version of an already existing data-resource, the authors certainly reach their aim and fulfil expectations that the reader might have. The content of the database is as good as the original data, collected from the published knowledge-database, sometimes with the help of the original authors, and the overall quality depends further on how the data are curated by the team of authors and many others who helped them. That process is briefly described and more details are available in descriptions of previous versions and on the website. The data extraction, examples of how data can be used, and the part on attempts to model the hippocampus are exciting and open doors to new and exciting research opportunities. Excellent description with many outlined opportunities. Nicely illustrated and inviting to explore the online database. Reviewer #1 (Public Review): Summary: The authors made significant updates to Hippacampome.org including 50 new cell types. Strengths: The authors have been thorough in basing their views on peer-reviewed literature. They have made the data highly accessible and the user has the ability to control what is included. Weaknesses: There are many inconsistencies in the literature regarding cell types and how these are incorporated into hippocampome.org is not clear. We agree with the Reviewer that there can be inconsistencies in the literature, especially when it comes to nomenclature. This is why for Hippocampome.org v1.0 we decided to focus on the morphologies, the distributions of axons and dendrites across the layers and parcels of the hippocampal formation, rather than the names authors have applied to the neurons they are studying. We have also clarified our stance on nomenclature in our Brain Informatics manuscript that accompanied v1.1. We will revise the manuscript to make these points explicit. Properties are often a result of modeling and not biological data, and caveats to this approach, and other assumptions are unclear. The foundation for Hippocampome.org has always been the data that are published in the literature. Those include, among others, the axonal and dendritic spans in each layer and subregion, the molecular expression patterns, the total neuron count by layer and subregion, the membrane properties, firing patterns, and experimental synaptic signals and corresponding covariates. For all of those, we do not depend on how the data are modeled, although there is always some level of interpretation of the data to make them machine readable and ready for incorporation into our database. However, some of the simulation- ready parameters now also included in Hippocampome.org are indeed the result of modeling, such as the neuronal input/output functions (Izhikevich model) and the unitary synaptic values (Tsodyks-Markram model). Other simulation-ready parameters are the result of specific analysis approaches, including the connection probabilities (axonal-dendritic spatial overlaps) and the neuron type census (numerical optimization of all constraints). We plan to explicitly distinguish among these various cases in the revised manuscript. Several interneuron subtypes in the dentate gyrus do not appear to be listed, such as neurogliaform cells. The neuron types listed in Figure 2 of the current manuscript are only the new additions to the catalog of neuron types at Hippocampome.org v2.0. DG Neurogliaform cells were included in our original eLife manuscript, which described the deployment of v1.0 of the website. Author Response: Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 44 of 47 Reviewer #2 (Public Review): Summary and strengths: The authors have developed a helpful resource for the community regarding hippocampal cell types and their interactions from many perspectives. There have been many updates to hippocampome v1.0 to v1.12, that are nicely summarized and explained (e.g., Table 1). The content and impact are also presented (Fig. 4). Weaknesses: My main comment is that it is not completely clear and/or it is a bit buried as to what makes this v2.0 (rather than v1.13). The title would seem to encompass it ('... enabling data-driven spiking neural network simulations...), but in the introduction, the authors seem to emphasize "50 newly identified neuron types...". Is it the case that launching network simulations (using CARLsim) was not possible up to v1.12? I don't think so? I think that this research advance is to announce and summarize the various updates and to demonstrate how network simulations can be easily done? If so, this should and could be made more clear so that the reader does not necessarily have to go through all the previous versions to understand what is 'special' or different about v2.0. This could perhaps be achieved by situating their tool and its goals relative to other efforts (e.g., blue brain project) that are mentioned in the Discussion? We thank the Reviewer for their helpful suggestions. Hippocampome.org v1.12 included the final piece needed, the synaptic physiology parameter values, to start fully simulating the hippocampal formation. In the revised manuscript, we will endeavor to emphasize more the specialness of v2.0 over the various v1.X in the Abstract, Introduction, and Discussion, in part by more fully describing the differences between our work and that of other efforts, such as the Blue Brain Project. Reviewer #1 (Public Review): As the amount of literature concerning relating to the other axes of the hippocampus continues to grow, we will gradually incorporate information along the added dimensions into our knowledge base. In the revised manuscript we intend to note this, and also stress the fact that Hippocampome.org contains knowledge from a mixture of sexes, and that whenever the original papers report the animal sex, so does our knowledge base. The revised manuscript will also mention that, whenever possible (e.g. synaptic physiology parameters), values are reported separately for males and females Hippocampome.org contains knowledge from a mixture of sexes, and that whenever the original papers report the animal sex, so does our knowledge base. The revised manuscript will also mention that, whenever possible (e.g. synaptic physiology parameters), values are reported separately for males and females. Reviewer #1 (Public Review): We will clarify this in the revisions. The nomenclature HIPROM should be distinguished or made synonymous with HIPP. Same for MOCAP and MOPP/HICAP. The Reviewer has referred to 5 separate neuron types in Hippocampome.org. Each neuron type has a unique distribution of axonal and dendritic invasions of the 26 layers and parcels of the hippocampal formation. For example, HIPROM cells have dendrites in the inner one- third of stratum moleculare, stratum granulosum, and hilus and axons in all four layers of the dentate gyrus in addition to axonal projections into CA3 stratum radiatum, stratum lucidum, stratum pyramidale, and stratum oriens. HIPP cells in contrast have dendrites only in the hilus and axons only in the outer two-thirds of stratum moleculare with no cross- Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 45 of 47 45 of 47 subregional projections. Similar considerations distinguish MOPP, MOCAP, and HICAP cells in Hippocampome.org. In expanding the nomenclature to include the neuron types we first described at Hippocampome.org, we attempted to mimic the styling of the already established neuron types of the DG: HIPROM (Hilar Interneuron with PRojections to the Outer Molecular layer), HIPP (HIlar Perforant Path-associated), MOCAP (MOlecular Commissural- Associational Pathway-related axons and dendrites), MOPP (MOlecular layer Perforant Path- associated), and HICAP (HIlar Commissural-Associational Pathway-related). We intend to insert a paragraph in the revised version to clarify these issues. Dorsal ventral and sex differences are not mentioned. We thank the Reviewer for pointing this out. As a result of the dearth of literature describing differences between dorsal and ventral hippocampus when we first assembled Hippocampome.org v1.0, we made the decision to focus solely on the distributions of the axons and dendrites along the depth, or layers, of the hippocampal formation. As the amount of literature concerning relating to the other axes of the hippocampus continues to grow, we will gradually incorporate information along the added dimensions into our knowledge base. In the revised manuscript we intend to note this, and also stress the fact that We thank the Reviewer for pointing this out. As a result of the dearth of literature describing differences between dorsal and ventral hippocampus when we first assembled Hippocampome.org v1.0, we made the decision to focus solely on the distributions of the axons and dendrites along the depth, or layers, of the hippocampal formation. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 Reviewer #3 (Public Review): Summary: The authors aim to provide a multidisciplinary resource on the structural and physiological organization of the hippocampal system and make the available Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 46 of 47 46 of 47 experimental data available for further theoretical work, providing tools to do so in a very flexible and user-friendly way. Since this is a new version of an already existing data- resource, the authors certainly reach their aim and fulfil expectations that the reader might have. The content of the database is as good as the original data, collected from the published knowledge-database, sometimes with the help of the original authors, and the overall quality depends further on how the data are curated by the team of authors and many others who helped them. That process is briefly described and more details are available in descriptions of previous versions and on the website. The data extraction, examples of how data can be used, and the part on attempts to model the hippocampus are exciting and open doors to new and exciting research opportunities. Strengths: Excellent description with many outlined opportunities. Nicely illustrated and inviting to explore the online database. Weaknesses: The figures are complex, containing a heavy information load with many abbreviations. You need some general knowledge of the system in order to grasp the enormous potential of what is provided. We agree with the Reviewer that we generously used abbreviations throughout our figures as a means of conserving limited space. We have attempted to balance that by providing a complete glossary of all the abbreviations used throughout the manuscript. However, we will make an effort to supply definitions of the abbreviations in the figure captions and at their first use in the manuscript, or even replacing the abbreviations altogether in key places in the figures. Diek W. Wheeler et al., 2023 eLife. https://doi.org/10.7554/eLife.90597.1 47 of 47
https://openalex.org/W2135342954	https://publikationen.bibliothek.kit.edu/1000049248/7297503	English	null	Thermal conductivity of isotopically controlled silicon nanostructures	New journal of physics	2,014	cc-by	10,625	To cite this article: H Bracht et al 2014 New J. Phys. 16 015021 View the article online for updates and enhancements. 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Phys. 16 015021 PAPER • OPEN ACCESS Related content Nanoscale thermal transport in self- organized epitaxial Ge nanostructures on Si(001) T Frigge, B Hafke, V Tinnemann et al. - Thermal conductivity of silicene nanosheets and the effect of isotopic doping Bo Liu, C D Reddy, Jinwu Jiang et al. - One dimensional Si/Ge nanowires and their heterostructures for multifunctional applications—a review Samit K Ray, Ajit K Katiyar and Arup K Raychaudhuri - 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. New Journal of Physics 16 (2014) 015021 1367-2630/14/015021+18$33.00 © 2014 IOP Publishing Ltd and Deutsche Physikalische Gesellscha H Bracht1,6, S Eon1, R Frieling1, A Plech2, D Issenmann2, D Wolf3, J Lundsgaard Hansen4, A Nylandsted Larsen4, J W Ager III5 and E E Haller5 H Bracht1,6, S Eon1, R Frieling1, A Plech2, D Issenmann2, D Wolf3, J Lundsgaard Hansen4, A Nylandsted Larsen4, J W Ager III5 and E E Haller5 and E E Haller5 1 Institute of Materials Physics, University of Muenster, Wilhelm-Klemm-Straße 10, D-48149 Muenster, Germany 2 Institute for Photon Science and Synchrotron Radiation/ANKA, Karlsruher Institute of Technology, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany 3 University Duisburg-Essen, Faculty of Physics, Lotharstrasse 1, D-47048 Duisburg, Germany 4 Department of Physics and Astronomy, Ny Munkegade 120, University of Aarhus, DK-8000 Aarhus, Denmark 5 L B k l N ti l L b t 1 C l t R d B k l CA 94720 USA Muenster, Germany 2 Institute for Photon Science and Synchrotron Radiation/ANKA, Karlsruher Institute of Technology, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany 3 University Duisburg-Essen, Faculty of Physics, Lotharstrasse 1, D-47048 Duisburg, Germany 4 Department of Physics and Astronomy, Ny Munkegade 120, University of Aarhus, DK-8000 Aarhus, Denmark 5 Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA E-mail: bracht@uni-muenster.de 5 Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA E-mail: bracht@uni-muenster.de Received 14 August 2013, revised 13 December 2013 Accepted for publication 17 December 2013 Published 17 January 2014 Received 14 August 2013, revised 13 December 2013 Accepted for publication 17 December 2013 Published 17 January 2014 New Journal of Physics 16 (2014) 015021 doi:10.1088/1367-2630/16/1/015021 Received 14 August 2013, revised 13 December 2013 Accepted for publication 17 December 2013 Published 17 January 2014 New Journal of Physics 16 (2014) 015021 doi:10.1088/1367-2630/16/1/015021 New Journal of Physics 16 (2014) 015021 doi:10.1088/1367-2630/16/1/015021 6 Author to whom any correspondence should be addressed. Recent citations Atom probe microscopy of zinc isotopic enrichment in ZnO nanorods C. N. Ironside et al - This content was downloaded from IP address 129.13.72.197 on 21/03/2018 at 10:09 This content was downloaded from IP address 129.13.72.197 on 21/03/2018 at 10:09 1. Introduction The efﬁcient conversion of energy in different energy forms is the grand challenge of the 21st century. Up to now our energy needs are still mainly covered by fossil resources, i.e. solar energy that has been stored during hundreds of millions of years, and by nuclear plants that use the energy stored in heavy uranium isotopes during their formation in e.g. supernova explosions. However, these resources are not only limited but also bear some serious risks as more and more carbon dioxide ends up in the atmosphere and nuclear waste, with long lifetimes, is produced. Sooner or later our energy requirements must be covered by energy plants operating in a sustainable manner. Some progress has been achieved over the past decades in solar energy, wind energy, and waste-to-energy plants that all describe an energy conversion system, that converts sunlight, wind, and the chemical energy of waste e.g. electric energy. Conversion to electricity is highly desirable as the energy can be easily distributed over short and long distances to the consumer, who again converts the electrical energy to e.g. mechanical or chemical energy. The conversion efﬁciency of fuel gas with the advantage of high energy density (chemical energy) to mechanical energy (potential and kinetic energy) is limited due to thermodynamic principles. These energy conversion systems produce waste heat that limits the efﬁciency of the process. Considering a general efﬁciency of 50–70% of today’s mechanical heat engines, 50– 30% of energy is lost as waste heat. Often waste heat from industry is used as long-distance heating for public and domestic use but waste heat incurred on the smaller scale is, in general, not used. This for example concerns the heat produced by car engines, lamps and even by the human body. Scalable systems would allow to regain energy in such places. Possibly small autonomous devices could proﬁt, where a supply of external energy is not possible in a sustainable manner. These could, for instance, be remote sensors or miniaturized devices with a need of process-compatible technologies, such as semiconductor thin-ﬁlm growth [1, 2]. Systems that directly convert heat to electricity are thermoelectric generators. As a solid state device these generators have no moving parts and thus are quite robust and long-lasting. However, the efﬁciency of such energy conversion systems is very low compared with steam turbines. Moreover, commercial thermoelectric devices are made of bismuth and tellurium. Abstract Nanostructured semiconductors open the opportunity to independently tailor electric and thermal conductivity by manipulation of the phonon transport. Nanostructuring of materials is a highly promising strategy for engineering thermoelectric devices with improved efﬁciency. The concept of reducing the thermal conductivity without degrading the electrical conductivity is most ideally realized by controlled isotope doping. This work reports on experimental and theoretical investigations on the thermal conductivity of isotopically modulated silicon nanostructures. State-of-the-art pump-and-probe experiments are conducted to determine the thermal conductivity of the different nanostructures of isotopically enriched silicon layers epitaxially grown on natural silicon substrates. Concomitant molecular dynamics calculations are performed to study the impact of the silicon isotope mass, isotope interfaces, and of the isotope layer ordering and thickness on the thermal conductivity. Engineering the isotope distribution is a striking concept to reduce the thermal 6 Author to whom any correspondence should be addressed. © 2014 IOP Publishing Ltd and Deutsche Physikalische Gesellschaft New J. Phys. 16 (2014) 015021 H Bracht et al conductivity of silicon without affecting its electronic properties. This approach, using isotopically engineered silicon, might pave the way for future commercial thermoelectric devices. 1. Introduction Both the elements are considered as critical raw materials due to their limited availability. In order to promote thermoelectric devices for applications serving our daily life that implies their large- scale production, devices should be fabricated from abundant raw materials and should have an acceptable energy conversion efﬁciency [3]. Today electronic devices based on silicon (Si) are produced in mass production with high throughput, reliability and performance, thanks to the progress achieved over the past decades in the processing of Si-based nanoelectronic devices. In small autonomous devices, such as remote sensors a high degree of integration and miniaturization is required, which favors process-related materials. Unfortunately, Si, which is one of the most abundant elements of the earth’s crust, is not well suited as thermoelectric material as its thermal conductivity is with 150 W m−1K−1 too high. The addition of the heavier element germanium (Ge) with up to 50% reduces the 2 New J. Phys. 16 (2014) 015021 H Bracht et al thermal conductivity by a factor of 10 [4]. This reduction is caused by alloy disorder scattering of the phonons due to the high mass difference between Si and Ge host atoms and their random distribution. With increasing Ge content also the electrical conductivity of the SiGe alloys decreases but less strongly than the thermal conductivity. These properties of SiGe alloys result in good ﬁgures of merit for thermoelectric applications at high temperatures. However, Ge tends to leave the material at high temperature due to its higher vapor pressure compared to Si. This favors a decomposition of the alloy although it is, in principle, completely miscible. Moreover dopant accumulation degrades the performance of SiGe-based thermoelectric devices. Similar to the disorder scattering of phonons in SiGe alloys, the thermal conductivity of natural silicon is disturbed by the isotope defects that describe variants of the same chemical element with different atomic mass due to different numbers of neutrons in the nucleus. Natural Si consists of an alloy of the three stable isotopes 28Si(92.2%), 29Si(4.7%) and 30Si(3.1%), where the number in bracket describes the isotopic abundance. In high isotopically enriched material the scattering of phonons at isotope defects is reduced and accordingly 28Si possesses a slightly higher thermal conductivity than natural Si [5–7]. Since the electronic properties of Si, i.e. 1. Introduction the band structure, carrier mobility and electronic defect states only weakly depend on the isotope mass [8–10], the thermal conductivity of Si due to phonon transport can be altered speciﬁcally by isotope defects without affecting the electronic properties. On the other hand the Seebeck coefﬁcient is not expected to change signiﬁcantly with isotopic composition for temperatures above room temperature because the electronic contribution to the Seebeck coefﬁcient exceeds the phonon contribution [11, 12]. In this work, the impact of controlled isotope doping on the thermal conductivity of Si is investigated experimentally and theoretically. Various isotopically modulated Si layers are studied including a high-purity 28Si layer and alternating layers of 28Si and 29Si or 30Si. The thermal conductivity of the layers is determined by means of time-resolved x-ray scattering (TRXS). Molecular dynamics calculations are performed to examine the impact of isotope doping, layer ordering and thickness on the thermal conductivity. It is evident that the properly designed isotopically modulated Si nanostructures can exhibit thermal conductivities well below those of isotopically pure and natural Si. 2.1. Sample preparation Silicon samples with different isotopically modulated layers were epitaxially grown by means of molecular beam epitaxy (MBE) and chemical vapor deposition (CVD) on (100) oriented Si wafers of natural isotopic abundance. Twenty undoped bilayer structures of (29Si/28Si)20 (sample #1) and (30Si/28Si)20 (sample #2) were grown by MBE on p-type substrates (boron doped) with a speciﬁc resistivity of ∼0.02 and ∼4 cm, respectively. The thickness of each individual isotope layer is about 10 nm resulting in a total thickness of about 400 nm for the entire layer structure. Figures 1(a) and (b) illustrate the concentration of the Si isotopes versus depth of samples #1 and #2, respectively. The isotope proﬁles were measured with secondary ion mass spectrometry (SIMS). In order to prove the signiﬁcance of isotopically modulated Si layers on the thermal conductivity, we also prepared by MBE 400 nm thick natural undoped Si layers on p-type Si wafer substrates with speciﬁc resistivities of ∼0.02 cm (sample #3) and ∼4 cm (sample #4). These samples, #3 and #4, with natural Si epilayers serve as reference 3 New J. Phys. 16 (2014) 015021 H Bracht et al y ( ) Figure 1. Concentration versus depth proﬁles of stable silicon isotopes in samples (a) #1 and (b) #2 measured with SIMS. Proﬁles of #1 and #2 represent isotope multilayer structures with 20 bilayers of (a) 29Si/28Si and (b) 30Si/28Si, respectively. Figure 1. Concentration versus depth proﬁles of stable silicon isotopes in samples (a) #1 and (b) #2 measured with SIMS. Proﬁles of #1 and #2 represent isotope multilayer structures with 20 bilayers of (a) 29Si/28Si and (b) 30Si/28Si, respectively. samples for the isotope samples #1 and #2, respectively. Moreover, we utilized a Si sample with a ∼90 µm thick enriched 28Si layer (sample #5) doped with boron during CVD growth. The speciﬁc resistivity of the epilayer and the boron doped substrate is about 1 and 0.007 cm, respectively. Samples with lateral dimensions of 5 × 5 mm2 were cut from the wafers #1–#5, cleaned in organic solvents, etched in diluted hydroﬂuoric acid to remove the native oxide, and purged in deionized water. Immediately after cleaning, the epitaxial layers of the samples were covered ﬁrst with a 2 nm thick chromium (Cr) layer and subsequently, i.e. without exposure to air, with a 28 nm thick gold (Au) layer by sputter deposition. 2.1. Sample preparation The Cr layer between Si and Au enhances the adhesion of the Au layer and reduces the thermal resistance between the metal/Si interface [13, 14]. Since the thermal interface resistance strongly affects the heat transport from the metal layer to the Si, the isotope sample #1 (#2) and its reference #3 (#4) were processed simultaneously in pairs, i.e. cleaned and metal coated. In this way it is ensured that the thermal interface resistance between the metal layer and Si are similar for #1 and #3 (#2 and #4). Also sample #5 with the top isotopically enriched thick 28Si layer and the polished backside of another piece of sample #5 were cleaned and coated simultaneously with a Au(28 nm)/Cr(2 nm) bilayer. The metal-coated natural Si back side of sample #5 serves as a reference sample (#6) for the top 28Si epilayer of sample #5. p p y p Gold layer thickness and ﬁlm quality have been determined by x-ray reﬂectivity at the synchrotron beamline SCD at ANKA (Karlsruhe). The thermal conductivity of the epitaxial layers of samples #1–#6 is investigated by means of TRXS following pulsed laser excitation. This technique is described in the following section. 2.2. Heat transport measurements by time-resolved x-ray scattering The thermal conductivity of the isotopically enriched epitaxial layers of samples #1, #2 and #5 is deduced from the cooling kinetics of the top metal layer in air after pulsed laser heating. This cooling behavior is affected by the thermal interface resistance and the thermal conductivity of the underlying material. The reference samples #3, #4 and #6, serve to determine the thermal interface resistance from the cooling behavior of the metal layer since the thermal conductivity 4 H Bracht et al New J. Phys. 16 (2014) 015021 Figure 2. Schematic representation of the TRXS experiment conducted at the beam- line ID09B of the ESRF in Grenoble to investigate the thermal conductivity of an epitaxial isotopically modulated Si layer on a natural Si substrate. In this pump- and-probe experiment the laser pulse heats the top metal Au(28 nm)/Cr(2 nm) layer and, subsequently, the cooling of this metal layer is probed by x-ray pulses. The chronological sequence of the laser and x-ray pulses with their corresponding pulse width is indicated in the inset. 1ti and 1ti+1 indicate the time delay between laser and x-ray pulses of two consecutive charge-coupled device (CCD) exposures. The time delay is varied up to 10 µs in order to record the cooling of the metal layer. Figure 2. Schematic representation of the TRXS experiment conducted at the beam- line ID09B of the ESRF in Grenoble to investigate the thermal conductivity of an epitaxial isotopically modulated Si layer on a natural Si substrate. In this pump- and-probe experiment the laser pulse heats the top metal Au(28 nm)/Cr(2 nm) layer and, subsequently, the cooling of this metal layer is probed by x-ray pulses. The chronological sequence of the laser and x-ray pulses with their corresponding pulse width is indicated in the inset. 1ti and 1ti+1 indicate the time delay between laser and x-ray pulses of two consecutive charge-coupled device (CCD) exposures. The time delay is varied up to 10 µs in order to record the cooling of the metal layer. of natural Si is well known. This interface resistance is then assumed to describe the cooling behavior of the metal layer on top of the isotope sample that was metal deposited in the same sputter process. The cooling of the metal layer after pulse-laser heating was followed by measuring the Au lattice constant with x-ray pulses as function of time. 2.2. Heat transport measurements by time-resolved x-ray scattering The utilized femtosecond laser system provides 600 fs pulses of 400 nm at a repetition rate of 1 kHz. The high thermal conductivity of the Au layer (∼320 W m−1K−1) ensures that the heat supplied by the laser pulse spreads within about 60 ps in the metal ﬁlm. X-ray pulses of 70 ps duration and 15 keV photon energy were reduced to the 1 kHz repetition rate of the laser by means of a rotating chopper. The laser pulses were synchronized to the x-rays to better than 5 ps. The two beams overlap spatially, i.e. the spot of the laser beam is about 500 µm in diameter while the width of the x-ray spot is about 100 µm. The pump-and-probe experiments were conducted at the beam-line ID09B of the European Synchrotron Radiation Facility (ESRF) in Grenoble [15, 16]. A silicon monochromator was used to select the 15 keV photon energy from the synchrotron beam. The time delay 1t between the two beams is varied up to 10 µs in order to record the cooling of the sample. The pump- and-probe experiment is schematically illustrated in ﬁgure 2. The incidence angle for the x-rays was the nominal Bragg angle for the Au (111) reﬂection at about 10◦, while the laser struck the sample at 18◦to the surface. The inset of ﬁgure 2 shows two consecutive laser pulses with the variable time delay 1ti between laser and x-ray pulses and their corresponding pulse duration. In practice, an integration over the scattering response of about 2000 consecutive pulses at ﬁxed delay was performed to increase the signal-to-noise ratio. The (111) Bragg peak of Au is 5 New J. Phys. 16 (2014) 015021 H Bracht et al Figure 3. Time-resolved relative lattice expansion 1a/a of a Au/Cr layer (symbols) deposited on top of different Si samples (#1–#6). The lattice expansion is measured by means of x-ray diffraction after heating the metal layer with a laser pulse. The left axis displays the relative lattice expansion 1a/a and the right axis the corresponding temperature 1T (t) = T −300 K. The solid lines represent best numerical ﬁts to the experimental results (symbols). Panels (a) and (b) illustrate the expansion of the metal layer on top of the (29Si/28Si)20 (sample # 1) and 30Si/28Si)20 (sample # 2) isotope multilayers in comparison to the reference samples #3 and #4, respectively. 2.2. Heat transport measurements by time-resolved x-ray scattering (c) Relative lattice expansion of the metal layer on top of a 90 µm thick isotopically enriched 28Si layer (sample #5) in comparison to the reference sample #6. Figure 3. Time-resolved relative lattice expansion 1a/a of a Au/Cr layer (symbols) deposited on top of different Si samples (#1–#6). The lattice expansion is measured by means of x-ray diffraction after heating the metal layer with a laser pulse. The left axis displays the relative lattice expansion 1a/a and the right axis the corresponding temperature 1T (t) = T −300 K. The solid lines represent best numerical ﬁts to the experimental results (symbols). Panels (a) and (b) illustrate the expansion of the metal layer on top of the (29Si/28Si)20 (sample # 1) and 30Si/28Si)20 (sample # 2) isotope multilayers in comparison to the reference samples #3 and #4, respectively. (c) Relative lattice expansion of the metal layer on top of a 90 µm thick isotopically enriched 28Si layer (sample #5) in comparison to the reference sample #6. recorded by a two-dimensional CCD detector. By measuring the angular position of the Bragg peak, it is possible to deduce the lattice expansion of the Au layer as function of time. The energy of the laser pulse that causes heating of the Au layer and thus the lattice expansion is adjusted in the linear response region, where a linear relationship between the pulse laser energy and the Au lattice expansion exists. The procedure is described in [13]. The temperature from the measured strain is estimated from the elastic uniaxial thermal expansion of a ﬁlm ﬁxed on a cold substrate. Figures 3(a)–(c) illustrate the time-resolved relative lattice expansion of the Au layer on top of samples #1, #2 and #5 in comparison to the behavior of the Au layer on top of the corresponding natural Si reference samples #3, #4 and #6, respectively. The temperature of the metal layer is illustrated by the right y-axis. The time resolution of the experiment is mainly determined by the width of the x-ray pulse since the laser pulse is short compared to the x-ray pulse (see inset of ﬁgure 2). In order to determine the thermal conductivity of the epitaxial Si layer beneath the metal layer, the cooling of the metal layer as function of time is described by means of a heat transport model that is introduced in the following section. 2.3. Continuum theoretical simulation of heat transport The structure considered for modeling the heat transport consists of a top metal layer deposited on an epitaxially grown Si layer. The epitaxial Si layer is grown on a natural Si substrate wafer and either represents the (29Si/28Si)20 multilayer of sample #1 or the (30Si/28Si)20 multilayer of sample #2 or the epitaxial natural Si layers of samples #3 and #4. In the case of the 90 µm thick 28Si layer on a natural Si wafer (sample #5), a two-layer structure is considered because the epitaxial layer is sufﬁciently thick to distribute the heat homogeneously. Conceptually, the entire multilayer structure is considered as a homogeneous material with an effective heat conductivity. This effective value will serve to describe the potential of conductivity tailoring. 6 New J. Phys. 16 (2014) 015021 H Bracht et al The metal coated backside of sample #5 representing the reference sample #6 is also described with a two-layer system consisting of the metal layer and the natural Si wafer substrate with a thickness ∼600 µm. The heat injected by the laser pulse spreads from the metal layer over the epilayer into the wafer substrate. A constant thermal boundary conductance σmetal/Si = 1/RK, whose reciprocal value equals the Kapitza resistance RK, is considered between the metal ﬁlm and Si. The smaller size of the x-ray spot compared to the laser spot justiﬁes to assume a one- dimensional heat transport from the metal layer into the Si sample. p y p The mathematical formulation of the heat transport problem is described by coupled partial differential equations with different heat diffusion coefﬁcients for the metal ﬁlm, epitaxial layer and the Si substrate in case of a three-layer system. For the two-layer system only two differential equations with appropriate heat diffusion coefﬁcients are sufﬁcient to describe the heat transport from the metal ﬁlm into the isotopically enriched thick 28Si layer or the natural Si substrate wafer. The differential equations are coupled via the boundary conditions established at the metal/Si interface. This heterointerface acts as a barrier for the heat transport. This is described by means of a Kapitza resistance RK. Details about the differential equations and boundary conditions describing heat transport in a three-layer system as well as the relation between the heat diffusion coefﬁcient and thermal conductivity λepi in the various epitaxial layers are given in [14]. A reduction to a two-layer system is straightforward and therefore not treated here. 2.3. Continuum theoretical simulation of heat transport Schematic illustration of the procedure to determine the thermal conductivity λepi of an isotopically controlled epitaxial Si layer. Isotope and reference samples, respectively, with and without an isotopically controlled Si layer are investigated in the experiment. The experimental results are described by means of numerical simulations of the heat transport problem. Modeling the results of the reference sample provides the thermal boundary conductance σmetal/Si at the metal/Si interface. This boundary conductance is then considered for modeling the cooling of the metal layer deposited on the isotope sample. Best ﬁts to the experimental results yield the thermal conductivity λepi of the isotopically controlled epitaxial Si layer. Figure 4. Schematic illustration of the procedure to determine the thermal conductivity λepi of an isotopically controlled epitaxial Si layer. Isotope and reference samples, respectively, with and without an isotopically controlled Si layer are investigated in the experiment. The experimental results are described by means of numerical simulations of the heat transport problem. Modeling the results of the reference sample provides the thermal boundary conductance σmetal/Si at the metal/Si interface. This boundary conductance is then considered for modeling the cooling of the metal layer deposited on the isotope sample. Best ﬁts to the experimental results yield the thermal conductivity λepi of the isotopically controlled epitaxial Si layer. at the metal/Si interface remains unaffected when Si of natural abundance is replaced with isotopically enriched Si. Figure 5 illustrates numerical simulations of heat transport in a three-layer system that consists of a metal layer, an epilayer, and a substrate as indicated in the inset. The laser pulse heats the metal layer and its cooling is recorded with time resolved measurements of the Au lattice expansion. The symbols shown in the right part of ﬁgure 5 demonstrate a typical relaxation of the Au lattice expansion as function of time obtained from the TRXS measurements of sample #1. The right y-axis illustrates the corresponding temperature 1T of the metal layer deduced from the linear relation between the relative lattice expansion 1a/a and 1T . The solid line describes the best ﬁt to the relaxation curve with λepi as a ﬁt parameter. Beforehand the thermal boundary conductance σmetal/Si was deduced from the relaxation curve of the reference sample. The left part of ﬁgure 5 gives more details on the heat transport simulation. 2.3. Continuum theoretical simulation of heat transport The system of differential equations is solved numerically considering the effective heat conductivity λepi in the isotopically modulated Si layers and the thermal boundary conductance σmetal/Si between metal/Si interface as free ﬁtting parameters. / / g p Previous experimental results and numerical calculations on heat transport in the three- layer system metal/Si-isotope-layer/Si-substrate reveal the importance of the thermal boundary conductance at the metal/Si heterointerface for an accurate determination of the heat conductivity λ of the isotopically modulated Si layer [14]. The higher the thermal boundary conductance the more sensitive is the cooling of the metal layer to the thermal conductivity of the subjacent isotopically controlled layer. This is demonstrated by TRXS experiments with Au- and Au/Cr-coated Si samples with and without an isotopically modulated layer [14]. For a Au-coated sample the thermal boundary conductance is about three times lower than that for a Au/Cr-coated sample. As a consequence the metal layer cooling of the Au-coated sample is less sensitive to the thermal conductivity of the isotope layer than in the case of the Au/Cr-coated sample. This demonstrates that the thermal boundary conductance σmetal/Si should be sufﬁciently high, i.e. σmetal/Si ≈108 W m−2 K−1, to be sensitive to the thermal conductivity of a layer with a thickness of only a few hundreds of nanometers. With respect to these results we deposited Au/Cr layers on Si samples to realize an acceptably high thermal boundary conductance. Moreover, Si samples of natural isotopic abundance that should be compared were coated in the same batch under equivalent conditions. These latter samples with known thermal conductivity serve as reference samples to determine σmetal/Si established in the sputter deposition process. This boundary conductance is then considered for Si samples with isotopically controlled layers that were metal deposited during the same sputter process. In this way only the thermal conductivity λepi of the isotope layer remains as the free-ﬁtting parameter to describe the cooling of the metal layer after pulse laser heating. This strategy is schematically illustrated in ﬁgure 4. Note that the correlation between reference and isotope samples is applicable because the bonds established at the metal/Si interface only depend on the chemical nature of the elements involved. As all Si isotopes possess identical chemical properties, the bond 7 7 H Bracht et al New J. Phys. 16 (2014) 015021 y ( ) Figure 4. 2.3. Continuum theoretical simulation of heat transport Considering the three-layer system indicated by the inset, the heat injected by the laser pulse spreads out within the metal layer almost instantaneously, i.e. in about a few tens of picoseconds thanks to the high thermal conductivity of Au (λAu = 320 W m−1K−1) and the boundary conductance at the metal/Si interface (σmetal/Si ≈108 W m−2 K−1). The value of σmetal/Si was deduced from the simulation of the lattice expansion of the Au layer deposited on the reference sample. The left part of ﬁgure 5 illustrates temperature versus depth x proﬁles (see black solid lines) for various times t starting at 10 ps up to 5000 ps. The simulations clearly resolve the temperature drop at the metal/Si interface that decreases with increasing time according to the thermal boundary conductance and the heat diffusivities of the material on both sides of the interface [14]. The red solid line in the left part of ﬁgure 5 shows the average temperature of the metal layer as function of time. This proﬁle is convolved with the resolution function of the x-ray pulse and compared to the experiment. This is indicated by the red solid line in the right part of ﬁgure 5. The simulations of the experimental results provide 8 New J. Phys. 16 (2014) 015021 H Bracht et al Figure 5. Illustration of the numerical calculation of heat transport in a three-layer system that consists of a metal layer, an epitaxial Si layer, and a Si substrate. The epitaxial layer represents an isotopically controlled Si layer or a Si layer of natural abundance. The three-layer structure is indicated by the inset on the left-hand side. The heat transport from the metal layer into the subjacent Si (see black solid lines on the left-hand side) is calculated for consecutive time steps until the temperature gradient at the metal/Si interface vanishes. The calculated average temperature of the Au layer and its cooling behavior indicated by the red solid line is compared on the right-hand side with the relative Au lattice expansion (+) measured after pulse laser heating of the metal layer. The lattice expansion is directly related to the temperature of the Au layer [13]. The temperature calculated for the Si side of the metal/Si interface is given by the red dashed line. 2.3. Continuum theoretical simulation of heat transport additional information about the temperature evolution at the Si side of the metal/Si interface. This temperature versus time proﬁle is illustrated by the red dashed line both in the left and right part of ﬁgure 5. Whereas the temperature of the Au layer changes during cooling from about 450 K to room temperature, the temperature at the Si side of the metal/Si interface only varies by about 25 K. In this temperature range the thermal conductivity of Si changes at most by 10 W m−1K−1 considering the temperature dependence of the thermal conductivity in natural and isotopically enriched 28Si reported in the literature [5–7]. Hence the variation in the thermal conductivity of Si is small during the pump-and-probe experiment. Therefore we can assume a temperature-independent thermal conductivity for the simulation of the heat transport in Si. 2.3. Continuum theoretical simulation of heat transport The temperature at the Si side of the interface varies by about 25 K with time whereas the temperature of the Au layer changes by about 150 K. To a good approximation the thermal conductivity of Si is independent of temperature during the cooling process. Figure 5. Illustration of the numerical calculation of heat transport in a three-layer system that consists of a metal layer, an epitaxial Si layer, and a Si substrate. The epitaxial layer represents an isotopically controlled Si layer or a Si layer of natural abundance. The three-layer structure is indicated by the inset on the left-hand side. The heat transport from the metal layer into the subjacent Si (see black solid lines on the left-hand side) is calculated for consecutive time steps until the temperature gradient at the metal/Si interface vanishes. The calculated average temperature of the Au layer and its cooling behavior indicated by the red solid line is compared on the right-hand side with the relative Au lattice expansion (+) measured after pulse laser heating of the metal layer. The lattice expansion is directly related to the temperature of the Au layer [13]. The temperature calculated for the Si side of the metal/Si interface is given by the red dashed line. The temperature at the Si side of the interface varies by about 25 K with time whereas the temperature of the Au layer changes by about 150 K. To a good approximation the thermal conductivity of Si is independent of temperature during the cooling process. additional information about the temperature evolution at the Si side of the metal/Si interface. This temperature versus time proﬁle is illustrated by the red dashed line both in the left and right part of ﬁgure 5. Whereas the temperature of the Au layer changes during cooling from about 450 K to room temperature, the temperature at the Si side of the metal/Si interface only varies by about 25 K. In this temperature range the thermal conductivity of Si changes at most by 10 W m−1K−1 considering the temperature dependence of the thermal conductivity in natural and isotopically enriched 28Si reported in the literature [5–7]. Hence the variation in the thermal conductivity of Si is small during the pump-and-probe experiment. Therefore we can assume a temperature-independent thermal conductivity for the simulation of the heat transport in Si. 2.4. Results The relative lattice expansion of Au layers after pulse laser heating was measured by TRXS for the metal deposited isotope samples #1, #2 and #5 and for the respective reference samples #3, #4 and #6. The best ﬁts are shown by the solid lines in ﬁgure 3. Table 1 summarizes the thermal conductivity and thermal boundary conductance that were determined from the pump-and-probe experiments with the isotope and reference samples #1–#6. 9 New J. Phys. 16 (2014) 015021 H Bracht et al Table 1. Thermal conductivity λepi of isotopically controlled silicon epitaxial layers (#1, #2, #5) determined from the pump-and-probe experiments described in this work. The respective reference samples (#3, #4 and #6) serve to deﬁne the thermal boundary conductance σmetal/Si at the metal/Si interface. No. Si material λepi (W m−1K−1) σmetal/Si (W m−2K−1) #1 (29Si(10 nm)/28Si(10 nm))20/natSi wafer 60 ± 7 7.6 × 107b #2 (30Si(10 nm)/28Si(10 nm))20/natSi wafer 58 ± 5 9.1 × 107b #3 natSi(400 nm)/natSi wafer (reference for #1) 149a (7.6 ± 0.2) × 107 #4 natSi(400 nm)/natSi wafer (reference for #2) 149a (9.1 ± 0.3) × 107 #5 28Si(90 µm)/natSi wafer 196 ± 19 6.6 × 107b #6 natSi wafer (backside of #5, reference for #5) 149a (6.6 ± 0.1) × 107 a Thermal conductivity of natural Si at 293 K [7]. b Set as equal to the value of the thermal boundary conductance of the respective reference sample. Table 1. Thermal conductivity λepi of isotopically controlled silicon epitaxial layers (#1, #2, #5) determined from the pump-and-probe experiments described in this work. The respective reference samples (#3, #4 and #6) serve to deﬁne the thermal boundary conductance σmetal/Si at the metal/Si interface. y [ ] b Set as equal to the value of the thermal boundary conductance of the respective reference sample. The thermal conductivity of the 90 µm thick highly enriched 28Si layer is with 196 W m−1K−1 higher than the thermal conductivity of Si with natural isotopic abundance. Within the experimental accuracy this supports the enhancement of about 10% reported in the literature for the thermal conductivity of pure 28Si compared to natural Si [5–7]. In contrast, the thermal conductivities of the isotopically modulated (29Si/28Si)20 (sample #1) and (30Si/28Si)20 (sample #2) multilayers are clearly lower than that of natural Si (see table 1). 2.4. Results The Kapitza resistance deduced from the analysis of the reference samples are equal within 20% for the different sputter deposition processes. The values given in table 1 indicate a similar reduction of the thermal conductivity for samples #1 and #2. Overall, the experimental results clearly demonstrate an impact of isotopic modulation on the thermal conductivity of Si and the question rises to which magnitude the thermal conductivity of Si can be altered by isotopic engineering. Since the preparation of Si isotope structures with different layer thicknesses, layer sequences, and layer numbers by means of MBE and their subsequent analysis with TRXS is by far too laborious and time consuming, we performed molecular dynamics (MD) calculations to determine the impact of various kinds of isotopic modulated Si layers on thermal conductivity. 3.1. Simulation setup In order to theoretically study the effect of isotopic composition on the thermal conductivity of Si reverse non-equilibrium MD simulations were performed using the LAMMPS MD code [17]. In a MD simulation Newton’s equations of motion ⃗F = m d2⃗r/dt2 = −⃗∇8(⃗r) are solved numerically and the positions and velocities of all atoms are determined. 8(⃗r) is a suitable interatomic potential. In our simulation we use the Stillinger–Weber potential for Si [18] with a timestep of 1 fs. As MD rely on classical mechanics, the simulations have to be performed at sufﬁciently high temperatures in order to avoid quantum effects [19]. In our case the average temperature of the sample is 400 K. The simulation cell consists of 28 × 10 × 10 unit cells of Si with a cell volume of 449 nm3 (22 400 atoms) and a lattice constant of 5.431 Å. Periodic boundary conditions are enforced in 10 H Bracht et al New J. Phys. 16 (2014) 015021 Figure 6. Sketch of the simulation cell and the isotopic modulation of the sample: The left half of the simulation cell consists of 28Si and the right half of 28Si, 29Si, 30Si or 44Si. The heat source is located at one quarter of the length of the simulation cell and the heat sink at three quarters of the simulation cell. Figure 6. Sketch of the simulation cell and the isotopic modulation of the sample: The left half of the simulation cell consists of 28Si and the right half of 28Si, 29Si, 30Si or 44Si. The heat source is located at one quarter of the length of the simulation cell and the heat sink at three quarters of the simulation cell. the x-direction (cross-plane direction of the layered structure) and free boundary conditions in the y- and z-direction (in-plane direction of the layered structure). The initial velocities of the atoms are assigned by a random velocity distribution. A temperature gradient along the sample in the x-direction is established by adding an amount of non-translational kinetic energy (i.e. heat, aggregate momentum is conserved) to the atoms in the hot reservoir at every time step and subtracting the same amount of energy from the atoms in the cold reservoir. 3.1. Simulation setup Due to periodic boundary conditions in the direction of the temperature gradient half of the heat Q/2 ﬂows to the left side of the heat source and the other half of the heat ﬂows to the right side of the heat source. Figure 7. Temperature proﬁle and isotopic modulation of a 28Si(7.60 nm)/ 30Si(7.60 nm) bilayer structure. The temperature gradient is determined from the temperature difference dT between heat source and heat sink divided by their distance dx. Due to periodic boundary conditions in the direction of the temperature gradient half of the heat Q/2 ﬂows to the left side of the heat source and the other half of the heat ﬂows to the right side of the heat source. 1T (m) is the temperature difference, if all atoms have the same mass m. It scales like √m so that 1T (m) = r m 281T (m = 28). (3) (3) Equation (2) is derived by assuming that the total thermal resistance, 1 λ 1x A , is a sum of three terms. The ﬁrst one, 1 λ(m=28) 1x/2 A , is the thermal resistance of the left half of the system, where the atomic mass is 28. The second one, 1 λ(m) 1x/2 A , is the thermal resistance of the right half. The interface contribution is described by the third term, RK. Inserting equation (1) to relate λ(m) to 1T (m) leads to equation (2). Equation (2) is derived by assuming that the total thermal resistance, 1 λ 1x A , is a sum of three terms. The ﬁrst one, 1 λ(m=28) 1x/2 A , is the thermal resistance of the left half of the system, where the atomic mass is 28. The second one, 1 λ(m) 1x/2 A , is the thermal resistance of the right half. The interface contribution is described by the third term, RK. Inserting equation (1) to relate λ(m) to 1T (m) leads to equation (2). Two sets of Si samples are investigated in this work. In the ﬁrst case the left half of the sample consists of 28Si and the right half consists of 28Si,29 Si, 30Si or 44Si. In this way the effect of mass difference on the thermal conductivity can be studied. In the second case, the impact of the superlattice period on thermal conductivity is examined. 3.1. Simulation setup As periodic boundary conditions are enforced in the direction of the temperature gradient, the hot reservoir is located at one quarter of the sample length and the cold reservoir at three quarters of the sample length. This is illustrated in ﬁgure 6. The size of each reservoir is one unit cell in the x-direction and ten unit cells in the y- and z-direction, the added/subtracted amount of kinetic energy per time step dE/dt is 20 eV ps−1. The temperature is determined locally for each atomic layer perpendicular to the x-direction. It is calculated from the kinetic energy assuming equipartition, T = ⟨mv2⟩/(3kB), averaging over the atoms in the respective layer and the simulation time. An effective thermal conductivity λ of the sample is calculated by means of Fourier’s law [20] from the added amount of energy per time step dE/dt, the cross section area A of the sample, the resulting temperature difference 1T , and the distance 1x between heat source and heat sink (see ﬁgure 7) λ = − dE dt 1x 2 A 1T . (1) The factor two in equation (1) arises from the fact that half of the heat ﬂows to the left and the other half to the right, because periodic boundary conditions are used (see ﬁgure 7) [21]. The effective thermal conductivity calculated from equation (1) is affected by the temperature anomalies at the heat source and sink, where the system, perturbed in every time step, is not able to relax into local equilibrium. These anomalies extend to about 2 nm on each side of the source and the sink. They could be neglected for much larger 1x than considered here. However, the boundary (or Kapitza) resistance RK between the isotopic layers can be considered as unaffected by these anomalies already for our system size. For the interface between regions with atomic masses 28 and m (in atomic mass units) it is given by R 21T −1T (m = 28) −1T (m) A (2) dE/dt 11 H Bracht et al New J. Phys. 16 (2014) 015021 Figure 7. Temperature proﬁle and isotopic modulation of a 28Si(7.60 nm)/ 30Si(7.60 nm) bilayer structure. The temperature gradient is determined from the temperature difference dT between heat source and heat sink divided by their distance dx. 3.1. Simulation setup The sample is built up by a varying number of 28Si/30Si bilayers at ﬁxed total sample length. The thermal conductivities calculated for the 28Si/30Si superlattice are compared with the thermal conductivities of natSi, 28Si and an 28Si0.5 30Si0.5 alloy. 3.2. Results 3.2.1. Effect of mass difference. Figure 8 shows the steady state temperature proﬁles for four bilayer systems 28Si/mSi with m = 28, 29, 30 and 44. Note the different temperature scales of the four plots. The sharp temperature drop at the interface (at ≈7.6 nm) visible for m ̸= 28 is due to the Kapitza resistance. It is evaluated according to equation (2) and plotted as a 12 New J. Phys. 16 (2014) 015021 H Bracht et al Figure 8. Temperature proﬁles for 28Si/28Si (top left), 28Si/29Si (top right), 28Si/30Si (bottom left) and 28Si/44Si (bottom right): the 28Si/28Si proﬁle shows no step change in temperature at the interface as expected. In the three other cases the Kapitza resistance at the interface increases with increasing mass difference of the Si isotopes. Figure 8. Temperature proﬁles for 28Si/28Si (top left), 28Si/29Si (top right), 28Si/30Si (bottom left) and 28Si/44Si (bottom right): the 28Si/28Si proﬁle shows no step change in temperature at the interface as expected. In the three other cases the Kapitza resistance at the interface increases with increasing mass difference of the Si isotopes. function of 1m = m −28 in ﬁgure 9. A linear regression of RK = a 1m gives the value a = 9.93 × 10−11K m2 W−1u−1. The simulation result for the thermal conductivity of the pure 28Si-system, λ(28) = 2.79 W m−1 K−1, is far below the experimental value for bulk silicon. The reason is that the boundary conditions imply that we simulate a nanowire, not a bulk lattice. Here a reduction of thermal conductivity due to size conﬁnement is indeed expected [22]. 3.2.2. Effect of superlattice period. In order to investigate the effect of interfaces and layer thickness on the thermal conductivity of a 28Si/30Si superlattice the bilayer thickness (superlattice period) is altered at a ﬁxed sample length. The longest period of about 15 nm is realized when one half of the simulation cell consists of 28Si and the other half of 30Si. The shortest period is 0.27 nm, i.e. the isotopic composition changes every monolayer. Figure 10 shows the thermal conductivity of a 28Si/30Si superlattice as function of the superlattice period. For comparison the thermal conductivities of natSi, 28Si and an 28Si0.5 30Si0.5 alloy are also displayed. Figure 10 reveals that a reduction in thermal conductivity of Si is possible by isotope doping. 3.2. Results The higher thermal conductivity of 28Si compared to natSi is due to the absence of 13 New J. Phys. 16 (2014) 015021 H Bracht et al Figure 9. Kapitza resistance calculated for the isotope bilayer structures shown in ﬁgure 8 as function of the mass difference of the Si atoms on the right and left side of the interface. The solid line shows the result of the linear regression analysis. Figure 9. Kapitza resistance calculated for the isotope bilayer structures shown in ﬁgure 8 as function of the mass difference of the Si atoms on the right and left side of the interface. The solid line shows the result of the linear regression analysis. phonon scattering by isotope defects. The 28Si/30Si superlattice and 28Si0.5 30Si0.5 alloy have the same stoichiometric composition but a different spatial isotopic distribution. In the alloy the lattice sites are randomly occupied by 28Si or 30Si atoms, in the superlattice the 28Si and 30Si atoms are arranged in layers. This leads to different phonon scattering mechanisms: in the alloy the phonons are scattered by randomly distributed isotope defects and in the superlattice the phonons are scattered at the interfaces between 28Si and 30Si. p The thermal conductivity of the 28Si/30Si superlattice has a distinct minimum at a period length of 1.09 nm. This minimum appears at a length that is of the order of the effective phonon mean free path [23]. Yang et al [24] reported qualitatively the same effect of superlattice period on the thermal conductivity of 28Si/29Si and 28Si/42Si superlattices. The period length with minimum thermal conductivity is exactly the same. The behavior can be qualitatively understood in the following way. First a periodic arrangement with superlattice periods of only a few layers appears to be a homogeneous medium for long-wavelength phonons. Therefore contribution to phonon scattering will be low and the effect on the thermal conductivity is less pronounced. Once the layers increase in thickness, the thermal conductivity will drop. Increasing the superlattice period at ﬁxed length of the simulation cell further will on the other hand, decrease the absolute number of scattering interfaces, consequently conductivity rises again. The inset of ﬁgure 10 illustrates an increase of the thermal resistivity with increasing number of interfaces for superlattice periods equal to and greater than 1.09 nm. 3.2. Results A linear increase is expected in case the thermal conductivity of the isotope superlattice is mainly given by serial connection of the interface resistances. However, our calculations clearly reveal a deviation from the linear dependence. The origin of this deviation is discussed in the next section. 14 New J. Phys. 16 (2014) 015021 H Bracht et al Figure 10. Thermal conductivity of an 28Si/30Si superlattice (red line and dots), natSi (dashed blue line), 28Si (dashed black line) and an 28Si0.5 30Si0.5 alloy (dashed green line). For the superlattice structure the thermal conductivity is plotted as a function of superlattice period, for the other three samples the lines are dashed as there is no isotopic periodicity within the samples. The inset demonstrates the thermal resistivity versus the number of interfaces. The calculations suggest an interrelation that clearly deviates from a linear dependence. Figure 10. Thermal conductivity of an 28Si/30Si superlattice (red line and dots), natSi (dashed blue line), 28Si (dashed black line) and an 28Si0.5 30Si0.5 alloy (dashed green line). For the superlattice structure the thermal conductivity is plotted as a function of superlattice period, for the other three samples the lines are dashed as there is no isotopic periodicity within the samples. The inset demonstrates the thermal resistivity versus the number of interfaces. The calculations suggest an interrelation that clearly deviates from a linear dependence. 4. Discussion Both experimental and theoretical results reveal a reduction in the thermal conductivity of silicon by isotopic modulation. The experiments indicate a factor of 2 to 3 lower thermal conductivity of the isotope multilayer structure compared with natural Si (see section 2.4 and [14]). MD simulations reveal a reduction of about 30% in thermal conductivity in comparison to the simulations of natSi. Thus the simulations predict a much smaller reduction. This is related to the fact that MD calculations represent the thermal properties of a silicon nanowire. The main reduction of the thermal conductivity compared to natural bulk Si is due to the lateral conﬁnement. An isotopic modulation gives only a modest further reduction. As isotope heterostructures represent chemical and electronic homostructures, the reduction in thermal conductivity must be due to phonon scattering at the isotope interfaces. The scattering process at interfaces can be explained either with a diffuse or acoustic mismatch model. In the ﬁrst case, the conductivity of a given multilayer is determined by the density of the interfaces, which scatter phonons incoherently. In the second case the coherent reﬂection of phonons will lead to complete stop bands for certain phonon wave vectors. A selective reﬂection or transmission of phonons has e.g. been reported for GaAs/AlGaAs superlattices by Narayanamurti et al [25]. The phonon scattering is similar to the reﬂection of electromagnetic waves at the interface between two optical media with different refractive indices [25]. Moreover, Spitzer et al [26] show clear evidence for optic phonon conﬁnement in isotopically modulated germanium superlattices. 15 New J. Phys. 16 (2014) 015021 H Bracht et al In the framework of the acoustic mismatch model, the phonons can be considered as waves whose mean free path is longer than the period length of the multilayer structure [27]. Within this limit the thermal conductivity of a multilayer structure will decrease with increasing superlattice period due to band-folding effects [27]. Band-folding reduces the phonon group velocities and thus the thermal conductivity [28, 29]. If the phonon mean free path is shorter than the period length, phonons can be treated as particles and diffuse scattering prevail. In this limit the thermal conductivity will increase with decreasing interface density. Accordingly, a minimum in thermal conductivity is expected as function of the superlattice period. This is supported by our MD simulation results shown in ﬁgure 10. 4. Discussion A minimum in the thermal conductivity as function of the period length is also conﬁrmed by MD calculations performed by Yang et al [24]. The minimum in thermal conductivity at a period length of 1.09 nm describes the crossover between a wave- and particle-type phonon transport [27], i.e. for period lengths below 1.09 nm the wave-like behavior and for period lengths above 1.09 nm the particle-like behavior of phonons prevails. In the ﬁrst case the phonons are fully coherent and give rise to phonon bands speciﬁc for the superlattice structure. In the latter case, the phonons are fully incoherent and the thermal conductivity can be calculated by means of the Boltzmann transport equation assuming the diffused scattering at the interfaces [30]. This impact of coherent and incoherent phonons on heat transport in superlattices is treated theoretically and discussed in the literature in detail (see e.g. [31–33]). The inset in ﬁgure 10 suggests a nonlinear dependence of the thermal resistivity as function of the number of interfaces for period lengths larger than 1.09 nm. This dependence indicates that phonon coherence still affects the thermal conductivity at this length scale. Although our MD calculations support a reduction in thermal conductivity by isotopic modulation, the absolute values obtained for the thermal conductivity of natural Si, isotopically enriched 28Si, the 28Si0.5 30Si0.5 alloy and of the isotope superlattice deviate from the experimental results listed in table 1. The calculated thermal conductivity is much lower compared to the experiment. This is a consequence of the limited size of the simulation cell both in vertical and parallel directions of heat ﬂow. In particular, the limited size affects the phonon mean free path, i.e. larger cells would yield larger phonon mean free path. Accordingly, phonons with long mean free path that signiﬁcantly contribute to the thermal conductivity in bulk silicon are not considered by MD simulations [34]. Moreover, our calculations assume perfect interfaces that are hardly realized experimentally. The impact of interfacial mixing on phonon transport in silicon/germanium superlattices has been studied by Landry et al [30] with MD simulations. They observe that phonon coherence vanishes in the case of interface mixing and, consequently, an increase in thermal conductivity with increasing period length is suggested indicating incoherent phonon transport [30]. The measurements of the two samples #1 and #2 with different isotopic compositions (28Si/29Si) and (28Si/30Si) indicate similar thermal conductivities. [1] Kishi M, Nemoto H, Hamao T, Yamamoto M, Sudou S, Mandai M and Yamamoto S 1999 Proc. 18th Int. Conf. on Thermoelectrics p 301 [2] Lay-Ekuakille A, Vendramin G, Trotta A and Mazzotta G 2009 Proc. 2009 IEEE Int. Workshop on Medical Measurements and Applications p 1 [3] Snyder G J and Toberer E S 2008 Nature Mater. 7 105 [4] Bhandari C 1994 CRC Handbook of Thermoelectrics ed D M Rowe (Boca Raton, FL: CRC) 4. Discussion The MD simulations reveal a further reduction of the thermal conductivity with increasing mass difference between the constituents of a superlattice in which the interfaces were assumed to be sharp (see ﬁgure 8). The comparison between experimental and theoretical results demonstrates the strong effect of the thermal interface resistance, which may cover the effect of the higher mass difference on the phonon transport in the experiment. This interface resistance is likely due to the interfacial mixing as discussed above. To understand more quantitatively the experimentally observed reduction in the thermal conductivity of silicon by isotopic modulation not only a mixing at the 16 New J. Phys. 16 (2014) 015021 H Bracht et al isotope interfaces but also appropriate procedures to eliminate the size effect in MD simulations must be considered in the future MD simulations. isotope interfaces but also appropriate procedures to eliminate the size effect in MD simulations must be considered in the future MD simulations. 5. Conclusion The experimental and theoretical results of this work lead to the conclusion that the acoustic mismatch model and the Fourier heat conduction theory based on bulk material properties do not explain in a satisfactory manner the effective thermal conductivity of isotopically modulated superlattice structures. The suggestion that the physical properties of the superlattice itself govern the thermal transport in such structures seems to be more convincing. Thus far the argumentation includes a diffuse and acoustic mismatch model. In the ﬁrst case, the conductivity of a given multilayer is determined by the density of the interfaces, which scatter phonons incoherently. In the latter case one may speculate about the inﬂuence of coherence on the phonon transport. In the optical picture [31, 35] of Bragg reﬂection of coherent phonons [32, 36] not only the presence of interfaces matters but also the periodic arrangement due to long mean free paths. A multilayer represents a Bragg grating that can efﬁciently block complete phonon bands. On the other hand a periodic lattice might not be the optimal structure, as compared to tailored aperiodic or chirped layer stacks. Such tailored structures will be investigated in future by combining predictions from MD simulations with experimental tests. These studies will help to identify more quantitatively the factors that determine the thermal conductivity in isotopically modulated silicon. Tailored silicon structures may ﬁnd applications as thermoelectric power generators or refrigerators in high temperature applications or highly integrated miniaturized devices. The ultimate power factor needs to be proven. Acknowledgments Provision of beamtime at the facilities ANKA and ESRF is gratefully acknowledged. We wish to thank for the excellent support by M Khakulin, M Wulff and G Buth. This work was funded by the Deutsche Forschungsgemeinschaft via the priority program SPP1386 ‘Nanostructured Thermoelectric Materials: Theory, Model Systems and Controlled Synthesis’ (BR 1520/13-1 and BR 1520/15-1) and individual grants for AP and within the Heisenberg program for HB (BR 1520/10-2). The isotopically enriched Si was developed by the Initiatives for Proliferation Prevention Program of the Ofﬁce of Nonproliferation Research and Engineering (NN-20) of the U.S. Department of Energy under contract DE-AC03-76SF00098. 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https://openalex.org/W4212816728	https://www.researchsquare.com/article/rs-13798/latest.pdf	English	null	Long noncoding RNA XIST suppresses tumorigenesis and enhances radiosensitivity in neuroblastoma cells through regulating miR-653-5p/HK2 axis	Research Square (Research Square)	2,020	cc-by	8,689	Long noncoding RNA XIST suppresses tumorigenesis and enhances radiosensitivity in neuroblastoma cells through regulating miR-653- 5p/HK2 axis Liping Mou People's Hospital of Rizhao Lili Wang People's Hospital of Rizhao Shaoming Zhang People's Hospital of Rizhao Qinghua Wang (  lwnl5ya@163.com People's Hospital of Rizhao https://o Primary research Page 1/22 Page 1/22 Page 1/22 Abstract Background: Abnormal expression of long noncoding RNAs (lncRNAs) was usually involved in tumorigenesis and radiosensitivity of various cancers. The aim of this study was to explore the biological function and regulatory mechanism of lncRNA X-inactive specific transcript (XIST) in tumorigenesis and radiosensitivity of neuroblastoma (NB). Methods: The expression levels of XIST, microRNA-653-5p (miR-653-5p) and hexokinase 2 (HK2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Methylthiazolyldiphenyl tetrazolium bromide (MTT) assay, colony formation assay and transwell assay were utilized to detect cell viability, colony formation and cell invasion abilities. Glucose consumption or lactate production was measured by glucose assay kit or lactate assay kit, respectively. The mice xenograft model was established to investigate the role of XIST in vivo. The interaction between miR-653-5p and XIST or HK2 was predicted by starBase v2.0 and verified by dual-luciferase reporter, RNA Immunoprecipitation (RIP) and RNA pull-down assays. Western blot was used to measure the protein expression of HK2. Results: XIST and HK2 were highly expressed whilst miR-653-5p was lowly expressed in NB tissues and cells. XIST knockdown inhibited tumorigenesis by repressing NB cell proliferation and invasion. Meanwhile, XIST downregulation increased the radiosensitivity via inhibiting colony formation rates and glycolysis. Moreover, miR-653-5p could bind to XIST and its downregulation reversed the effects of XIST knockdown on tumorigenesis and radiosensitivity. Additionally, HK2 was a direct target of miR-653-5p and its overexpression attenuated the effects of miR-653-5p restoration on suppression of tumorigenesis and promotion of radiosensitivity. Besides, XIST functioned as a molecular sponge of miR-653-5p to regulate HK2 expression. Furthermore, XIST knockdown also suppressed tumor growth by upregulating miR-653-5p and downregulating HK2 in vivo. Results: XIST and HK2 were highly expressed whilst miR-653-5p was lowly expressed in NB tissues and cells. XIST knockdown inhibited tumorigenesis by repressing NB cell proliferation and invasion. Conclusion: XIST interference inhibited tumorigenesis and increased radiosensitivity in NB by regulating miR-653-5p/HK2 axis, providing a novel therapeutic strategy for NB. Background Neuroblastoma (NB) is one of the most frequent extracranial childhood tumors and originates from primitive neural crest cells of the sympathetic nervous system, accounting for 7% of malignant tumors in children and approximately 15% of all childhood cancer deaths [1, 2]. NB is notable for its phenotypic diversity, from undifferentiated tumors to tumors containing a neural crest-derived differentiated cell range [3]. Although advances in treatment have improved the survival times of NB patients, children with regional or distant metastatic disease still have a poor prognosis [4]. Radiotherapy is the main treatment for tumors, but the efficacy of radiotherapy is limited due to obtaining radioresistance in the treatment of NB [5]. Thus, it is necessary to explain the molecular mechanisms of NB and identify more effective therapeutic targets to increase the radiosensitivity. Page 2/22 Page 2/22 Long noncoding RNAs (lncRNAs) are highly conserved transcripts (>200 nucleotides) with limited or without protein-coding ability and play regulatory roles in various physiopathology processes [6]. Currently, numerous studies have demonstrated that lncRNAs are abnormally expressed in a variety of cancers, including NB [7]. For instance, lncRNA NBAT-1 downregulation promoted aggressive NB through enhancing proliferation and attenuating differentiation of neuronal precursors [8]. Moreover, lncRNA RMRP was overexpressed in NB tissues and its knockdown suppressed the progression of NB [9]. X inactive-speciﬁc transcript (XIST) belongs to a class of RNA molecules known as non-coding transcripts. XIST gene is located in the X inactivation center, and its product is transcribed from the inactive X chromosome [10]. Besides, XIST plays critical roles in the differentiation, proliferation, and genome maintenance of human cells. Speciﬁcally, it has been suggested that dysregulation of XIST is tightly associated with the progression of multiple cancers, such as lung cancer [11], hepatocellular carcinoma [12] and gastric cancer [13]. Besides, pervious study uncovered that XIST was highly expressed in NB tissues and its expression was related to NB development [14]. However, the underlying mechanisms by which XIST is involved in regulating NB progression and radiosensitivity remain largely unknown. Long noncoding RNAs (lncRNAs) are highly conserved transcripts (>200 nucleotides) with limited or without protein-coding ability and play regulatory roles in various physiopathology processes [6]. Currently, numerous studies have demonstrated that lncRNAs are abnormally expressed in a variety of cancers, including NB [7]. For instance, lncRNA NBAT-1 downregulation promoted aggressive NB through enhancing proliferation and attenuating differentiation of neuronal precursors [8]. Moreover, lncRNA RMRP was overexpressed in NB tissues and its knockdown suppressed the progression of NB [9]. X inactive-speciﬁc transcript (XIST) belongs to a class of RNA molecules known as non-coding transcripts. XIST gene is located in the X inactivation center, and its product is transcribed from the inactive X chromosome [10]. Besides, XIST plays critical roles in the differentiation, proliferation, and genome maintenance of human cells. Speciﬁcally, it has been suggested that dysregulation of XIST is tightly associated with the progression of multiple cancers, such as lung cancer [11], hepatocellular carcinoma [12] and gastric cancer [13]. Besides, pervious study uncovered that XIST was highly expressed in NB tissues and its expression was related to NB development [14]. However, the underlying mechanisms by which XIST is involved in regulating NB progression and radiosensitivity remain largely unknown. Cell culture and transfection Human embryonic kidney (HEK293) and NB cell line (SK-N-BE(2)) were purchased from BeNa Culture Collection (Beijing, China). The NB cell line (GI-LI-N) was obtained from Conservation Genetics CAS Kunming Cell Bank (Yunnan, China). These cells were cultured in Dulbecco’s modified eagle medium (DMEM; Invitrogen, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin in an incubator with 5% CO2 at 37℃. For this study, small interfering RNA against XIST (si-XIST) and the corresponding control (si-NC), miR- 653-5p mimic (miR-653-5p) and the corresponding control (miR-NC), miR-653-5p inhibitor (anti-miR-653- 5p) and the corresponding control (anti-miR-NC), XIST or HK2 overexpression plasmid (XIST or HK2) and the corresponding control (pcDNA) were purchased from GenePharma (Shanghai, China). Lentivirus- mediated shRNA interference targeting XIST (sh-XIST) and the corresponding control (sh-NC) were constructed by Genechem (Shanghai, China). GI-LI-N and SK-N-BE(2) cells with 50–60% confluence were transfected with oligonucleotide (50 nM) or plasmid (2 μg) using Lipofectamine 3000 (Invitrogen). Clinical tissue samples In this study, thirty NB tissues and paired normal tissues were obtained from patients undergoing surgery at People's Hospital of Rizhao. The patients did not receive any treatment before surgery. Each patient has signed written informed consent. Excised tissues were collected and promptly frozen in liquid nitrogen, and then cryopreserved at -80℃ for subsequent study. Page 2/22 Increasing evidence has shown that lncRNAs serve as competing endogenous RNAs (ceRNAs) to modulate gene expression through sponging microRNA (miRNA) [15]. MiRNAs are usually small RNAs (18-25 nucleotides) that lack protein-coding potential and negatively gene expression via combining with complementary sequences on target mRNA [16, 17]. Currently, accumulating evidence demonstrated that aberrant expression miRNA was closely related to the development and progression of cancers, and could affect the cell’s response to radiation [18, 19]. MiR-653-5p targets 615 transcripts with conserved sites, containing a total of 662 conserved sites and 487 poorly conserved sites, and miR-653-5p can exert regulatory functions in mammalian evolution [20]. MiR-653-5p has been suggested to function as a tumor suppressor or tumor promoter in different cancers [21, 22]. Moreover, miR-653-5p was also reported to be lowly expressed in NB [23]. Nevertheless, the biological functions of miR-653-5p in the tumorigenesis and radiosensitivity of NB are poorly understood. Several miRNAs target hexokinase 2 (HK2; a metabolism-related factor) to influence the progression of multiple types of cancer [24, 25]. HK2, a member of the human glandular kallikrein family, is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA) [26]. HK2 has been shown to act as a critical regulator in cell growth, invasion and glycolysis [27]. Moreover, HK2 was reported to be upregulated in NB and can act as a target of miR-143- 3p in NB [28]. However, there is no report on the interaction between miR-653-5p and HK2, and the exact roles of HK2 in NB progression and radiosensitivity should be explored. Interestingly, starBase v2.0 software online predicted that XIST and HK2 shared the complementary binding sites of miR-653-5p. Hence, we hypothesized XIST might regulate the tumorigenesis and radiosensitivity of NB by acting as a sponge of miR-653-5p to modulate HK2 expression. In the present study, the expression levels of XIST, miR-653-5p and HK2 were analyzed in NB tissues and cells. In addition, we explored their effects on proliferation, invasion, glycolysis, and radiosensitivity, and investigated the regulatory network of XIST/miR-653-5p/HK2 in NB cells. The aim of this study was to Page 3/22 Page 3/22 identify promising therapeutic targets for treatment of NB and explore a novel mechanism for better understanding the pathogenesis of NB. identify promising therapeutic targets for treatment of NB and explore a novel mechanism for better understanding the pathogenesis of NB. Cell viability assay Methylthiazolyldiphenyl tetrazolium bromide (MTT) was utilized for detecting cell viability. In brief, GI-LI-N and SK-N-BE(2) cells (100 μL) were seeded in 96-well plates at a density of 1 × 104/mL and then transfected with si-XIST, si-XIST + anti-miR-653-5p, miR-653-5p, miR-653-5p + HK2, or matched controls. MTT reagent (5mg/mL, 10 μL, Beyotime, Shanghai, China) was added to each well after transfection for 24, 48 h, or 72 h. After incubation for 4 h, the cultured medium was carefully discarded and dimethyl sulfoxide (DMSO; 150 μL) was added to per well. Lastly, the absorbance was evaluated at 490 nm under a microplate reader (Bio-Rad, Hercules, CA, USA). Transwell assay Transwell assay was performed using a 24-well plate inserts with 8 μm pores (Corning Incorporated, Corning, NY, USA) to evaluate cell invasion capacity. GI-LI-N and SK-N-BE(2) cells (2×104 cells/well) re- suspended in DMEM medium alone (100 μL) were seeded into the top chamber pre-coated with Matrigel (BD Bioscience, Franklin Lakes, NJ, USA). To induce cells invading through the membrane, the bottom chamber was filled with DMEM containing 10% FBS (600 μL). After incubation for 24 h, non-invaded cells were carefully removed with a cotton bud, and invaded cells were fixed with 95% ethanol and then stained with 0.1% crystal violet. Finally, invaded cells from five random fields were photographed and counted by a microscope. Irradiation (IR) and colony formation assay After transfection for 48 h, GI-LI-N and SK-N-BE(2) cells (150 cells per well) were seeded into six-well plates and the medium was updated every three days. For treatment of IR, cells were exposed to different doses of radiation by a linear accelerator (Varian, Palo Alto, CA, USA) at a dose rate of 3.5 Gy/min. After incubation for 2 weeks, GI-LI-N and SK-N-BE(2) cells were carefully washed with cold phosphate-buffered saline (PBS; pH=7.2) and subsequently fixed with 4% paraformaldehyde for 30 min at 4℃. After that, GI- LI-N and SK-N-BE(2) cells were washed by PBS and then stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). Colonies containing more than 50 cells were counted by a microscope (Olympus, Tokyo, Japan). The survival fraction was calculated as previously described [29]. Quantitative real-time polymerase chain reaction (qRT-PCR) TRIzol reagent (Invitrogen) was applied to isolate total RNA from tissues (NB tissues and normal tissues and cells (HEK293, GI-LI-N and SK-N-BE(2)). Next, the first strand of complementary DNA (cDNA) was synthesized with a High-Capacity cDNA Reverse Transcription Kit and TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The qRT-PCR was carried out using the SYBR Green PCR kit (Thermo Fisher Scientific) on the ABI 7300 system (Thermo Fisher Scientific). Primers used for qRT-PCR were exhibited as below: XIST (Forward, 5’-CCTCTCCACATACCTCAGT-3’; Reverse, 5’-ACATAATCACACGCATACCA-3’); miR-653-5p (Forward, 5’- CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGTAGAG-3’; Reverse, 5’- ACACTCCAGCTGGGGTGTTGAAACAATCT-3’); HK2 (Forward, 5’-ACAGCCTGGACGAGAGCATC-3’; Reverse, 5’-AGGTCAAACTCCTCTCGCCG-3’); 18S RNA (Forward, 5’-CGAACGTCTGCCCTATCAACTT-3’; Reverse, 5’- ACCCGTGGTCACCATGGTA-3’), U6 snRNA (Forward, 5’-CTCGCTTCGGCAGCACATATACT-3’; Reverse, 5’- TRIzol reagent (Invitrogen) was applied to isolate total RNA from tissues (NB tissues and normal tissues) and cells (HEK293, GI-LI-N and SK-N-BE(2)). Next, the first strand of complementary DNA (cDNA) was synthesized with a High-Capacity cDNA Reverse Transcription Kit and TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The qRT-PCR was carried out using the SYBR Green PCR kit (Thermo Fisher Scientific) on the ABI 7300 system (Thermo Fisher Scientific). Primers used for qRT-PCR were exhibited as below: XIST (Forward, 5’-CCTCTCCACATACCTCAGT-3’; Reverse, 5’-ACATAATCACACGCATACCA-3’); miR-653-5p (Forward, 5’- CAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGTAGAG-3’; Reverse, 5’- ACACTCCAGCTGGGGTGTTGAAACAATCT-3’); HK2 (Forward, 5’-ACAGCCTGGACGAGAGCATC-3’; Reverse, 5’-AGGTCAAACTCCTCTCGCCG-3’); 18S RNA (Forward, 5’-CGAACGTCTGCCCTATCAACTT-3’; Reverse, 5’- ACCCGTGGTCACCATGGTA-3’), U6 snRNA (Forward, 5’-CTCGCTTCGGCAGCACATATACT-3’; Reverse, 5’- ACACTCCAGCTGGGGTGTTGAAACAATCT-3’); HK2 (Forward, 5’-ACAGCCTGGACGAGAGCATC-3’; Reverse, 5’-AGGTCAAACTCCTCTCGCCG-3’); 18S RNA (Forward, 5’-CGAACGTCTGCCCTATCAACTT-3’; Reverse, 5’- ACCCGTGGTCACCATGGTA-3’), U6 snRNA (Forward, 5’-CTCGCTTCGGCAGCACATATACT-3’; Reverse, 5’- Page 4/22 Page 4/22 ACGCTTCACGAATTTGCGTGTC-3’). The expression levels of XIST, HK2 and miR-653-5p were calculated with the 2-ΔΔCt method, followed by normalizing to GAPDH or U6 snRNA, respectively. ACGCTTCACGAATTTGCGTGTC-3’). The expression levels of XIST, HK2 and miR-653-5p were calculated with the 2-ΔΔCt method, followed by normalizing to GAPDH or U6 snRNA, respectively. Measurement of glucose consumption and lactate production GI-LI-N and SK-N-BE(2) cells (5×104 cells/well) were seeded into six-well plates and transfected with oligonucleotide or/and plasmid. Cell culture media were collected 48 h after the transfection. In accordance with the manufacturer’s instructions, the glucose consumption and lactate production were detected with the glucose assay kit (Sigma-Aldrich) and lactate assay kit (BioVision, Mountain View, CA, Page 5/22 Page 5/22 USA), respectively. A standard calibration curve was used for determining the data, followed by normalizing to the amount of total protein. Dual-luciferase reporter assay The possible binding sites of miR-653-5p and XIST or HK2 were predicted by starBase v2.0. The XIST and HK2 3’UTR fragments containing wild-type (WT) or mutant (MUT) binding sites of miR-653-5p were amplified and inserted into pmirGlO luciferase reporter vector (Promega, Madison, WI, USA) to create the WT plasmids (WT-XIST, HK2 3’UTR-WT) or MUT plasmids (MUT-XIST, HK2 3’UTR-MUT). Afterward, GI-LI-N and SK-N-BE(2) cells were seeded in 24-well plates (2×104 cells/well) and were co-transfected with reporter plasmids and miR-653-5p or miR-NC. After incubating the cells for 48 h, the luciferase activity was estimated via Dual-Luciferase Reporter assay system (Promega). RNA pull-down assay RNA pull-down assay was performed to verify the endogenous interaction between miR-653-5p and XIST or HK2 in NB cells. Biotinylated miRNAs (bio-miR-NC and bio- miR-653-5p) were obtained from Sangon Biotech (Shanghai, China). In brief, cells transfected with bio-miR-NC or bio- miR-653-5p were lysed by lysis buffer. The cell lysates were incubated with Dynabeads M-280 Streptavidin (Thermo Fisher Scientific). Finally, the enrichment of XIST and HK2 was detected by qRT-PCR. RNA immunoprecipitation (RIP) assay EZ-Magna RIP Kit (Millipore, Billerica, MA, USA) was used to validate the interaction between miR-653-5p and XIST or HK2. In brief, cells were lysed by complete RIP lysis buffer. After that, whole cell extract (100 µL) was incubated by RIP buffer containing magnetic beads coated with human anti-argonaute2 (Ago2) or anti-IgG antibody (as control). Next, samples were digested using the proteinase K. Finally, immunoprecipitated RNA was isolated, and the expression levels of XIST, miR-653-5p and HK2 were examined by qRT-PCR. In vivo tumor growth assay BALB/c nude mice (male, 5-week-old) were purchased from Huafukang (Beijing, China). The sh-XIST or sh-NC was transfected into SK-N-BE(2) cells. Subsequently, stably transfected cells (3×106) were subcutaneously injected in BALB/c nude mice. From the 8th day, tumor volume was examined using a caliper every 4 days and calculated with the formula: length × width2/2. After injection for 10 days, sh- XIST group or sh-NC group were randomly divided into two groups. One group was irradiated with 6Gy X- ray once a week, and the other group served as control. All mice were sacrificed after injection for 4 weeks, and tumor samples were weighted and collected for detection of XIST, miR-653-5p and HK2 expression levels. Western blot assay GI-LI-N and SK-N-BE(2) cells (1×105 cells/well) were seeded into six-well plates and transfected with oligonucleotide or/and plasmid. After transfection for 48h, cells were lysed using RIPA lysis buffer (Sigma-Aldrich) supplemented with 1mM phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich) to extract the total protein, followed by being centrifuged at 12 000 × g at 4℃ for 15 min. After quantification by using bicinchoninic acid protein assay kit (Tanon, Shanghai, China), 30 μg of protein in each group was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then and then transferred to polyvinylidene fluoride membranes (Beyotime). After blocking with 5% non-fat milk in PBS containing 0.05% Tween 20 (PBST), the membranes were immunoblotted by primary antibody against HK2 (1:5000, ab227198, Abcam, Cambridge, UK) or GAPDH (1:2500, ab9485, Abcam) for 12-16 h at 4℃. After washing with PBST, membranes were incubated in horseradish peroxidase- Page 6/22 Page 6/22 conjugated anti-rabbit IgG (1:4000, D110058, Sangon Biotech) for 2 h at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL; Tanon) reagent following the manufacturer’s protocol. ImageJ software was utilized to quantify the protein expression. GAPDH was used as an internal control. Statistical analysis All data in this study were displayed as mean ± standard deviation (SD) and all experiments were repeated at least 3 times. Correlation between miR-653-5p and XIST or HK2 was detected by Spearman rank correlation. The results from different groups were assessed using the two-tailed Student’s t-test (for two groups) and a one-way analysis of variance (ANOVA; for more than two groups). Statistical analyses were performed using Graphpad Prism version 6.0 software (GraphPad Software, San Diego California, USA). P<0.05 indicates a statistically significant result. (P<0.05, P<0.01, P<0.001, P<0.0001). XIST was upregulated and miR-653-5p was downregulated in NB tissues and cells Firstly, we explored the expression of XIST and miR-653-5p in NB tissues and cells by qRT-PCR. As presented in Figure 1A and 1B, XIST was overexpressed in NB tissues and cells in contrast to their matched controls. Moreover, we found that the abundance of miR-653-5p was decreased in NB tissues and cells with respect to their corresponding controls (Figure 1C and 1D). Besides, the correlation between of XIST and miR-653-5p was analyzed in NB tissues. As depicted in Figure 1E, XIST expression was negatively correlated with miR-653-5p expression in NB tissues (r=-0.8114, p<0.001). These data indicated that XIST and miR-653-5p might be involved in the pathogenesis of NB. XIST knockdown inhibited cell proliferation and invasion, and enhanced radiosensitivity by inhibiting glycolysis in NB cells Next, the effects of XIST on cell proliferation, invasion, glycolysis, and radiosensitivity were investigated using loss-function experiments. The transfection efficiency of si-XIST was determined by qRT-PCR. The Page 7/22 Page 7/22 Page 7/22 results showed that the expression of XIST was decreased in GI-LI-N, SK-N-BE(2) and HEK293 cells transfected with si-XIST relative to those cells transfected with si-NC or control cells (Figure 2A, Supplementary Figure 1A), suggesting that si-XIST was successfully transfected into GI-LI-N, SK-N-BE(2) and HEK293 cells. MTT analysis revealed that XIST interference inhibited the viability of GI-LI-N and SK- N-BE(2) cells (Figure 2B). However, downregulation of XIST did not significantly inhibit the viability of HEK293 cells (Supplementary Figure 1B), indicating that XIST knockdown had little effect on HEK293 cells. Moreover, colony formation assay indicated that XIST deficiency decreased the number of colonies in GI-LI-N and SK-N-BE(2) cells (Figure 2C). Furthermore, GI-LI-N and SK-N-BE(2) cell invasion were inhibited by silencing XIST (Figure 2D). GI-LI-N and SK-N-BE(2) cells were transfected with si-XIST or si-NC and then irradiated with 0 Gy to 8 Gy to explore the effect of XIST on radiosensitivity. The data showed that XIST downregulation strikingly decreased the survival fraction of GI-LI-N and SK-N-BE(2) cells exposed to radiation compared to si-NC or control group (Figure 2E). Most cancer cells mainly rely on aerobic glycolysis to produce the energy needed for cellular processes [30]. This aerobic glycolysis will result in increased glucose consumption and lactate production. We observed that knockdown of XIST inhibited glucose consumption and lactate production in GI-LI-N and SK-N-BE(2) cells (Figure 2F and 2G). Moreover, we found that glucose consumption and lactate production were decreased in GI-LI-N and SK- N-BE(2) cells treated with 2-deoxyglucose (2-DG; glycolytic inhibitor) (Figure 2H and 2I). QRT-PCR analysis showed that the expression of XIST was enhanced in cells transfected with XIST compared to pcDNA group (Figure 2J). Besides, the promoting effect of XIST on cell survival fraction was overturned by treatment with 2-DG (Figure 2K), suggesting that XIST knockdown enhanced the radiosensitivity through inhibition of glycolysis. Taken together, these results demonstrated that XIST interference inhibited the tumorigenesis and increased the radiosensitivity in NB cells. XIST directly interacted with miR-653-5p in NB cells In view of the alteration of XIST and miR-653-5p in NB tissues and cell lines, as well as the negative correction between XIST and miR-653-5p expression, we wondered whether the function of XIST was mediated by miR-653-5p through complementary binding sites. StarBase v2.0 predicted that XIST contained putative binding sites of miR-653-5p (Figure 3A). To verify whether miR-653-5p was a direct target of XIST, the dual-luciferase reporter assay, RIP assay and RNA pull-down assay were performed. As presented in Figure 3B, overexpression of miR-653-5p led to a significant decrease in the luciferase activity of WT-XIST, while luciferase activity of MUT-XIST was not evidently affected after transfection with miR-653-5p in GI-LI-N and SK-N-BE(2) cells. RIP assay revealed that both miR-653-5p and XIST were immunoprecipitated in Ago2 group instead of IgG group (Figure 3C). RNA pull-down assay showed that the expression of XIST was significantly elevated when GI-LI-N and SK-N-BE(2) cells were transfected with bio-miR-653-5p (Figure 3D), suggesting that miR-653-5p could pull down XIST. Moreover, the expression of XIST was reduced in GI-LI-N and SK-N-BE(2) cells transfected with si-XIST, while its expression was elevated in cells transfected with XIST (Figure 3E). Next, we explored the effect of XIST on expression of miR-653-5p. As expected, knockdown of XIST increased the expression of miR-653-5p, whereas overexpression of XIST presented an opposite effect (Figure 3F), suggesting that miR-653-5p was negatively regulated by XIST. In addition, XIST downregulation also enhanced the expression of miR-653- Page 8/22 Page 8/22 Page 8/22 5p in HEK293 cells (Supplementary Figure 1C). At the same time, we observed that co-transfection of anti-miR-653-5p and si-XIST abated the effect of si-XIST on promotion of miR-653-5p expression (Figure 3G). Additionally, deficiency of miR-653-5p reversed the inhibitory effects of XIST knockdown on viability, colony formation and invasion of GI-LI-N and SK-N-BE(2) cells (Figure 3H-3J). Moreover, the suppressive effect of XIST interference on the survival fraction was abolished by downregulation of miR-653-5p in GI- LI-N and SK-N-BE(2) cells exposed to IR (Figure 3K). Furthermore, miR-653-5p silence abated the inhibitory effect of XIST knockdown on glucose consumption and lactate production (Figure 3L and 3M). Our findings suggested that miR-653-5p could bind to XIST and its knockdown reversed the effects of XIST interference on tumorigenesis and radiosensitivity in NB cells. HK2 was a direct target of miR-653-5p in NB cells To explore the underlying mechanism of miR-653-5p in progression of NB, the function targets of miR- 653-5p were searched by starBase v2.0. As presented in Figure 4A, miR-653-5p and HK2 3’UTR had complementary binding sequence, indicating that HK2 might be a target of miR-653-5p. Dual-luciferase reporter assay displayed that the luciferase activity of HK2 3’UTR-WT was obviously inhibited by transfection of miR-653-5p, whereas no clear change of the luciferase activity of HK2 3’UTR-MUT was found (Figure 4B). RIP assay demonstrated that miR-653-5p and HK2 were obviouly enriched in the Ago2 immunoprecipitation compared with the IgG immunoprecipitation (Figure 4C). Moreover, HK2 was strikingly enriched in GI-LI-N and SK-N-BE(2) cells transfected with bio- miR-653-5p (Figure 4D). Next, we detected the expression of HK2 in NB tissues and cells. The results suggested that the mRNA expression and protein expression of HK2 were increased in NB tissues in comparison with normal tissues (Figure 4E and 4F). Additionally, HK2 expression was negatively correlated with miR-653-5p level in NB tissues (r=-0.5985, p<0.001) (Figure 4G). Likewise, the mRNA expression and protein expression of HK2 were also enhanced in NB cells (GI-LI-N and SK-N-BE(2)) compared to HEK293 cells (Figure 4H). These data illustrated that miR-653-5p directly interacted with HK2. Downregulation of XIST inhibited tumor growth and increased radiosensitivity by regulating miR-653-5p To explore the impact of XIST on tumor growth and radiation response in vivo, we established a xenograft model in which the SK-N-BE(2) cells stably transfected with sh-XIST or sh-NC were subcutaneously injected into BALB/c nude mice and irradiated with 6Gy X-ray once a week or didn’t irradiate. In line with in vitro results, XIST knockdown or IR treatment suppressed tumor volume and weight, and combination of XIST interference and IR treatment significantly inhibited tumor volume and weight in xenograft model compared with only sh-XIST group or IR group (Figure 7A and 7B). In addition, knockdown of XIST decreased the expression of XIST and HK2 while increased the expression of miR- 653-5p in excised tumor masses (Figure 7C and 7D). These above findings indicated that downregulation of XIST could enhance the radiosensitivity to inhibit tumor growth in vivo. Overexpression of HK2 reversed the effects of miR-653-5p on inhibition of tumorigenesis and promotion of radiosensitivity in NB cells To determine whether the effects of miR-653-5p were regulated by HK2 expression, GI-LI-N and SK-N- BE(2) cells were transfected with miR-NC, miR-653-5p, miR-653-5p + pcDNA, or miR-653-5p + HK2. Western blot showed that HK2 protein expression was decreased in cells transfected with miR-653-5p, while the effect was abated by co-transfection of HK2 (Figure 5A). Additionally, overexpression of miR- 653-5p reduced the cell viability, colony formation and invasion of GI-LI-N and SK-N-BE(2) cells, which was reversed by upregulating HK2 (Figure 5B-5D). Moreover, the inhibitory effects of miR-653-5p upregulation on survival fraction and glycolysis were also overturned by overexpression of HK2 (Figure 5E-5H). Collectively, these findings demonstrated that miR-653-5p exerted its biological functions through regulating HK2 expression. HK2 was regulated by XIST and miR-653-5p in NB cells Page 9/22 Next, knockdown efficiency of miR-653-5p was tested by qRT-PCR. As shown in Figure 6A, the expression of miR-653-5p was significantly reduced in GI-LI-N and SK-N-BE(2) cells transfected with anti-miR-653-5p compared to those cells transfected with anti-miR-NC or control group. To explore whether XIST served as a ceRNA of miR-653-5p to modulate HK2 expression, GI-LI-N and SK-N-BE(2) cells were transfected with si-NC, si-XIST, si-XIST + anti-miR-NC, or si-XIST + anti-miR-653-5p. Western blot assay demonstrated that the protein level of HK2 was decreased after transfection with si-XIST, while the effect was reversed by downregulating miR-653-5p (Figure 6B). Meanwhile, knockdown of XIST also decreased the protein expression of HK2 in HEK293 cells (Supplementary Figure 1D). In a word, our results proved that XIST regulated HK2 expression by sponging miR-653-5p in NB cells. Downregulation of XIST inhibited tumor growth and increased radiosensitivity by regulating miR-653-5p and HK2 in vivo Downregulation of XIST inhibited tumor growth and increased radiosensitivity by regulating miR-653-5p and HK2 in vivo Discussion NB is a common and aggressive malignancy in children. Recently, it has been increasingly recognized that dysregulation of lncRNAs is involved in the progression of NB [31]. Radiotherapy has become an effective strategy for treatment of NB; however, the influence of lncRNAs on radiosensitivity of NB cells is not well elucidated. The purpose of this study was to explore the effects of XIST on NB progression and radiosensitivity. Up to now, a growing number of studies have shown that XIST serves as an oncogene and plays essential roles in cellular behaviors, such as cell growth, cell cycle, metastasis and apoptosis [32, 33]. Besides, Song et al. pointed out that XIST was overexpressed in nasopharyngeal carcinoma tissues, and accelerated nasopharyngeal carcinoma cell growth through targeting miR-34a-5p [34]. Moreover, Chen et al. uncovered that XIST knockdown inhibited colorectal cancer cell proliferation and metastasis via sponging miR-200b-3p to decrease ZEB1 expression [35]. In our research, it was found that XIST abundance was elevated in NB tissues and cells. Additionally, interference of XIST suppressed cell proliferation and invasion, and elevated radiosensitivity via suppressing glycolysis in NB cells. In agreement with our findings, Zhang et al. disclosed that XIST expression was markedly enhanced in NB tissues and its knockdown inhibited cell proliferation and metastasis in NB cells by regulating H3 histone Page 10/22 Page 10/22 methylation of DKK1 [14]. These results revealed that XIST was involved in the tumorigenesis and radiosensitivity of NB. Previous studies showed that miR-653-5p played pivotal roles in the regulating cellular behaviors, such as proliferation, cell cycle and apoptosis [36]. It has recently been identified as a cancer-related miRNA for several cancers. For example, Fu et al. declared that miR-653-5p was highly expressed in prostate cancer tissues, and miR-653-5p knockdown limited prostate cancer cell proliferation and metastasis via inhibiting Wnt/β-catenin signaling [37]. Han, et al. proved that miR-653-5p acted as a tumor-suppressive miRNA through targeting TIAM1 to suppress lung cancer cell proliferation and invasion [38]. These findings suggested that miR-653-5p could exert a tumor-suppressive or tumor-promotive function depending on the type of cancer. More importantly, Chi et al. showed that miR-653-5p expression was reduced in NB tissues, and miR-653-5p knockdown reversed the inhibitory abilities of cell proliferation, migration, and invasion induced by downregulating SNHG7 in NB cells, suggesting that miR-653-5p might act as a tumor suppressor in NB [23]. Consent for publication Not applicable. Authors' contributions Liping Mou participated in the conception and design of the study. Lili Wang performed the analysis and interpretation of data. Shaoming Zhang contributed to drafting the manuscript. Qinghua Wang contributed the methodology and analyzed the data. All authors read and approved the final manuscript. Conclusion XIST and HK2 were upregulated and miR-653-5p was downregulated in NB tissues and cells. Knockdown of XIST inhibited the tumorigenesis and enhanced the radiosensitivity in NB cells by regulating miR-653- 5p and HK2 expression. These findings might provide a potential therapeutic strategy for NB. Funding None. Availability of data and materials The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Discussion Here, we found that miR-653-5p abundance was declined in NB tissues and cells, and miR-653-5p expression was inversely correlated with XIST. Previous studies have demonstrated that lncRNAs can execute their functions through binding with their downstream miRNAs [39]. To further explore the relationship between XIST and miR-653-5p, starBase v2.0 was used to predict the targeting relationship. Interestingly, starBase v2.0 showed that miR-653-5p contained predicted binding sites with XIST. Next, the prediction was validated through dual-luciferase reporter assay. In addition, rescue experiments demonstrated that miR-653-5p knockdown could reverse the impact of XIST interference on cell proliferation, invasion, radiosensitivity, and glycolysis in NB cells. Collectively, our data indicated that XIST exerted its function through regulating miR-653-5p expression. It is well known that the miRNAs exerted their biological functions via modulating the expression of target mRNAs, so the potential target genes for miR-653-5p were analyzed in further analysis. Despite the fact that numerous tumor-associated genes were predicted using starBase v2.0, HK2 was selected as the candidate target gene of miR-653-5p due to its tumor-promoting effect. Further, dual-luciferase reporter assay proved that miR-653-5p directly targeted HK2. HK2 (a major type of hexokinase family) was reported to be overexpressed and facilitated rates of glucose metabolism necessary for tumor growth in multiple cancers [40, 41]. In addition, Botzer et al. reported that high expression of HK2 promoted NB cell metastasis [42]. Besides, Cen et al. revealed that HK2 was upregulated in NB tissues and cells, and HK2 accumulation weakened the repressive effects of miR-143-3p on progression of NB [28]. In line with previous findings, we also demonstrated that HK2 expression was increased in NB tissues and cells. Moreover, HK2 upregulation reversed the effect of miR-653-5p overexpression on inhibition of tumorigenesis and promotion of radiosensitivity in NB cells. Furthermore, we uncovered that XIST acted as a molecular sponge of miR-653-5p to modulate HK2 expression. Besides, knockdown of XIST restrained tumor growth and enhanced radiosensitivity by upregulating miR-653-5p and downregulating HK2 in vivo. In a word, these findings disclosed that XIST regulated the progression and radiosensitivity of NB cells by sponging miR-653-5p to modulate HK2 expression. 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Abbreviations lncRNAs: long noncoding RNAs; XIST: X-inactive specific transcript; miR-653-5p: microRNA-653-5p; HK2: hexokinase 2; qRT-PCR: quantitative real-time polymerase chain reaction; MTT: Methylthiazolyldiphenyl tetrazolium bromide; ceRNAs: competing endogenous RNAs; miRNA: microRNA; PBS: phosphate‐buffered saline; WT: wild-type; MUT: mutant Acknowledgement Thanks for all participants involved in this study. Page 12/22 References A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature. 1991; 349(6304):38-44. 11. Fang J, Sun CC, Gong C. Long noncoding RNA XIST acts as an oncogene in non-small cell lung cancer by epigenetically repressing KLF2 expression. Biochem Biophys Res Commun. 2016;478(2):811-7. 12. Zhuang LK, Yang YT, Ma X, Han B, Wang ZS, Zhao QY, Wu LQ, Qu ZQ. MicroRNA-92b promotes hepatocellular carcinoma progression by targeting Smad7 and is mediated by long non-coding RNA XIST. 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SOX30, a target gene of miR-653-5p, represses the proliferation and invasion of prostate cancer cells through inhibition of Wnt/β-catenin signaling. Cell Mol Biol Lett. 2019;24(1):1-13. 38. Han W, Wang L, Zhang L, Wang Y, Li Y. Circular RNA circ-RAD23B promotes cell growth and invasion by miR-593–3p/CCND2 and miR-653–5p/TIAM1 pathways in non-small cell lung cancer. Biochem Biophys Res Commun. 2019;510(3):462-6. 38. Han W, Wang L, Zhang L, Wang Y, Li Y. Circular RNA circ-RAD23B promotes cell growth and invasion by miR-593–3p/CCND2 and miR-653–5p/TIAM1 pathways in non-small cell lung cancer. Biochem Biophys Res Commun. 2019;510(3):462-6. 39. Jin B, Jin H, Wu HB, Xu JJ, Li B. Long non-coding RNA SNHG15 promotes CDK14 expression via miR- 486 to accelerate non-small cell lung cancer cells progression and metastasis. J Cell Physiol. 2018;233(9):7164-72. 40. He HC, Bi XC, Zheng ZW, Dai QS, Han ZD, Liang YX, Ye YK, Zeng Gh, Zhu G. Real-time quantitative RT- PCR assessment of PIM-1 and hK2 mRNA expression in benign prostate hyperplasia and prostate cancer. Med Oncol. 2009;26(3):303-8. 41. Mathupala S, Ko Ya, Pedersen PL. Hexokinase II: cancer's double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria. Oncogene. 2006;25(34):4777- 86. 42. Botzer LE, Maman S, Sagi-Assif O, Meshel T, Nevo I, Yron I, Witz IP. Figures Page 15/22 Page 15/22 Figure 1 XIST was upregulated while miR-653-5p was downregulated in NB tissues and cell lines. (A and B) Relative XIST expression was detected by qRT-PCR in NB tissues, normal tissues, NB cells (GI-LI-N and S N-BE(2)) and HEK293 cells. (C and D) Relative miR-653-5p expression was measured by qRT-PCR in NB tissues, normal tissues, NB cells (GI-LI-N and SK-N-BE(2)) and HEK293 cells. (E) Correlation between XIS and miR-653-5p expression was verified by Spearman rank correlation. P<0.001, *P<0.0001. Figure 1 Figure 1 XIST was upregulated while miR-653-5p was downregulated in NB tissues and cell lines. (A and B) Relative XIST expression was detected by qRT-PCR in NB tissues, normal tissues, NB cells (GI-LI-N and SK- N-BE(2)) and HEK293 cells. (C and D) Relative miR-653-5p expression was measured by qRT-PCR in NB tissues, normal tissues, NB cells (GI-LI-N and SK-N-BE(2)) and HEK293 cells. (E) Correlation between XIST and miR-653-5p expression was verified by Spearman rank correlation. P<0.001, **P<0.0001. Page 16/22 Page 16/22 Figure 2 XIST knockdown suppressed cell proliferation and invasion, and promoted radiosensitivity t Page 17/22 Figure 2 XIST knockdown suppressed cell proliferation and invasion, and promoted radiosensitivity through inhibiting glycolysis in NB cells. (A-D) GI-LI-N and SK-N-BE(2) cells were divided into 3 groups: Control, si- NC, and si-XIST. (A) The expression of XIST was examined using qRT-PCR analysis. (B) MTT assay was employed to analyze cell viability. (C) Colony formation assay was utilized to determine the number of colonies. (D) Cell invasion was detected by transwell assay. (E) Cell survival fraction was detected by Figure 2 Page 17/22 XIST knockdown suppressed cell proliferation and invasion, and promoted radiosensitivity through inhibiting glycolysis in NB cells. (A-D) GI-LI-N and SK-N-BE(2) cells were divided into 3 groups: Control, si- NC, and si-XIST. (A) The expression of XIST was examined using qRT-PCR analysis. (B) MTT assay was employed to analyze cell viability. (C) Colony formation assay was utilized to determine the number of colonies. (D) Cell invasion was detected by transwell assay. (E) Cell survival fraction was detected by Page 17/22 Page 17/22 colony formation assay in GI-LI-N and SK-N-BE(2) cells transfected with si-NC or si-XIST and exposed to radiation. (F-I) Glucose consumption and lactate production were measured in GI-LI-N and SK-N-BE(2) cells transfected with si-NC or si-XIST as well as the cells treated with 2-DG by glucose assay kit and lactate assay kit, respectively. (J) The expression of XIST was analyzed in GI-LI-N and SK-N-BE(2) cells transfected with pcDNA or XIST using qRT-PCR analysis. (K) Colony formation assay was applied to determine cell survival fraction in LI-N and SK-N-BE(2) cells transfected with si-NC or si-XIST or si-XIST + 2-DG under radiation condition. P<0.05, ***P<0.0001. Page 18/22 transfected with pcDNA or XIST using qRT-PCR analysis. (K) Colony formation assay was applied to determine cell survival fraction in LI-N and SK-N-BE(2) cells transfected with si-NC or si-XIST or si-XIST + 2-DG under radiation condition. P<0.05, ***P<0.0001. 2 DG under radiation condition. P<0.05, P<0.0001. , Page 18/22 Page 18/22 Page 18/22 Figure 3 XIST knockdown inhibited tumorigenesis and promoted radiosensitivity in NB cells by sponging miR-653- 5p. (A) Predicted binding sites between miR-653-5p and XIST were shown. (B) Dual-luciferase reporter assay was conducted to determine luciferase activity in GI-LI-N and SK-N-BE(2) cells co-transfected with miR-653-5p or miR-NC and WT-XIST or MUT-XIST. (C) The levels of miR-653-5p and XIST were measured by RIP assay in GI-LI-N and SK-N-BE(2) incubated with Ago2 or IgG. (D) The level of XIST was examined in GI-LI-N and SK-N-BE(2) cells transfected with bio-miR-653-5p or bio-miR-NC by RNA pull-down assay. (E and F) XIST and miR-653-5p expression were detected using qRT-PCR analysis in GI-LI-N and SK-N-BE(2) cells transfected with si-NC, si-XIST, pcDNA, or XIST. (E-K) GI-LI-N and SK-N-BE(2) cells were transfected with si-NC, si-XIST, si-XIST + anti-miR-NC, or si-XIST + anti-miR-653-5p. (G) The abundance of miR-653-5p was measured by qRT-PCR. (H) MTT assay was performed to examine cell viability. (I) The number of colonies was determined by colony formation assay. (J) Transwell assay was carried out to detect the number of invaded cells. (K) Cell survival fraction was determined by colony formation assay under radiation condition. (L and M) Glucose consumption or lactate production was measured by glucose assay kit or lactate assay kit, respectively. P<0.05, P<0.01, P<0.001, **P<0.00001. XIST knockdown inhibited tumorigenesis and promoted radiosensitivity in NB cells by sponging miR-653- 5p. (A) Predicted binding sites between miR-653-5p and XIST were shown. (B) Dual-luciferase reporter assay was conducted to determine luciferase activity in GI-LI-N and SK-N-BE(2) cells co-transfected with miR-653-5p or miR-NC and WT-XIST or MUT-XIST. (C) The levels of miR-653-5p and XIST were measured by RIP assay in GI-LI-N and SK-N-BE(2) incubated with Ago2 or IgG. (D) The level of XIST was examined in GI-LI-N and SK-N-BE(2) cells transfected with bio-miR-653-5p or bio-miR-NC by RNA pull-down assay. (E and F) XIST and miR-653-5p expression were detected using qRT-PCR analysis in GI-LI-N and SK-N-BE(2) cells transfected with si-NC, si-XIST, pcDNA, or XIST. (E-K) GI-LI-N and SK-N-BE(2) cells were transfected with si-NC, si-XIST, si-XIST + anti-miR-NC, or si-XIST + anti-miR-653-5p. (G) The abundance of miR-653-5p was measured by qRT-PCR. (H) MTT assay was performed to examine cell viability. (I) The number of colonies was determined by colony formation assay. (J) Transwell assay was carried out to detect the number of invaded cells. (K) Cell survival fraction was determined by colony formation assay under radiation condition. Figure 3 (L and M) Glucose consumption or lactate production was measured by glucose assay kit or lactate assay kit, respectively. P<0.05, P<0.01, P<0.001, *P<0.00001. Figure 4 HK2 was a downstream target of miR-653-5p in NB cells. (A) The putative binding sites between miR-653 5p and HK2 were predicted by starBase v2.0. (B) Relative luciferase activity was determined in GI-LI-N an SK-N-BE(2) cells co-transfected with HK2 3’UTR-WT or HK2 3’UTR-MUT and miR-653-5p or miR-NC. (C) Figure 4 HK2 was a downstream target of miR-653-5p in NB cells. (A) The putative binding sites between miR-653- 5p and HK2 were predicted by starBase v2.0. (B) Relative luciferase activity was determined in GI-LI-N and SK-N-BE(2) cells co-transfected with HK2 3’UTR-WT or HK2 3’UTR-MUT and miR-653-5p or miR-NC. (C) Page 19/22 Page 19/22 RIP assay was used to explore the interaction between HK2 and miR-653-5p in GI-LI-N and SK-N-BE(2) cells. (D) RNA pull-down assay was performed to confirm the targeted relationship between miR-653-5p and HK2 in GI-LI-N and SK-N-BE(2) cells. (E and F) The mRNA and protein expression of HK2 were evaluated in NB tissues and normal tissues by miR-653-5p and western blot analysis, respectively. (G) Correlation between HK2 and miR-653-5p expression was determined by Spearman rank correlation. (H) The mRNA and protein expression of HK2 were assessed in NB cells (GI-LI-N and SK-N-BE(2)) and HEK293 cells by qRT-PCR and western blot, respectively. P<0.001, **P<0.0001. Figure 5 Overexpression of miR-653-5p suppressed tumorigenesis and increased radiosensitivity in NB cells by ( ) Figure 5 Figure 5 Page 20/22 Overexpression of miR-653-5p suppressed tumorigenesis and increased radiosensitivity in NB cells by downregulating HK2. GI-LI-N and SK-N-BE(2) cells were divided into 5 groups: Control, miR-NC, miR-653- 5p, miR-653-5p + pcDNA, and miR-653-5p + HK2. (A) The protein abundance of HK2 was detected by western blot assay. (B) Cell viability was evaluated by MTT assay. (C) The number of colonies was determined using colony formation assay. (D) Transwell assay was performed to assess cell invasion ability. (E and F) Cell survival fraction was measured by colony formation assay under radiation condition. (G and H) Glucose consumption and lactate production were measured using glucose assay kit and lactate assay kit, respectively. P<0.05, *P<0.001, P<0.0001. Figure 7 XIST positively regulated HK2 expression via sponging miR-653-5p. (A) Knockdown efficiency of miR-653- 5p was determined by qRT-PCR. (B) Weston bolt assay was conducted to measure the protein abundance of HK2 in GI-LI-N and SK-N-BE(2) cells transfected with si-NC, si-XIST, si-XIST + anti-miR-NC, or si-XIST + anti-miR-653-5p. P<0.001, *P<0.0001. XIST positively regulated HK2 expression via sponging miR-653-5p. (A) Knockdown efficiency of miR-653- 5p was determined by qRT-PCR. (B) Weston bolt assay was conducted to measure the protein abundance of HK2 in GI-LI-N and SK-N-BE(2) cells transfected with si-NC, si-XIST, si-XIST + anti-miR-NC, or si-XIST + anti-miR-653-5p. P<0.001, **P<0.0001. Figure 6 Interference of XIST treatment of IR inhibited tumor growth by upregulating miR-653-5p and downregulating HK2 in vivo. SK-N-BE(2) cells transfected with sh-NC or sh-XIST were injected subcutaneously into nude mice, and the mice were treated with IR or without IR. (A and B) Tumor volume and weight were detected. (C) The expression levels of XIST and miR-653-5p were analyzed by qRT-PCR in resected tumor tissues. (D) Western blot assay was used to measure the protein expression of HK2 in resected tumor tissues. P<0.05, P<0.01, P<0.001, **P<0.0001. Interference of XIST treatment of IR inhibited tumor growth by upregulating miR-653-5p and downregulating HK2 in vivo. SK-N-BE(2) cells transfected with sh-NC or sh-XIST were injected subcutaneously into nude mice, and the mice were treated with IR or without IR. (A and B) Tumor volume and weight were detected. (C) The expression levels of XIST and miR-653-5p were analyzed by qRT-PCR in resected tumor tissues. (D) Western blot assay was used to measure the protein expression of HK2 in resected tumor tissues. P<0.05, P<0.01, P<0.001, *P<0.0001. Page 21/22 Figure 7 XIST positively regulated HK2 expression via sponging miR-653-5p. (A) Knockdown efficiency of miR-653- 5p was determined by qRT-PCR. (B) Weston bolt assay was conducted to measure the protein abundance of HK2 in GI-LI-N and SK-N-BE(2) cells transfected with si-NC, si-XIST, si-XIST + anti-miR-NC, or si-XIST + anti-miR-653-5p. P<0.001, ***P<0.0001. SupplementaryFigure1revised.tif Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 22/22 Page 22/22
https://openalex.org/W4313901689	https://ems.press/content/serial-article-files/26427	English	null	On the Quotients of the Maximal Unramified 2-Extension of a Number Field	Documenta mathematica	2,018	cc-by	13,519	1263 1263 Documenta Math. Documenta Math. Communicated by Otmar Venjakob Abstract. Let K be a totally imaginary number ﬁeld. Denote by Gur K (2) the Galois group of the maximal unramiﬁed pro-2 extension of K. By using cup-products in étale cohomology of SpecOK we study situations where Gur K (2) has no quotient of cohomological dimension 2. For example, in the family of imaginary quadratic ﬁelds K, the group Gur K (2) almost never has a quotient of cohomological dimension 2 and of maximal 2-rank. We also give a relation between this question and that of the 4-rank of the class group of K, showing in particular that when ordered by absolute value of the discriminant, more than 99% of imaginary quadratic ﬁelds satisfy an alternative (but equivalent) form of the unramiﬁed Fontaine-Mazur conjecture (at p = 2). 2010 Mathematics Subject Classiﬁcation: 11R37, 11R29. 2010 Mathematics Subject Classiﬁcation: 11R37, 11R29. Keywords and Phrases: Unramiﬁed extensions, cohomological dimen- sion, uniform pro-2 groups. Keywords and Phrases: Unramiﬁed extensions, cohomological dimen- sion, uniform pro-2 groups. Received: June 14, 2018 Revised: July 26, 2018 Received: June 14, 2018 Revised: July 26, 2018 On the Quotients of the Maximal Unramified 2-Extension of a Number Field Christian Maire Documenta Mathematica 23 (2018) 1263–1290 1.1 On the cohomological dimension Let S be a ﬁnite set of places of K. Denote by KS(p) the maximal unramiﬁed outside S pro-p extension of K; put GK,S = Gal(KS(p)/K). For p = 2 and for a real archimedean place v not in S, we assume that v splits totally in KS(p)/K. Hence, Kur(p) = K∅(p), Gur K (p) = GK,∅(p), and by class ﬁeld theory the p-Sylow ClK of the class group of K is isomorphic to the abelianization of Gur K (p). When S contains the set Sp of all p-adic places, and when moreover K is totally imaginary for p = 2, it has been known for a long time that the cohomological dimension cd(GK,S(p)) of the groups GK,S(p) is at most 2 (See for example [12], [24, Chapter X, §2], etc.). In the two last decades, various results have completed this fact. −First, when Sp ⊂S, Schmidt in [29] completed the Galois cohomological study of GK,S(p) at p = 2, proving in particular that if S contains a real place, then the group GK,S(p) has some torsion (and then cd(GK,S(p)) = ∞). −First, when Sp ⊂S, Schmidt in [29] completed the Galois cohomological study of GK,S(p) at p = 2, proving in particular that if S contains a real place, then the group GK,S(p) has some torsion (and then cd(GK,S(p)) = ∞). −A short while later, Labute in [17] made a real breakthrough by giving some examples of pro-p groups of cohomological dimension 2 when S∩Sp = ∅. Recall that in this tame context, the cohomological dimension cd(GK,S(p)) of GK,S(p) is always > 1 (when GK,S(p) is not trivial). Methods and ideas of Labute have been extended by many authors: Labute-Mináˇc [18], Schmidt [28] [30], Vogel [34], Forré [6], Gärtner [9], etc. Let us give probably the most complete result known due to Schmidt [30]: Given a ﬁnite set T of places of K with T ∩Sp = ∅, there exist inﬁnitely many ﬁnite sets S of places of K with T ⊂S and S∩Sp = ∅, such that cd(GK,S(p)) = 2. −A short while later, Labute in [17] made a real breakthrough by giving some examples of pro-p groups of cohomological dimension 2 when S∩Sp = ∅. Recall that in this tame context, the cohomological dimension cd(GK,S(p)) of GK,S(p) is always > 1 (when GK,S(p) is not trivial). 1 Introduction Let K be a number ﬁeld. Given a prime number p, denote by Kur(p) the max- imal unramiﬁed pro-p extension of K; put Gur K (p) := Gal(Kur(p)/K). Here we are interested in the quotients of Gur K (p), more precisely in their cohomolog- ical dimension, and in the comparison to their structure with those of p-adic analytic groups, a question which is related to the unramiﬁed Fontaine-Mazur conjecture. Documenta Mathematica 23 (2018) 1263–1290 1264 Christian Maire 1.1 On the cohomological dimension Methods and ideas of Labute have been extended by many authors: Labute-Mináˇc [18], Schmidt [28] [30], Vogel [34], Forré [6], Gärtner [9], etc. Let us give probably the most complete result known due to Schmidt [30]: Given a ﬁnite set T of places of K with T ∩Sp = ∅, there exist inﬁnitely many ﬁnite sets S of places of K with T ⊂S and S∩Sp = ∅, such that cd(GK,S(p)) = 2. In this work, when p = 2, we propose to give some families of imaginary quadratic number ﬁelds K for which Gur K (2) has no quotient of cohomological dimension 2 and large 2-rank. The starting point comes from the fact that H3 et(SpecOK, F2) is not trivial. Then we exploit a computation of cup-products in H3 et(SpecOK, F2) made by Carlson and Schlank in [3], thanks to the relationship between cohomology of number ﬁelds and étale cohomology (see the work of Mazur [23]). We ﬁrst prove that in the family of imaginary quadratic ﬁelds F−, there are few ﬁelds K for which Gur K (2) has a quotient G of maximal 2-rank and of cohomological dimension 2, in particular, such that cd(Gur K (2)) = 2. Indeed, for X ≥2 denote by for X ≥2 denote by F− X := {K ∈F−, \|discK\| ≤X}, and D− X = {K ∈F− X, Gur K (2) has no quotient G s.t. cd(G) = 2 & d2G = d2Gur K (2)}. D− X = {K ∈F− X, Gur K (2) has no quotient G s.t. cd(G) = 2 & d2G = d2Gur K (2)}. Theorem 1. One has: Theorem 1. One has: 0 ≤1 −#D− X #F− X ≤C log log X √log X , where C is an absolute constant. where C is an absolute constant. Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 1265 Unramified 2-Extensions of a Number Field Unramified 2-Extensions of a Number Field cd(G) = 2 & d2G ≥d}, D(d) n,X := {K ∈Fn,X, Gur K (2) has no quotient G s.t. cd(G) = 2 & d2G ≥d}, and consider the limit: D(d) n := lim inf X→+∞ #D(d) n,X #Fn,X . In the family of imaginary quadratic ﬁelds K where the 2-rank of the class group is equal to n, the quantity D(d) n measures the proportion of ﬁelds K for which Gur K (2) has no quotient G of cohomological dimension 2 and of 2-rank d2G ≥d. In the family of imaginary quadratic ﬁelds K where the 2-rank of the class group is equal to n, the quantity D(d) n measures the proportion of ﬁelds K for which Gur K (2) has no quotient G of cohomological dimension 2 and of 2-rank d2G ≥d. Then [10] allows us to obtain the following densities: Then [10] allows us to obtain the following densities: Corollary i. One has: Unramified 2-Extensions of a Number Field Next, we extend this result by using a bilinear form that appears in the study of the 4-rank of the class group of number ﬁelds. Let us be more precise. Let (xi)i=1,··· ,n be an F2-basis of H1(Gur K (2), F2) ≃H1 et(SpecOK, F2), and con- sider the n × n-square matrix MK := (ai,j)i,j with coeﬃcients in F2, where the ai,j’s are cup-products ai,j = xi ∪xi ∪xj, thanks to the fact that here H3 et(SpecOK, F2) ≃F2. As we will see, this is the Gram matrix of a certain bilinear form deﬁned, via the Artin symbol, on the Kummer radical of the 2- elementary abelian maximal unramiﬁed extension Kur,2/K of K. We also will see that for imaginary quadratic number ﬁelds, this matrix is often of large rank. Next, we extend this result by using a bilinear form that appears in the study of the 4-rank of the class group of number ﬁelds. Let us be more precise. Let (xi)i=1,··· ,n be an F2-basis of H1(Gur K (2), F2) ≃H1 et(SpecOK, F2), and con- sider the n × n-square matrix MK := (ai,j)i,j with coeﬃcients in F2, where the ai,j’s are cup-products ai,j = xi ∪xi ∪xj, thanks to the fact that here H3 et(SpecOK, F2) ≃F2. As we will see, this is the Gram matrix of a certain bilinear form deﬁned, via the Artin symbol, on the Kummer radical of the 2- elementary abelian maximal unramiﬁed extension Kur,2/K of K. We also will see that for imaginary quadratic number ﬁelds, this matrix is often of large rank. For a proﬁnite group G, as usual we denote by dpG := dimFp H1(G, Fp) the p-rank of G. We can now present the second result of our work: We can now present the second result of our work: Theorem 2. Let K be a totally imaginary number ﬁeld. Then the pro-2 group Gur K (2) has no quotient G for which cd(G) = 2 and d2G > d2ClK −1 2rk(MK). Now the key fact is the following: by relating the matrix MK to a Rédei-matrix type, and thanks to the work of Gerth [10] and Fouvry-Klüners [8], one can also deduce some density information when K varies in F−. For n, d, X ≥0, denote by Fn,X := {K ∈F− X, d2ClK = n}, D(d) n,X := {K ∈Fn,X, Gur K (2) has no quotient G s.t. Corollary i. One has: (i) D(3) 5 ≥.33129, D(4) 5 ≥.99062, D(5) 5 ≥.99999, (ii) D(4) 6 ≥.86718, D(5) 6 ≥.99925, D(6) 6 ≥1 −5.2 · 10−8. Moreover, for large n, D(2+n/2) n ≥.99995. (i) D(3) 5 ≥.33129, D(4) 5 ≥.99062, D(5) 5 ≥.99999, (ii) D(4) 6 ≥.86718, D(5) 6 ≥.99925, D(6) 6 ≥1 −5.2 · 10−8. Moreover, for large n, D(2+n/2) n ≥.99995. To conclude, let us mention a general asymptotic estimate thanks to [8]. Put D[i] X := {K ∈F− X, Gur K (2) has no quotient G s.t. cd(G) = 2 & d2G ≥i+d2ClK 2 } D[i] X := {K ∈F− X, Gur K (2) has no quotient G s.t. cd(G) = 2 & d2G ≥i+d2ClK 2 } Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 1266 Christian Maire and D[i] := lim inf X→+∞ #D[i] X #F− X . Our work allows us to obtain: Corollary ii. One has: D[1] ≥.28878, D[2] ≥0.99471, and D[3] ≥1 −9.7 · 10−8. Remark. At this point, one should make three observations. 1) Perhaps for many K ∈F3,X and F4,X, the pro-2 group Gur K (2) is ﬁnite but, by the Theorem of Golod-Shafarevich (see for example [16]), for every K ∈Fn,X, n ≥5, the pro-2 group Gur K (2) is inﬁnite. 2) In our work, it will appear that we have no information about the cohomo- logical dimension of the quotients of Gur K (2) for number ﬁelds K for which the 4-rank of the class group is large. Typically, in the estimates of D(∗) i one keeps out all the number ﬁelds having maximal 4-rank. 3) A part of the computation of [3] has been extended in [1] by Bleher, Chinburg, Greenberg, Kakde, Pappas and Taylor, at all p and number ﬁelds K containing µp. 1.2 On the unramified Fontaine-Mazur conjecture at p = 2 Now we also propose to give a relation between the unramiﬁed Fontaine-Mazur conjecture (conjecture (5b) of [5]) and the matrix MK deﬁned before. More precisely, here we are interested in uniform quotients of Gur K (p) (see section 2.2 for deﬁnition) which are related to the unramiﬁed Fontaine-Mazur conjecture thanks to the following equivalent version: Conjecture 1.1. Every uniform quotient G of Gur K (p) is trivial. Conjecture 1.1. Every uniform quotient G of Gur K (p) is trivial. Remark that Conjecture 1.1 can be rephrased as follows: the pro-p group Gur K (p) has no uniform quotient G of dimension d for all d > 0. As we will see, the matrix MK dectects the 4-rank of the class group of K, and the 4-rank is a ﬁrst test for Conjecture 1.1. We also obtain: Theorem 3. Let K/Q be a number ﬁeld. Theorem 3. Let K/Q be a number ﬁeld. (i) Suppose that the 4-rank of the class group of K is at most 2. Then Con- jecture 1.1 holds for K (at p = 2). (ii) Suppose K is totally imaginary. Then the pro-2 group Gur K (2) has no uniform quotient of dimension d > d2ClK −rk(MK). In particular, Con- jecture 1.1 holds (for Gur K (2)) when rk(MK) ≥d2ClK −2. (ii) Suppose K is totally imaginary. Then the pro-2 group Gur K (2) has no uniform quotient of dimension d > d2ClK −rk(MK). In particular, Con- jecture 1.1 holds (for Gur K (2)) when rk(MK) ≥d2ClK −2. (ii) Suppose K is totally imaginary. Then the pro-2 group Gur K (2) has no uniform quotient of dimension d > d2ClK −rk(MK). In particular, Con- jecture 1.1 holds (for Gur K (2)) when rk(MK) ≥d2ClK −2. Now as for the cohomological dimension, one can also deduce a general asymp- totic estimate when K varies in the family F−(resp. F+) of imaginary (resp. Documenta Mathematica 23 (2018) 1263–1290 1267 Unramified 2-Extensions of a Number Field real) quadratic ﬁelds. For n, d, X ≥0, denote by F± X := {K ∈F±, \|discK\| X}. Put FM± X := {K ∈F± X, Conjecture 1.1 holds for K } real) quadratic ﬁelds. For n, d, X ≥0, denote by F± X := {K ∈F±, \|discK\| ≤ and FM± := lim inf X→+∞ #FM± X #F± X . Corollary iii. One has Corollary iii. One has FM−≥.99471, and FM+ ≥.99929. This paper has ﬁve sections. In Section 2, we give the elementary tools con- cerning the étale cohomology of number ﬁelds and p-adic analytic groups. In section 3, we develop some basic facts about bilinear forms over F2, specially for the form introduced in our study (which is deﬁned on a certain Kummer radical). In particular, we emphasize the role played by totally isotropic sub- spaces. Section 4 is devoted to considerations of cohomological dimension. After proving Theorem 1 and Theorem 2 we consider a relation with a Rédei matrix that allows us to obtain density information. In Section 5, we consider the unramiﬁed Fontaine-Mazur conjecture at p = 2 by showing the relation with the bilinear form studied in the previous sections. We ﬁnish this section by giving a computational approach of this conjecture. Documenta Mathematica 23 (2018) 1263–1290 2.1 Cup-products and etale cohomology: what we need Assume K is totally imaginary when p = 2, and put XK = SpecOK. We use the formalism of étale cohomology Hi et of the site XK that we can ﬁnd for example in [23] (see also for example [28], [29]). The Hochschild-Serre spectral sequence gives for every i ≥1 a map αi : Hi(Gur K (p)) −→Hi et(XK), where the coeﬃcients are in Fp (meaning the constant sheaf for the étale site XK). Remark that α1 is an isomorphism. As H1 et(XKur(p)) is trivial, one obtains the long exact sequence (see for example [24, Chapter I, Proposition 1.6.7]): H2(Gur K (p)) ֒→H2 et(XK) −→H2 et(XKur(p))Gur K (p) −→H3(Gur K (p)) −→H3 et(Xk) (1) where H3 et(Xk) ≃(µK,p)∨, here (µK,p)∨is the Pontryagin dual of the group of pth-roots of unity in K. p y For the two next subsections, take p = 2. For the two next subsections, take p = 2. Notations • Let p be a prime number and let K be a number ﬁeld. Denote by • Let p be a prime number and let K be a number ﬁeld. Denote by −p∗= (−1)(p−1)/2p, when p is odd; −p∗= (−1)(p−1)/2p, when p is odd; −OK the ring of integers of K; −ClK the p-Sylow of the class group of OK; −dpClK the p-rank of ClK: it is the dimension over Fp of Fp ⊗ClK; −Kur(p) the maximal pro-p extension of K unramiﬁed everywhere. Put Gur K (p) := Gal(Kur(p)/K). −Kur(p) the maximal pro-p extension of K unramiﬁed everywhere. Put Gur K (p) := Gal(Kur(p)/K). Recall that the group Gur K (p) is a ﬁnitely presented pro-p group (due to Koch [16]). See also for example [24, Chapter X, §7] or [11, Appendix]. Moreover ClK is isomorphic to the abelianization of Gur K (p). In particular it implies that every open subgroup H of Gur K (p) has ﬁnite abelianization: this property is known as "FAb". • If G is a ﬁnitely generated pro-p group, denote by • If G is a ﬁnitely generated pro-p group, denote by −Hi(G) := Hi(G, Fp), i ∈Z≥0; −Hi(G) := Hi(G, Fp), i ∈Z≥0; −Hi(G) := Hi(G, Fp), i ∈Z≥0; −dpG = dimFp H1(G) the p-rank of G; −dpG = dimFp H1(G) the p-rank of G; 1268 Christian Maire −cd(G) the cohomological dimension of G: it is the smallest integer n ≥0 such that Hn+m(G) = {0}, ∀m > 0. Acknowledgements. This work has been done during a visiting scholar position at Cornell University for the academic year 2017-18, and funded by the program "Mobilité sortante" of the Région Bourgogne Franche-Comté. The author thanks the Department of Mathematics at Cornell University for provid- ing a beautiful research atmosphere. He also thanks Georges Gras and Farshid Hajir for encouragement and useful remarks, Ravi Ramakrishna for very in- spiring discussions, Alexander Schmidt for helpful comments, and construc- tive observations concerning the cup-products, Etienne Fouvry for stimulating exchanges and the computation of Proposition 4.7 which is central for Theo- rem 1, Magnus Carlson for his interest and comments, Christophe Delaunay for some references, and Bill Allombert for the support concerning the use of GP-Pari [25]. Documenta Mathematica 23 (2018) 1263–1290 Unramified 2-Extensions of a Number Field 1269 Proof. See for example [31, Chapter 1, §1.2]. Take x a non-trivial character of H1(Gur(p)) ≃H1 et(XK). Put Kx = (Kur)ker(x). Hence the previous proposition allows us to recover the follow- ing corollary of Carlson and Schlank [3, Corollary 5.14]: Corollary 2.2. The cup-product x ∪x ∈H2 et(XK) is trivial if and only, the quadratic extension Kx/K embeds in an unramiﬁed degree 4 cyclic extension. Proof. Indeed, as H2(Gur K (2)) ֒→H2 et(XK), see (1), the cup-product x ∪x vanishes in H2(Gur K (2)) if and only if, it vanishes in H2 et(XK). Then, apply Proposition 2.1 to G = Gur K (2). Proof. Indeed, as H2(Gur K (2)) ֒→H2 et(XK), see (1), the cup-product x ∪x vanishes in H2(Gur K (2)) if and only if, it vanishes in H2 et(XK). Then, apply Proposition 2.1 to G = Gur K (2). Remark 2.3. It is not diﬃcult to see that Corollary 2.2 allows us to obtain the following equivalence (see [3]): x ∪x = 0 ∈H2 et(XK) if and only if, for all h ∈ClK[2], h is a norm in Kx/K. Here ClK[2] denotes the kernel of the map ClK h7→h2 −→ClK. Remark 2.3. It is not diﬃcult to see that Corollary 2.2 allows us to obtain the following equivalence (see [3]): x ∪x = 0 ∈H2 et(XK) if and only if, for all h ∈ClK[2], h is a norm in Kx/K. Here ClK[2] denotes the kernel of the map ClK h7→h2 −→ClK. ClK h7→h2 −→ClK. 2.1.2 Cup-products in H3 et Take x and y two non-trivial characters of H1(Gur(p)) ≃H1 et(X). By Kummer theory, there exist ax, ay ∈K×/(K×)2 such that Kx = K(√ax) and Ky = K(√ay). As the extension Ky/K is unramiﬁed, for every prime ideal p of OK, the p-valuation vp(ay) is even, and then p (ay) is well-deﬁned (as an ideal of OK). Let us write OK). Let us write q (ay) := Y i (py,i)ey,i. Denote by Ix the set of prime ideals p of OK such that p is inert in Kx/K (or equivalently, Ix is the set of primes of K such that the Frobenius at p generates Gal(Kx/K)). In [3, Proposition 3.3], Carlson and Schlank prove: Proposition 2.4. The cup-product x ∪x ∪y ∈H3 et(X) is non-zero if and only if, X p ∈I ey,i is odd. py,i∈Ix Remark 2.5. The condition of Proposition 2.4 is equivalent to the triviality of K /K ! Remark 2.5. The condition of Proposition 2.4 is equivalent to the triviality of / ! the Artin symbol Kx/K p (ay) ! . 2.1.1 Cup-products in H2 et Recall a result concerning cup-products in H2. Recall a result concerning cup-products in H2. Proposition 2.1. Let G be a ﬁnitely presented pro-2 group. Take x ∈H1(G). Then x lifts in H1(G, Z/4Z) if and only if, x ∪x = 0 ∈H2(G). In particular, the cup-product H1(G) ⊗H1(G) →H2(G) is alternating (x ∪x = 0 for all x ∈H1(G)) if and only if, G/G4[G, G] ≃(Z/4Z)n, where n = d2G. Proposition 2.1. Let G be a ﬁnitely presented pro-2 group. Take x ∈H1(G). Then x lifts in H1(G, Z/4Z) if and only if, x ∪x = 0 ∈H2(G). In particular, the cup-product H1(G) ⊗H1(G) →H2(G) is alternating (x ∪x = 0 for all x ∈H1(G)) if and only if, G/G4[G, G] ≃(Z/4Z)n, where n = d2G. Documenta Mathematica 23 (2018) 1263–1290 Unramified 2-Extensions of a Number Field 2.2.1 Let us recall the p-central descending series (Gi)i of a pro-p group G: et us recall the p-central descending series (Gi)i of a pro-p group G: G1 = G, Gi+1 = Gp i [Gi, G], i ≥1. Let us give the deﬁnition of a uniform pro-p group (see for example [4, Chapter 4, §4.1]). Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 1270 Christian Maire Definition 2.6. Let G be a ﬁnitely generated pro-p group. We say that G is uniform if: (i) [G, G] ⊂G2p, and (ii) for i ≥1, [Gi+1 : Gi] = [G2 : G]. Remark 2.7. For a uniform group G, the p-rank of G coincides with the di- mension of G (as p-adic manifold). See [4, Chapter 4 and 8]. The uniform pro-p groups play a central role in the study of analytic pro-p groups, indeed: Theorem 2.8 (Lazard [19]). Let G be a proﬁnite group. Then G is p-adic analytic i.e. G embeds as a closed subgroup of Glm(Zp) for some positive integer m, if and only if, G contains an open uniform subgroup H. For diﬀerent equivalent deﬁnitions of p-adic analytic groups, see [4, Interlude A]. See also [22]. 2.2.2 Cohomology Recall by Lazard [19] (see also [33] for an alternative proof): Recall by Lazard [19] (see also [33] for an alternative proof): Theorem 2.9 (Lazard). Let G be a uniform pro-p group (of dimension d > 0). Then for all i ≥1, one has: Hi(G) ≃ i^ (H1(G)), where here the exterior product is induced by the cup-product. where here the exterior product is induced by the cup-product. where here the exterior product is induced by the cup-product. As consequence, one has immediately: Corollary 2.10. Let G be a uniform pro-2 group of dimension d. Then for all x ∈H1(G), one has x ∪x = 0 ∈H2(G). In particular, Gab ։ (Z/4Z)d. Corollary 2.10. Let G be a uniform pro-2 group of dimension d. Then for all x ∈H1(G), one has x ∪x = 0 ∈H2(G). In particular, Gab ։ (Z/4Z)d. Proof. By Theorem 2.9, the cup-product form on H1(G) is alternating, then apply Proposition 2.1. Remark 2.11. For p > 2, Theorem 2.9 is an equivalence: a pro-p group G is uniform if and only if, for i ≥1, Hi(G) ≃Vi(H1(G). (See [33, Corollary 5.1.7].) Let us mention another consequence useful in our context: Let us mention another consequence useful in our context: Let us mention another consequence useful in our context: Corollary 2.12. Let G be a FAb uniform pro-p group of dimension d > 0. Then d ≥3. Documenta Mathematica 23 (2018) 1263–1290 1271 Unramified 2-Extensions of a Number Field Proof. This is well-known. Let G be a uniform pro-p group of dimension d. If dim G = 1, then G ≃Zp (G is pro-p free) and, if dim G = 2, then by Theorem 2.9, H2(G) ≃Fp and then, by taking homology long exact sequence of the exact sequence 1 −→Zp −→Zp −→Fp −→1, one gets · · · −→H2(G, Fp) −→Gab −→Gab −→Gab/p −→1 and then dp(Gab[p]) ≤1, and Gab ։ Zp. Hence, if we assume moreover that G is FAb, then dim G should be at least 3. and then dp(Gab[p]) ≤1, and Gab ։ Zp. Hence, if we assume moreover that G is FAb, then dim G should be at least 3. 2.2.3 Filtrations Let us start with a ﬁnitely generated pro-p group G with its p-central descend- ing series (Gi)i. Usually one has [Gi, Gj] ⊂Gi+j, but when p = 2 and when G is uniform, let us give a reﬁnement of this inclusion. To do this, we need the following result (see [4, Chapter 3, Theorem 3.6]): Proposition 2.13. When G is uniform, one has for i, k ≥1, Gi+k = Gpk i . Proposition 2.13. When G is uniform, one has for i, k ≥1, Gi+k = Gpk i . a bilinear form B is at ubspace of V of dim V. It is then easy to s −2i. al as we can achieve it The bilinear form H s called alternating if nating. 3.1 Bilinear forms over F2 Let B be a bilinear form over an F2-vector space V of ﬁnite dimension. Denote by n the dimension of V and by rk(B) the rank of B. Definition 3.1. Given a bilinear form B, one deﬁnes the index ν(B) of B by ν(B) := max W⊂V{dim W, B(W, W) = 0}. One has: Proposition 3.2. The index ν(B) of a bilinear form B is at most n −1 2rk(B). Proposition 2.13. When G is uniform, one has for i, k ≥1, Gi+k = Gpk i . Here as usual, for a pro-p group G, we note by Gpk the subgroup of G generated by the pk-powers. One can now deduce the following: Corollary 2.14. Let G be an uniform pro-2 group. Then, for all i, j ≥1, one has: [Gi, Gj] ⊂Gi+j+1. Proof. We prove it by induction. When i = j = 1, [G, G] ⊂G4 = G3, and the statement holds. Suppose now that [Gi, Gj] ⊂Gi+j+1, and let us look at [Gi+1, Gj]. Let us apply Hall’s formula: [xy, z] = y−1[x, z]y[x, z]−1[x, z][y, z] = [y, [x, z]−1][x, z][y, z], to note that [xy, z] ∈Gi+j+2 when [x, z] and [y, z] are in Gi+j+2. Moreover, for x ∈Gi and z ∈Gj, one has [x, [x, z]−1] ∈G2i+j+1 and [x, z]2 ∈Gi+j+2, by hypothesis. As [x2, z] = [x, [x, z]−1][x, z]2, one obtains [x2, z] ∈Gi+j+2 when x ∈Gi and z ∈Gj. One conludes thanks to the fact that Gi+1 = G2 i . In [14], the authors show that when G is uniform of dimension d then Gi/Gpi i [Gi, Gi] ≃(Z/piZ)d. In fact, for p = 2, one can say a little bit more: Proposition 2.15. Let G be an uniform pro-2 group of dimension d. Then for i ≥1, one has Gi/G2i+1 i [Gi, Gi] ≃(Z/2i+1)d. Gi/G2i+1 i [Gi, Gi] ≃(Z/2i+1)d. When i = 1, it is an alternative proof to Corollary 2.10. Proof. Let S = {g1, · · · , gd} be a set of generators of G. Then the elements g2i−1 j , j = 1, · · · , d, generate the uniform group Gi. Let g ∈S and suppose that (g2i−1)2i ∈[Gi, Gi]. As [Gi, Gi] ⊂G2i+1 by Corollary 2.14, one gets Documenta Mathematica 23 (2018) 1263–1290 1272 Christian Maire g22i−1 ∈G2i+1. Now let us recall that z 7→z22i−1 induces an isomorphism ϕi between G/G2 and G2i/G2i+1 (see [4, Chapter 4, §4.1]). But as ϕi(g) = 0, one has g = 0 which is a contraction. Hence, for j = 1, · · · , d, the element g2i−1 j is of order at least 2i+1 in Gi/[Gi, Gi]. Chapter 4, §4.1]). But e, for j = 1, · · · , d, th ementary maximal tor space V of ﬁnite d the rank of B. m B, one deﬁnes the in W, B(W, W) = 0}. Proposition 3.2. The index ν(B) of a bilinear form B is at most n −1 2rk(B). Proof. Let W be a totally isotropic subspace of V of dimension i. Let us complete a basis of W to a basis B of V. It is then easy to see that the Gram matrix of B in B is of rank at most 2n −2i. This bound is in a certain sense optimal as we can achieve it in the symmetric case. Definition 3.3. (i) Given a ∈F2. The bilinear form H(a) with matrix a 1 1 0 is called a metabolic plane. (ii) A symmetric bilinear form (V, B) is called alternating if B(x, x) = 0 for all x ∈V . Otherwise B is called nonalternating. (ii) A symmetric bilinear form (V, B) is called alternating if B(x, x) = 0 for all x ∈V . Otherwise B is called nonalternating. Recall the Witt decomposition Theorem for symmetric bilinear forms over F2 (see for example [15, Chapter I, §2, Remark 5 of Theorem 1]). Theorem 3.4. Let (V, B) be a symmetric bilinear form of dimension n over F2. Denote by r the rank of B. Write r = 2r0 +δ, with δ = 0 or 1, and r0 ∈N. (i) If B is nonalternating, then (V, B) is isometric to r0 z }\| { H(1)⊥· · · ⊥H(1) ⊥ δ z}\|{ ⟨1⟩⊥ n−r z }\| { ⟨0⟩⊥· · · ⊥⟨0⟩≃ r z }\| { ⟨1⟩⊥· · · ⊥⟨1⟩⊥ n−r z }\| { ⟨0⟩⊥· · · ⊥⟨0⟩; (ii) If B is alternating, then B is isometric to r0 z }\| { H(0)⊥· · · ⊥H(0) ⊥ n−r z }\| { ⟨0⟩⊥· · · ⊥⟨0⟩. Documenta Mathematica 23 (2018) 1263–1290 1273 Unramified 2-Extensions of a Number Field 1273 Unramified 2-Extensions of a Number Field Moreover, ν(B) = n −r + r0 = n −r0 −δ. Moreover, ν(B) = n −r + r0 = n −r0 −δ. Moreover, ν(B) = n −r + r0 = n −r0 −δ. When (V, B) is not necessary symmetric, let us introduce the symmetrization Bsym of B by Bsym(x, y) = B(x, y) + B(y, x), ∀x, y ∈V. One has: Corollary 3.5. Let (V, B) be a bilinear form of dimension n over F2. Then ν(B) ≥n −⌊1 2rk(Bsym)⌋−⌊1 2rk(B)⌋. In particular, ν(B) ≥min{n −3 2rk(B), 1 2(n −rk(B)}. Proof. It is easy. Proposition 3.2. The index ν(B) of a bilinear form B is at most n −1 2rk(B). Let us start with a maximal totally isotropic subspace W of (V, Bsym). Then B\|W is symmetric: indeed, for any two x, y ∈W, we get 0 = Bsym(x, y) = B(x, y) + B(y, x), and then B(x, y) = B(y, x) (recall that V is deﬁned over F2). Hence by Theorem 3.4, B\|W has a totally isotropic subspace of dimension ν(B\|W) = dim W−⌊1 2rk(B\|W)⌋. As dim W = n−⌊1 2rk(Bsym)⌋(by Theorem 3.4), one obtains the ﬁrst assertion. For the second one, it is enough to note that rk(Bsym) ≤max{2rk(B), n}. 3.2 Bilinear form and Kummer radical Let us start with a number ﬁeld K. Denote by n the 2-rank of Gur K (2), in other words, n = d2ClK. Let V = ⟨a1, · · · , an⟩(K×)2 ∈K×/(K×)2 be the Kummer radical of the 2- elementary abelian maximal unramiﬁed extension Kur,2/K. Then V is an F2- vector space of dimension n. For a ∈V , denote Ka := K(√a), and a(a) := p (a) ∈OK (see section 2.1.2). We can now introduce the bilinear form BK that plays a central role in our work. Definition 3.6. For a, b ∈V , put: BK(a, b) = Ka/K a(b) · √a , √a ∈F2, Documenta Mathematica 23 (2018) 1263–1290 where here we use the additive notation. where here we use the additive notation. Of course, we have: Of course, we have: Lemma 3.7. The application BK : V × V →F2 is a bilinear form on V . Lemma 3.7. The application BK : V × V →F2 is a bilinear form on V . Proof. The linearity on the right comes from the linearity of the Artin symbol. Let us show that BK is linear on the left. Take a1 ̸= a2 ∈V, and let p be a prime ideal of OK. Let us remark the following: Documenta Mathematica 23 (2018) 1263–1290 1274 Christian Maire • If p splits in Ka1/K and in Ka2/K, then p splits in Ka1a2/K; • If p splits in Ka1/K and in Ka2/K, then p splits in Ka1a2/K; • If p is inert in Ka1/K but splits in Ka2/K, then p is inert in Ka1a2/K. Hence for each case one has Ka1a2/K p = Ka1/K p + Ka2/K p , and we conclude by using again the linearity of the Artin symbol. • If p is inert in Ka1/K but splits in Ka2/K, then p is inert in Ka1a2/K. Hence for each case one has Ka1a2/K p = Ka1/K p + Ka2/K p , and we l d b h l f h A b l The bilinear form BK is not necessarily symmetric, but we will give later some situations where BK is symmetric. The bilinear form BK is not necessarily symmetric, but we will give later some situations where BK is symmetric. Remark 3.8. Assume K is totally imaginary. If we denote by xi a generator of H1(Gal(K(√ai)/K)), then the Gram matrix of the bilinear form BK in the basis {a1(K×)2, · · · , an(K×)2} is exactly the matrix (xi ∪xi ∪xj)i,j of the cup- products in H3 et(SpecOK). See Proposition 2.4 and Remark 2.5. Hence the bilinear form BK coincides with the bilinear form Bet K on H1 et(SpecOK) deﬁned by Bet K(x, y) = x ∪x ∪y ∈H3 et(SpecOK). Recall that the right-radical (resp. left-radical) Radr (resp. Radl) of a bilinear form B on V is the subspace deﬁned by: Radr := {x ∈V, B(V, x) = 0} (resp. Radl := {x ∈V, B(x, V ) = 0}. Of course one always has dim B = rk(B) + dim(Radr) = rk(B) + dim(Radl). Documenta Mathematica 23 (2018) 1263–1290 Definition 3.12. The index ν(K) of K is the index of the bilinear form BK. Of course, if the form BK is non-degenerate, one has: ν(K) ≤1 2d2ClK. Thus one says that ClK is non-degenerate if the form BK is non-degenerate. To ﬁnish this part, let us give the relation with the 4-rank RK,4 of the class group ClK deﬁned as follows: RK,4 := dimF2 ClK[4]/ClK[2], where ClK[m] = {c ∈ClK, cm = 1}. One has immediately: Proposition 3.13. Let K be a totally imaginary number ﬁeld. Then RK,4 ≤ d2ClK −rk(BK). Proof. Indeed, the subspace of characters of H1(Gur K (2)) corresponding to the unramiﬁed degree 4 cyclic extensions of K is a subspace of the left-radical of BK (see Proposition 3.11), and then RK,4 ≤dim Radl. To conclude, use the fact that n = d2ClK = dim Radl + rk(BK). the Remark 3.14. Of course, Proposition 3.13 can be made more precise in many cases. Typically, in the quadratic case thanks to the Rédei matrix (see Section 4.3.4). See also the generalization of Yue [36]. where here we use the additive notation. And, remark moreover that the restriction of B at Radr (resp. Radl) produces a totally isotropic subspace of V. Let us come back to the bilinear form BK on the Kummer radical of Kur,2/K, and let us give now three types of totally isotropic subspaces W that may appear. Proposition 3.9. Let W := ⟨ε1, · · · , εr⟩(K×)2 ⊂V be an F2-subspace of di- mension r, generated by some units εi ∈O× K. Then W ⊂Radr, and thus (V, BK) contains W as a totally isotropic subspace of dimension r. Proof. Indeed, here a(εi) = OK for i = 1, · · · , r. Proof. Indeed, here a(εi) = OK for i = 1, · · · , r. Proposition 3.10. Let K = k( √ b) be a quadratic extension. Suppose that there exist a1, · · · , ar ∈k such that the extensions k(√ai)/k are independent and unramiﬁed everywhere. Suppose moreover that b /∈⟨a1, · · · , ar⟩(k×)2. Then W := ⟨a1, · · · , ar⟩(K×)2 is a totally isotropic subspace of dimension r. Proof. Let p ⊂Ok be a prime ideal of Ok. It is suﬃcient to prove that Kai/K p is trivial. Let us study all the possibilities. p • If p is inert in K/k, then as K(√ai)/K is unramiﬁed at p, p must split in K(√ai)/K and then Kai/K p is trivial. • If p = P2 is ramiﬁed in K/k, then Kai/K p = Kai/K P 2 is trivial. Documenta Mathematica 23 (2018) 1263–1290 1275 Unramified 2-Extensions of a Number Field • If p = P1P2 splits, then obviously Kai/K P1 = Kai/K P2 , and then Kai/K p is trivial. Proposition 3.11. Suppose K is totally imaginary. Let W := ⟨a1, · · · , ar⟩(K×)2 ⊂V be a subspace of V such that each quadratic ex- tension K(√ai)/K, i = 1, · · · , r, embeds in an unramiﬁed degree 4 cyclic extension. Then W ⊂Radl. Proof. Denote by xi a generator of H1(Gal(Kai/K)). By Proposition 2.2, we get xi ∪xi = 0 ∈H2 et(Gur K (2)), and then BK(ai, V ) = 0 by Remark 3.8. It is then natural to deﬁne the index of K as follows: It is then natural to deﬁne the index of K as follows: Definition 3.12. where here we use the additive notation. The index ν(K) of K is the index of the bilinear form BK. Documenta Mathematica 23 (2018) 1263–1290 4.1 A first observation As the extension L/K1 is ramiﬁed at the odd prime p3, the fundamental unit of K1 is also the fundamental unit of O× L . As NK1/Qε = +1, one concludes that −1 is not an universel norm in Kur(2)/K. 4.1 A first observation For basic facts concerning the cohomological dimension of a pro-p group we refer for example to [24, Chapter III]. For basic facts concerning the cohomological dimension of a pro-p group we refer for example to [24, Chapter III]. Before developing a general setting, let us start with the following observation. Let O× Kur(p) be the group generated by the units of the ring of integers of all subextensions F/K of Kur(p)/K. Let us recall the following result due to Wingberg [35, Theorem 1.1] (see also [24, Chapter VIII, §8, Corollary 8.8.3]): Before developing a general setting, let us start with the following observation. Let O× Kur(p) be the group generated by the units of the ring of integers of all subextensions F/K of Kur(p)/K. Let us recall the following result due to Wingberg [35, Theorem 1.1] (see also [24, Chapter VIII, §8, Corollary 8.8.3]): Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 1276 Christian Maire Theorem 4.1 (Wingberg). There are canonical isomorphims: Wingberg). There are canonical isomorphims: ˆHi(Gur K (p), O× Kur(p)) ≃ˆH3−i(Gur K (p), Z)∨, for all i ∈Z. Hence, the exact sequence 0 −→Z/pZ−→Q/Z p −→Q/Z −→0 allows us to obtain: Hence, the exact sequence 0 −→Z/pZ−→Q/Z p −→Q/Z −→0 allows us to obtain: H3(Gur K (p), Fp) −→ˆH0(GK(p), O× Kur(p)) p −→ˆH0(GK(p), O× Kur(p)) By using the isomorphism ˆH0(GK(p), O× Kur(p)) ≃lim ← F O× K/NF/K(O× F ), where F runs through the ﬁnite extensions of K in Kur(p), we then have H3(GK(p), Fp) ։ lim ←− F O× K/NF/K(O× F ) [p]. (2) (2) Take now p = 2. Thanks to (2), we note that if −1 is not a universal norm (of a unit) in Kur(2)/K, then H3(G, F2) ̸= {0}, showing that cd(Gur K (2)) ̸= 2. This condition about −1 is sometimes not so diﬃcult to test, indeed: K This condition about −1 is sometimes not so diﬃcult to test, indeed: Proposition 4.2. Let K be an imaginary quadratic ﬁeld such that the discrim- inant discK of K is divisible by at least three odd primes pi, with pi ≡3(mod 4), i = 1, 2, 3. Then cd(Gur K (2)) ̸= 2. Proof. Put L = K(√p1p2) and K1 = Q(√p1p2). Then the biquadratic ﬁeld L is an unramiﬁed extension of K. 4.2 General statement Let W be the Kummer radical of H1(G)∨; here W is a subspace of the Kummer radical V of Kur,2/K. As d > ν(K), the space W is not totally isotropic. Then, one can ﬁnd x, y ∈H1(G) ⊂H1(XK) such that x ∪x ∪y ∈H3 et(XK) is not zero (by Proposition 2.4). See also Remark 3.8. And thanks to the stategy developed just before, we are done for the ﬁrst part ot the theorem. For the second part, one has just to note that in this case ν(K) ≤d2ClK −1 2rk(BK) by Proposition 3.2. Corollary 4.5. If ClK is non-degenerate, then Gur K (2) has no quotient G of cohomological dimension 2 and of 2-rank d2G > 1 2d2ClK. roof. In this case, rk(BK) = d2ClK Proof. In this case, rk(BK) = d2ClK 4.2 General statement The strategy here is more or less the one used in [3] to disprove the existence of unramiﬁed embeddings. Here we apply their idea to disprove the apparence of quotients of Gur K (2) of cohomological dimension 2 in a more systematic way, by using the bilinear form BK. From now on we assume that K is totally imaginary (and that p = 2). 1 1 From now on we assume that K is totally imaginary (and that p 2). Suppose given G a quotient of Gur K (2). Then one has H1(G) ֒→H1(Gur K (2)). Now take x, y ∈H1(Gur K (2)) coming from H1(G). Then, the cup-product x∪x∪y ∈H3(Gur K (2)) comes from H3(G) by the inﬂation map. Now, one may use the computation of Carlson-Schlank : if x ∪x ∪y is non-zero in H3 et(XK), then H3(G) ̸= 0, and then G is not of cohomological dimension 2. Remark 4.3. If we want x ∪x ∪y ̸= 0, then we need the cup-product x ∪x non-trivial in H2 et(XK), which is equivalent to the condition that Kx/K can not be embedded in an unramiﬁed degree 4 cyclic extension of K. Let us recall Theorem 2 (we use the notations of Section 3): Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 1277 Unramified 2-Extensions of a Number Field Theorem 4.4. Let K/Q be a totally imaginary number ﬁeld. Then Gur K (2) has no quotient G of cohomological dimension 2 and of 2-rank d2G > ν(K). In particular, Gur K (2) has no quotient G of cohomological dimension 2 and of 2-rank d2G > d2ClK −1 2rk(BK). Proof. Let G be a non-trivial uniform quotient of Gur K (2) of dimension d > 0. Let W be the Kummer radical of H1(G)∨; here W is a subspace of the Kummer radical V of Kur,2/K. As d > ν(K), the space W is not totally isotropic. Then, one can ﬁnd x, y ∈H1(G) ⊂H1(XK) such that x ∪x ∪y ∈H3 et(XK) is not zero (by Proposition 2.4). See also Remark 3.8. And thanks to the stategy developed just before, we are done for the ﬁrst part ot the theorem. For the second part, one has just to note that in this case ν(K) ≤d2ClK −1 2rk(BK) by Proposition 3.2. Proof. Let G be a non-trivial uniform quotient of Gur K (2) of dimension d > 0. 4.3.1 A first density estimate Let us give now a density estimate for imaginary quadratic ﬁelds. Recall that we denote by F−the set of imaginary quadratic ﬁelds and by F− X := {K ∈ F−, \|discK\| ≤X}. Put also D− X = {K ∈F− X, Gur K (2) has no quotient G s.t. d2G = d2Gur K (2) & cd(G) = 2}. Theorem 4.6 (Theorem 1). One has: 0 ≤1 −#D− X #F− X ≤C log log X √log X , where C is an absolute constant. where C is an absolute constant. Proof. The proof is based on the two following things: (i) on an analytic esti- mate; (ii) on our strategy and on a computation of [3]. Let us start the analytic tools. For X ≥2, denote by T − X = {K ∈F− X, ∃odd primes p ̸= q, pq\|discK, p∗ q = −1}. Remark at this point that {K ∈F− X, ∃odd primes p ̸= q, p ≡q ≡3 (mod 4), pq\|discK} ⊂T − X ; {K ∈F− X, ∃odd primes p ̸= q, p ≡q ≡3 (mod 4), pq\|discK} ⊂T − X ; indeed, by Legendre formula, p∗ q q∗ p = −1. Denote by E− X the comple- ment of T − X in F− X. ment of T − X in F− X. Documenta Mathematica 23 (2018) 1263–1290 1278 Christian Maire Proposition 4.7. The set E− X is of zero density. More precisely, one has Proposition 4.7. The set E− X is of zero density. More precisely, one has #E− X = O X log log X √log X . Proof. Note by Ei,X the set of square-free integers n ≤X, having exactly i prime factors ≡3 (mod 4), and put EX = E0,X∪E1,X. Clearly, #E− X = O(#EX). We will use the following: Lemma 4.8. Uniformly for X ≥2, one has Lemma 4.8. Uniformly for X ≥2, one has #E0,X = O X √log X . Proof. Indeed, every integer n ∈E0,X is a sum of two squares. A famous result of Landau (see for example [21, Chapter 7, Theorem 7.28]) states that the number of integers n ≤X which are sum of two squares is asymptotic to X C′ X √log X , for some absolute positive C′. C′ X √log X , for some absolute positive C′. We want to prove the bound We want to prove the bound E1,X = O X log log X √log X (3) (3) uniformly for X ≥2, as a consequence of Lemma 4.8. Since we are searching an upper bound, we may suppose that X = 2κ, where κ > 1 is an integer. We start from the decomposion formula #E1,X = X p≡3(mod 4) p≤X/2 #E0,X/p + O(π(X)). where C is an absolute constant. Put Y = 2t with t = 1, 2, · · · , κ −1 and split the above formula as Put Y = 2t with t = 1, 2, · · · , κ −1 and split the above formula as Put Y = 2t with t = 1, 2, · · · , κ −1 and split the above formula as #E1,X = X t X Y/2<p≤Y #E0,X/p + O X log X . (4) (4) We deduce from Lemma 4.8 that, uniformly for Y/2 < p ≤Y and Y = 2t, and 0 ≤t ≤κ −1, we have the bound #E0,X/p = O X Y √κ −t , and by Tchebychev’s bound we deduce and by Tchebychev’s bound we deduce X Y/2<p≤Y #E0,X/p = O X t√κ −t . Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 Unramified 2-Extensions of a Number Field 1279 Inserting this into (4), we obtain #E1,X = O X X 1≤t≤κ−1 1 t√κ −t . To evaluate the sum over t, we split it according to 1 ≤t < κ/ log κ and κ/ log κ ≤t ≤κ −1 leading to X 1≤t≤κ−1 1 t√κ −t = O 1 √κ X 1≤t≤κ 1 t + log κ κ X 1≤u≤κ 1 √u , which gives (3), since κ = O(log X). This completes the proof of Proposi- tion 4.7. which gives (3), since κ = O(log X). This completes the proof of Proposi- tion 4.7. Now let us give the tool from étale cohomology. Now let us give the tool from étale cohomology. Lemma 4.9. Let K/Q be an imaginary quadratic ﬁeld. Suppose that there exist two distinct odd prime numbers p and q such that pq\|discK and p∗ q = −1. Then there exist x, y ∈H1(Gur K (2)) such that x ∪x ∪y ̸= 0 ∈H3(Gur K (2)). Proof. Take Kx = K(√p∗) and Ky = K(√q∗). Then, by Proposition 2.4, the cup-product x ∪x ∪y ∈H3 et(X) is not trivial, and then non-trivial in H3(Gur K (2)). Lemma 4.9. Let K/Q be an imaginary quadratic ﬁeld. Suppose that there exist two distinct odd prime numbers p and q such that pq\|discK and p∗ q = −1. Then there exist x, y ∈H1(Gur K (2)) such that x ∪x ∪y ̸= 0 ∈H3(Gur K (2)). where C is an absolute constant. We now ﬁnish the proof of Theorem 4.6. Take K ∈T − K , and let us consider a quotient Gur K (2) ։ G such that H1(Gur K (2)) ≃H1(G). But by Lemma 4.9, there exists x, y ∈H1(GK(2)) such that x ∪x ∪y ̸= 0 ∈H3 et(XK), and then not trivial in H3(Gur K (2)) and ﬁnally not trivial in H3(G). Hence, T − X ⊂D− X, and #F− X −#D− X ≤E− X; to conclude, let us recall the well-known estimate: 3 #F− X = 3 π2 X + O( √ X). #F− X = 3 π2 X + O( √ X). Now, we would like to extend this density estimate. Now, we would like to extend this density estimate. Documenta Mathematica 23 (2018) 1263–1290 4.3.2 The context Let us consider an imaginary quadratic ﬁeld K = Q( √ D), D ∈Z<0 square- free. Let p1, · · · , pk+1 be the odd prime numbers dividing D (we assume D ̸= −1, −2). Let us write the discriminant discK of K as: discK = p∗ 0 · p∗ 1 · · · p∗ k+1, where p∗ 0 ∈{1, −4, ±8}. Denote by n the 2-rank of ClK: −if 2 is unramiﬁed in K/Q, i.e. p∗ 0 = 1, then n = k and V =< p∗ 1, · · · , p∗ k > (K×)2 ⊂K× is the Kummer radical of Kur,2/K; −if 2 is unramiﬁed in K/Q, i.e. p∗ 0 = 1, then n = k and V =< p∗ 1, · · · , p∗ k > (K×)2 ⊂K× is the Kummer radical of Kur,2/K; −is 2 is ramiﬁed in K/Q, i.e. p∗ 0 = −4 or ±8, then n = k + 1 and V =< p∗ 1, · · · , p∗ k+1 > (K×)2 ⊂K× is the Kummer radical of Kur,2/K. −is 2 is ramiﬁed in K/Q, i.e. p∗ 0 = −4 or ±8, then n = k + 1 and V =< p∗ 1, · · · , p∗ k+1 > (K×)2 ⊂K× is the Kummer radical of Kur,2/K. −is 2 is ramiﬁed in K/Q, i.e. p∗ 0 = −4 or ±8, then n = k + 1 and V =< p∗ 1, · · · , p∗ k+1 > (K×)2 ⊂K× is the Kummer radical of Kur,2/K. Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 1280 Christian Maire We denote by F = {p∗ 1, · · · , p∗ n} the F2-basis of V , where here n = k or k + 1. Lemma 4.10. (i) For p∗̸= q∗∈F, one has: BK(p∗, q∗) = 0 if and only if, p∗ q = 1. We denote by F = {p∗ 1, · · · , p∗ n} the F2-basis of V , where here n = k or k + 1. Lemma 4.10. (i) For p∗̸= q∗∈F, one has: BK(p∗, q∗) = 0 if and only if, p∗ q = 1. q (ii) For p\|D, put Dp := D/p∗. Then for p∗ ∈ F, one has: BK(p∗, p∗) := Dp p . q (ii) For p\|D, put Dp := D/p∗. 4.3.2 The context Then for p∗ ∈ F, one has: BK(p∗, p∗) := Dp p . Proof. Obvious. Proof. Obvious. Proof. Obvious. Hence the matrix of the bilinear form BK in the basis F is a square n × n Rédei-matrix type MK = (mi,j)i,j, where mi,j =        p∗ i pj if i ̸= j, Dpi pi if i = j. mi,j =        p∗ i pj if i ̸= j, Dpi pi if i = j. Here as usual, one uses the additive notation (the 1’s are replaced by 0’s and the −1’s by 1’s). Here as usual, one uses the additive notation (the 1’s are replaced by 0’s and the −1’s by 1’s). Example 4.11. Take K = Q(√−4 · 3 · 5 · 7 · 13). This quadratic ﬁeld has a root discriminant \|discK\|1/2 ≈73.89, and the 2-rank of GK(2) is actually 4 but we don’t know if Gur K (2) is ﬁnite or not; see the recent works of Boston and Wang [2]. Take F = {−3, −5, −7, −13}. Then the Gram matrix of BK in F is: MK =     1 1 1 0 1 1 1 1 0 1 1 1 0 1 1 0    . Hence rk(BK) = 3 and ν(K) ≤4−3 2 = 2.5. By Theorem 5.1, one concludes that Gur K (2) has no quotient G of cohomological dimension 2 and 2-rank d2G ≥3. By Corollay 3.5, remark that here one has ν(K) = 2. Hence rk(BK) = 3 and ν(K) ≤4−3 2 = 2.5. By Theorem 5.1, one concludes that Gur K (2) has no quotient G of cohomological dimension 2 and 2-rank d2G ≥3. By Corollay 3.5, remark that here one has ν(K) = 2. Unramified 2-Extensions of a Number Field • p1 ≡· · · pk ≡1(mod 4) and pk+1 ≡3(mod 4); • for 1 ≤i < j ≤k, pi pj = 1; • for i = 1, · · · , k, pi pk+1 = −1 • p1 ≡· · · pk ≡1(mod 4) and pk+1 ≡3(mod 4); • p1 ≡· · · pk ≡1(mod 4) and pk+1 ≡3(mod 4); • for 1 ≤i < j ≤k, pi pj = 1; • for i = 1, · · · , k, pi pk+1 = −1 • for 1 ≤i < j ≤k, pi pj = 1; Put K = Q(√−p1 · · · pk+1). In this case the matrix of the bilinear form BK in the basis (pi)1≤k is the identity matrix of dimension k × k and, ν(K) = ⌊k 2⌋. Hence, Gur K (2) has no quotient G of cohomological dimension 2 and of 2-rank d2G ≥⌊k 2⌋+ 1. xample 4.14. Take 2m + 1 prime numbers p1, · · · , p2m+1, such that Example 4.14. Take 2m + 1 prime numbers p1, · · · , p2m+1, such that • p1 ≡· · · p2m ≡1(mod 4) and p2m+1 ≡3(mod 4); • p1 ≡· · · p2m ≡1(mod 4) and p2m+1 ≡3(mod 4); • p1 p2 = p3 p4 = · · · = p2m−1 p2m = −1; • for the other indices 1 ≤i < j ≤2m, pi pj = 1; • for the other indices 1 ≤i < j ≤2m, pi pj = 1; • for i = 1, · · · , 2m, pi p2m+1 = −1 Put K = Q(√−p1 · · · p2m+1). In this case the bilinear form BK is non- m z }\| { Put K = Q(√−p1 · · · p2m+1). In this case the bilinear form BK is non- m z }\| { degenerate and alternating, then isometric to z { H(0)⊥· · · ⊥H(0). Hence, ν(K) = m, and Gur K (2) has no quotient of cohomological dimension 2 and of 2-rank at least m + 1. Documenta Mathematica 23 (2018) 1263–1290 4.3.3 Symmetric bilinear forms. Examples Let us conserve the context of the previous section 4.3.2. Then, thanks to the quadratic reciprocity law, one gets: Proposition 4.12. The bilinear form BK : V × V →F2 is symmetric, if and only if, there is at most one prime p ≡3( mod 4) dividing D. Proposition 4.12. The bilinear form BK : V × V →F2 is symmetric, if and only if, there is at most one prime p ≡3( mod 4) dividing D. Proof. Obvious. Let us give some examples. Let us give some examples. Example 4.13. Take k + 1 prime numbers p1, · · · , pk+1, such that Example 4.13. Take k + 1 prime numbers p1, · · · , pk+1, such that Documenta Mathematica 23 (2018) 1263–1290 1281 Unramified 2-Extensions of a Number Field 4.3.4 Relation with the 4-rank of the class group The study of the 4-rank of the class group of quadratic number ﬁelds started with the work of Rédei [26] (see also [27]). Since, then many authors have contribued to its extensions, generalizations and applications. Let us cite an article of Lemmermeyer [20] where one can ﬁnd a large literature about the question. See also a paper of Stevenhagen [32], and the work of Gerth [10] and Fouvry-Klüners [8] concerning the density question. Let us conserve the context and the notations of the section 4.3.2: here K = Q( √ D) is an imaginary quadratic ﬁeld of discrimant discK, D ∈Z<0 square- free. Denote by {q1, · · · qn+1} the set of prime numbers that ramify in K/Q; d2ClK = n. Here we can take qi = pi for 1 ≤i ≤n, and qn = pk+1 or qn = 2 following the ramiﬁcation at 2. Then, consider the Rédei matrix M′ K = (mi,j)i,j of size (n + 1) × (n + 1) with coeﬃcients in F2, where mi,j =        q∗ i qj if i ̸= j, Dqi qi if i = j. 1282 Christian Maire It is not diﬃcult to see that the sum of the rows is zero, hence the rank of M′ K is smaller than n. Theorem 4.15 (Rédei). Let K be an imaginary quadratic number ﬁeld. Then RK,4 = d2ClK −rk(M′ K). Remark 4.16. The strategy of Rédei is to construct for every couple (D1, D2) "of second kind", a degree 4 cyclic unramiﬁed extension of K. Here to be of second kind means that discK = D1D2, where Di are fundamental discrimi- nants such that D1 p2 = D2 p1 = 1, for every prime pi\|Di, i = 1, 2. And clearly, this condition corresponds exactly to the existence of orthogonal sub- spaces Wi of the Kummer radical V, i = 1, 2, generated by the p∗ i , for all pi\|Di: BK(W1, W2) = BK(W2, W1) = {0}. Such orthogonal subspaces allow us to con- struct totally isotropic subspaces. And then, the larger the 4-rank of ClK, the larger ν(K) must be (as noted by Proposition 3.2 and 3.13). Consider now the matrix M′′ K obtained from M′ K after deleting the last row. Hence, rk(BK) + 1 ≥rk(M′ K) = rk(M′′ K) ≥rk(BK). Remark that in example 4.11, rk(BK) = 3 and rk(M′ K) = 4. But sometimes one has rk(M′ K) = rk(Bk), as for example: Remark that in example 4.11, rk(BK) = 3 and rk(M′ K) = 4. But sometimes one has rk(M′ K) = rk(Bk), as for example: (A): when: p0 = 1 (the set of primes pi ≡3(mod 4) is odd); (A): when: p0 = 1 (the set of primes pi ≡3(mod 4) is odd); (B): or, when BK is non-degenerate. (B): or, when BK is non-degenerate. For situation (A), it suﬃces to note that the sum of the columns is zero (thanks to the properties of the Legendre symbol). 4.3.4 Relation with the 4-rank of the class group Remark here that MK (which is the Gram matrix of BK) is a submatrix of the Rédei matrix M′′ K: Rédei matrix M′′ K: M′′ K =    MK ∗ ... ∗    Hence, Unramified 2-Extensions of a Number Field Theorem 4.17 (Gerth [10]). The limits lim X→∞ \|An,r,X\| \|An,X\| and lim X→∞ \|Fn,r,X\| \|Fn,X\| exist and are equal. Denote by dn,r this quantity. Then dn,r can be estimated and, d∞,r := lim n→∞dn,r = 2−r2 Q∞ k=1(1 −2−k) Qr k=1(1 −2−k) . Recall also the following quantities introduced at the beginning of our work: Recall also the following quantities introduced at the beginning of our work: D(d) n,X := {K ∈Fn,X, Gur K (2) has no quotient G s.t. cd(G) = 2 & d2(G) ≥d}, and consider the limit: and consider the limit: (d) D(d) n := lim inf X→+∞ #D(d) n,X #Fn,X . After combining all our observations, we obtain (see also Corollary i): 8. For n+1 2 ≤d ≤n, one has D(d) n ≥dn,0 + dn,1 + · · · + dn,2d−n−1. Corollary 4.18. For n+1 2 ≤d ≤n, one has Corollary 4.18. For n+1 2 ≤d ≤n, one has D(d) n ≥dn,0 + dn,1 + · · · + dn,2d−n−1. D(d) n ≥dn,0 + dn,1 + · · · + dn,2d−n−1. In particular: (i) D(3) 5 ≥.33129, D(4) 5 ≥.99062, D(5) 5 ≥.99999; (ii) D(4) 6 ≥.86718, D(5) 6 ≥.99925, D(6) 6 ≥1 −5.2 · 10−8; Moreover, for large n, D(2+n/2) n ≥.99995. Proof. As noted by Gerth in [10], the dominating set in the density compu- tation is the set An,X of imaginary quadratric number ﬁelds K = Q( √ D) satisfying (A). But for K in An,X, one has rk(BK) = rk(MK) = n −RK,4. Hence for K ∈An,X,r, by Proposition 3.2 ν(K) ≤n −1 2 n −RK,4 = 1 2 n + RK,4 . Hence Gur K (2) has no quotient G of cohomological dimension 2 and 2-rank d when RK,4 < 2d −n. In particular, when 2d −n ≥1, one has D(d) n ≥dn,0 + dn,1 + · · · + dn,2d−n−1. 4.3.5 From now on we follow the work of Gerth [10]. Recall that we denote by F− the set of imaginary quadratic number ﬁelds, and for 0 ≤r ≤n and X ≥0, F− X = K ∈F−, \|discK\| ≤X , Fn,X = K ∈F− X, d2ClK = n , Fn,r,X = K ∈Fn,X, RK,4 = r . Denote also F− X = K ∈F−, \|discK\| ≤X , Denote also AX = K ∈F− X, satisfying (A) , AX = K ∈F− X, satisfying (A) , An,X = K ∈AX, d2ClK = n , An,r,X = K ∈An,X, RK,4 = r . One has the following density theorem due to Gerth: An,X = K ∈AX, d2ClK = n , An,r,X = K ∈An,X, RK,4 = r . An,X = K ∈AX, d2ClK = n , An,r,X = K ∈An,X, RK,4 = r One has the following density theorem due to Gerth: One has the following density theorem due to Gerth: One has the following density theorem due to Gerth: One has the following density theorem due to Gerth: Documenta Mathematica 23 (2018) 1263–1290 Unramified 2-Extensions of a Number Field 1283 Corollary 4.19. For i ≥1, one has: Corollary 4.19. For i ≥1, one has: D[i] ≥d∞,0 + d∞,1 + · · · + d∞,2i−2. In particular, D[1] ≥.28878, D[2] ≥.99471, and D[3] ≥1 −9.7 · 10−8. Proof. By Fouvry-Klüners [8], the density of imaginary quadratic ﬁelds for which RK,4 = r, is equal to d∞,r. Recall that for K ∈F−, one has rk(BK) ≥ rk(M′ K) −1. Then thanks to Proposition 3.2 and Theorem 4.15, we get ν(K) ≤1 2d2ClK + 1 2 + 1 2RK,4. Putting this fact together with Theorem 4.4, we obtain that Gur K (2) has no quotient G of cohomological dimension 2 and 2-rank d2G > 1 2d2ClK+ 1 2 + 1 2RK,4. Then for i ≥1, the proportion of the ﬁelds K in D[i] is at least the proportion of K ∈F−for which RK,4 < 2i −1, hence at least d∞,0 + d∞,1 + · · · + d∞,2i−2 by [8]. To conclude: D[1] ≥d∞,0 ≈.28878 To conclude: D[1] ≥d∞,0 ≈.28878 D[2] ≥d∞,0 + d∞,1 + d∞,2 ≈.99471 D[3] ≥d∞,0 + d∞,1 + d∞,2 + d∞,3 + d∞,4 ≈1 −9.7 · 10−8. D[2] ≥d∞,0 + d∞,1 + d∞,2 ≈.99471 D[3] ≥d∞,0 + d∞,1 + d∞,2 + d∞,3 + d∞,4 ≈1 −9.7 · 10−8. Unramified 2-Extensions of a Number Field Now one uses the estimates of Gerth in [10], to obtain: (i) D(3) 5 ≥d5,0 ≈.33129, D(4) 5 ≥d5,0 + d5,1 + d5,2 ≈.99062, D(5) 5 ≥d5,0 + d5,1 + d5,2 + d5,3 + d5,4 ≈.99999, (i) D(3) 5 ≥d5,0 ≈.33129, D(4) 5 ≥d5,0 + d5,1 + d5,2 ≈.99062, D(5) 5 ≥d5,0 + d5,1 + d5,2 + d5,3 + d5,4 ≈.99999, (ii) D(4) 6 ≥d6,0 + d6,1 ≈.86718, D(5) 6 ≥d6,0 + d6,1 + d6,2 + d6,3 ≈.99925, D(6) 6 ≥1 −d6,6 ≈1 −5.2 · 10−8, (ii) D(4) 6 ≥d6,0 + d6,1 ≈.86718, D(5) 6 ≥d6,0 + d6,1 + d6,2 + d6,3 ≈.99925, D(6) 6 ≥1 −d6,6 ≈1 −5.2 · 10−8, Documenta Mathematica 23 (2018) 1263–1290 284 Christian Maire 1284 Christian Maire In the spirit of the Cohen-Lenstra heuristics, the work of Gerth has been im- proved by Fouvry-Klüners [7], [8]. This work allows us to give a more general density estimation as announced in the Introduction. Recall D[i] X := {K ∈FX, Gur K (2) has no quotient G s.t. cd(G) = 2 & d2G ≥i+d2ClK 2 } and and D[i] := lim inf X→+∞ #D[i] X #F− X . Our work allows us to obtain (see Corollary ii): Our work allows us to obtain (see Corollary ii): Corollary 4.19. For i ≥1, one has: 5.1 General result The unramiﬁed Fontaine-Mazur at p = 2 has a ﬁrst evidence just by looking at the 2-part of the class group and the matrix of the bilinear form BK. Indeed, one has very easily: Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 1285 Unramified 2-Extensions of a Number Field Theorem 5.1 (Theorem 3). Let K/Q be a number ﬁeld. (i) Suppose that the 4-rank of the class group of K is at most 2. Then Con- jecture 1.1 holds for K (at p = 2). (ii) Suppose K is totally imaginary. Then Gur K (2) has no uniform quotient of dimension d > d2ClK −rk(BK). In particular, Conjecture 1.1 holds for K (and p = 2) when rk(BK) ≥d2ClK −2. Proof. (i) Let G be a non-trivial uniform quotient of Gur K (2) of dimension d. By class ﬁeld theory, the group G is FAb, and then by Corollary 2.12 the dimension of G must verify d ≥3. Then by Proposition 2.15, Gab ։ (Z/4Z)3, which implies RK,4 ≥3. (ii) If G is a uniform quotient of Gur K (2) of dimension d then RK,4 ≥d, but by Proposition 3.13, RK,4 ≤d2ClK −rk(BK). For the second part, recall that as G must be FAb then d ≥3. Proof. (i) Let G be a non-trivial uniform quotient of Gur K (2) of dimension d. By class ﬁeld theory, the group G is FAb, and then by Corollary 2.12 the dimension of G must verify d ≥3. Then by Proposition 2.15, Gab ։ (Z/4Z)3, which implies RK,4 ≥3. Proof. (i) Let G be a non-trivial uniform quotient of Gur K (2) of dimension d. By class ﬁeld theory, the group G is FAb, and then by Corollary 2.12 the dimension of G must verify d ≥3. Then by Proposition 2.15, Gab ։ (Z/4Z)3, which implies RK,4 ≥3. , (ii) If G is a uniform quotient of Gur K (2) of dimension d then RK,4 ≥d, but by Proposition 3.13, RK,4 ≤d2ClK −rk(BK). For the second part, recall that as G must be FAb then d ≥3. , (ii) If G is a uniform quotient of Gur K (2) of dimension d then RK,4 ≥d, but by Proposition 3.13, RK,4 ≤d2ClK −rk(BK). For the second part, recall that as G must be FAb then d ≥3. Remark 5.2. One of the main draw backs of the bilinear form BK is the appear- ance of totally isotropic subspaces (following Proposition 3.9 and Proposition 3.10). Here is a situation where such phenomena do not occur. Take a CM- extension K/k such that (i) the 2-rank of the class group of k in the narrow sense is odd and µK,2 = {±1}, (ii) the extension K/k is unramiﬁed at every prime p\|2. 5.2 Climbing in Gur K (2) Suppose that Gur K (2) has a non-trivial uniform quotient G of dimension d > 1. Let L/K be the subextension of Kur(2)/K with Galois group G. Consider the p-central decending series (Gi)i of G, and for i ≥1, denote by Ki the ﬁxed ﬁeld by the group Gi. Hence, K2 ⊂K2,el. By Proposition 2.15, Lemma 5.4. One has: ClK2 ։ (Z/8Z)d. In particular, the 8-rank RK2,8 of ClK2 should be at least d. In fact, one can say a little bit more. Indeed, by Chebotarev’s density theorem there exist prime ideals p1, · · · , pd of OK such that the Frobenius gi of pi (with an obvious abuse of notation) in L/K generate G. As G is uniform, the elements g2i−1 1 , . . . , g2i−1 d generate the subgroups Gi, for all i ≥1. For i = 1, · · · , d, let us choose now Pi ⊂OK2 a prime ideal of K2 above pi. Then, as pi does not totally split in K2/K, and thanks to the property of the global Frobenius, we get that the Frobenius of the primes Pi in L/K2 (or a Gal(K2/K)-conjugate of it) is equal to g2 i . These Frobenius elements generate the Galois group Gal(L/K2) which is of p-rank d, and in particular, the class of Pi in ClK2 is of order at least 8 by Lemma 5.4. This observation is then a new obstruction to test Conjecture 1.1. Let us explain it with an example. Example 5.5. Take K = Q(√−2 · 31 · 41 · 113). Here ClK ≃(Z/4Z)3. The 2-class group ClK of K is generated by the classes of the prime ideals p29, p823, and p211: by Burnside’s lemma the Frobenius of these elements generate Gur K (2) (and then every 2-adic analytic quotient of Gur K (2)). Note that here K2 = K(√−31, √ 41, √ 113), and ClK2 ≃(Z/16Z)3×(Z/8Z)2×Z/4Z×(Z/2Z)4. One can also verify that, in ClK2, the class of P29 is of order 16, the class of P823 is of order 16, and the class of P211 is of order 4. Hence, thanks to the prime number 211, Conjecture 1.1 holds for K. To ﬁnish, let us look at the family of imaginary quadratic ﬁelds K for which ClK ≃(Z/4Z)3. For X ≥0, denote by NX = {K ∈F− X, ClK ≃(Z/4Z)3}. (ii) the extension K/k is unramiﬁed at every prime p\|2. (ii) the extension K/k is unramiﬁed at every prime p\|2. Then there is no totally isotropic subspaces coming from units (see Proposition 3.9). As consequence of Theorem 5.1, one can give some density estimates following the work of Fouvry-Klueners. Recall also the following quantities introduced at the beginning of our work: for n, d, X ≥0, denote by F± X := {K ∈F±, \|discK\| ≤X}, FM± X := {K ∈F± X, Conjecture 1.1 holds for K}, F± X := {K ∈F±, \|discK\| ≤X}, and put FM± := lim inf X→+∞ #FM± X #F± X . FM± := lim inf X→+∞ #FM± X #F± X . This work allows us to give a more general density estimation as announced in the Introduction. Corollary 5.3. One has FM−≥d∞,0 + d∞,1 + d∞,2 ≈.99471, and FM+ ≥.99929. Proof. It is a consequence of Theorem 5.1, and the work of Fouvry-Klueners [8]. See also [10] for the computation of the densities. Documenta Mathematica 23 (2018) 1263–1290 Documenta Mathematica 23 (2018) 1263–1290 1286 Christian Maire Proof. It is a consequence of the fact that NF/KClF = (ClK)2. Hence, the criteria above the Frobenius will give nothing when the elements h1, · · · , hs are all trivial in ClK. And this condition is very easy to test thanks to GP-Pari. As before, take X = 25 ·106; in NX, we ﬁnd 37 number ﬁelds (out of 459), for which we are guaranteed that the criteria with the Frobenius will give nothing at the stage F. For the rest, take X = 107; one has \|NX\| = 120. Now, we use the strategy developped before. For every class h of ClK/Cl2 K, take a prime ideal p ⊂OK that corresponds to h (typically the prime having the smallest norm). Then, we look at the order the Frobenius of P\|p in ClF. If this order is less than 4, then Conjecture 1.1 holds. For 86 of these number ﬁelds, the computations ﬁnish, and for 10 of these ﬁelds, the strategy concerning the Frobenius holds; here is the list of these ﬁelds (with the prime): (1148984, p211), (1316755, p109), (1466643, p1721), (1934859, p127), (1148984, p211), (1316755, p109), (1466643, p1721), (1934859, p127), (1972191, p197), (2585464, p43), (3388855, p151), (4200655, p1303), (7089476, p953), (8139027, p1181). (1972191, p197), (2585464, p43), (3388855, p151), (4200655, p1303), (7089476, p953), (8139027, p1181). Remark 5.7. It woud be interesting to develop in a systematic way the test im- plying the Frobenius elements. For example, by studying the action of Gal(F/K) on ClF. 5.2 Climbing in Gur K (2) By a result of Koch (see Hajir [13]), one knows that for each K ∈NX, the pro-2 group Gur K (2) is inﬁnite. Let us see the diﬀerent tests for Conjecture 1.1. Denote by F the unramiﬁed 2-elementary maximal extension of K. (i) First, let us see the criteria concerning the 8-rank of ClF. When X = 25 · 106, we ﬁnd #NX = 459, and 7 number ﬁelds in NX are such that RF,8 ≤ 2 (in fact = 2), where RF,8 denotes the 8-rank of the class group of F. Here is the list of these ﬁelds: \|discK\| ∈ {9384952, 11577476, 13478584, 14524408, 17765944, 20167563, 21799304}. Th f th b ﬁld C j t 1 1 h ld Then, for these number ﬁelds, Conjecture 1.1 holds. Then, for these number ﬁelds, Conjecture 1.1 holds. (ii) Now we will test the condition implying the Frobenius. First, remark that one should exclude quickly some number ﬁelds. Let us be more precise. Take a Documenta Mathematica 23 (2018) 1263–1290 Unramified 2-Extensions of a Number Field 1287 set of generators {H1, · · · , Hr} of ClF[4]. One can assume that for i = 1, · · · , s, the elements Hi are not in (ClF)2, and for i = s+1, · · · , r, the elements Hi are in (ClF)2. For i = 1, · · · , r, put hi = NF/KHi. Let us make an easy observation: set of generators {H1, · · · , Hr} of ClF[4]. One can assume that for i = 1, · · · , s, the elements Hi are not in (ClF)2, and for i = s+1, · · · , r, the elements Hi are in (ClF)2. For i = 1, · · · , r, put hi = NF/KHi. Let us make an easy observation: Proposition 5.6. Suppose that there exists a prime ideal p ∈OK such that clK(p) ∈ClK −(ClK)2 and clF(P) ∈ClF[4], where P\|p, P ⊂OF. Then there exists i0 ∈{1, · · · , s} such that hi0 ̸= 1. Proof. 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Documenta Mathematica 23 (2018) 1263–1290 1290 Christian Maire Laboratoire de Mathématiques de Besançon (UMR 6623) Université Bourgogne Franche-Comté et CNRS 16 route de Gray 25030 Besançon cédex, France christian.maire@univ-fcomte.fr Christian Maire Laboratoire de Mathématiques de Besançon (UMR 6623) Université Bourgogne Franche-Comté et CNRS 16 route de Gray 25030 Besançon cédex, France christian.maire@univ-fcomte.fr
https://openalex.org/W4313465213	https://jse.rezkimedia.org/index.php/jse/article/download/162/40	English	null	Development of application-based badminton material teaching media for middle school level PJOK teachers	Journal of Science and Education	2,022	cc-by-sa	6,026	Journal of Science and Education (JSE) Vol. 3, No. 2, 2022, 82-93 DOI: 10.56003/jse.v3i2.162 ISSN: 2745-5351 Development of application-based badminton material teaching media for middle school level PJOK teachers Ari Wibowo Kurniawan1, Lokananta Teguh Hari Wiguno2, M. Helmy Firman Amrullah3 1,2,3 Physical Education, Health and Recreation, Faculty of Sports Science, Universitas Negeri Malang, Jl. Semarang No 5, Malang City, East Java Province, Indonesia E-mail: ari.wibowo.fik@um.ac.id Received: 27 September 2022 Accepted: 23 November 2022 Published: 12 December 2022 Journal of Science and Education (JSE) Vol. 3, No. 2, 2022, 82-93 DOI: 10.56003/jse.v3i2.162 ISSN: 2745-5351 Development of application-based badminton material teaching media for middle school level PJOK teachers Ari Wibowo Kurniawan1, Lokananta Teguh Hari Wiguno2, M. Helmy Firman Amrullah3 1,2,3 Physical Education, Health and Recreation, Faculty of Sports Science, Universitas Negeri Malang, Jl. Semarang No 5, Malang City, East Java Province, Indonesia E-mail: ari.wibowo.fik@um.ac.id Received: 27 September 2022 Accepted: 23 November 2022 Published: 12 December 2022 Journal of Science and Education (JSE) Vol. 3, No. 2, 2022, 82-93 DOI: 10.56003/jse.v3i2.162 ISSN: 2745-5351 Ari Wibowo Kurniawan1, Lokananta Teguh Hari Wiguno2, M. Helmy Firman Amrulla 1,2,3 Physical Education, Health and Recreation, Faculty of Sports Science, Universitas Negeri Malang Jl. Semarang No 5, Malang City, East Java Province, Indonesia E-mail: ari.wibowo.fik@um.ac.id Abstract: This development research has the goal of developing application-based badminton game learning using the Java programming language which is used as a supporting tool for learning badminton game material for Middle School PJOK Teachers in Kediri Regency as well as being a reference and development for further research. This research method refers to the development research method of Sugiono, which. The test subjects in this study involved 40 SMP PJOK teachers in Kediri Regency, consisting of 10 teachers involved in the small group trial and 30 teachers involved in the large group trial. The results of the analysis carried out by experts who have carried out the validity of this application product, the percentage of learning expert evaluations is obtained, which means it is very valid, the results of the evaluation of badminton playing experts mean it is very valid, the results of the evaluation of media experts mean it is very valid. So based on these results, the research product of this badminton material learning application application was declared "fit for use" for learning badminton game material for junior high school PJOK teachers in Kediri Regency. Keywords: learning; application media; badminton. How to cite: Kurniawan, A. W., Wiguno, L. T. H., & Amrullah, M. H. F. (2022). Development of application-based badminton material teaching media for middle school level PJOK teachers. Journal of Science and Education (JSE), 3(2): 82-93. https://doi.org/10.56003/jse.v3i2.162 How to cite: Kurniawan, A. W., Wiguno, L. T. H., & Amrullah, M. H. F. (2022). Development of application-based badminton material teaching media for middle school level PJOK teachers. Journal of Science and Education (JSE), 3(2): 82-93. https://doi.org/10.56003/jse.v3i2.162 How to cite: Kurniawan, A. W., Wiguno, L. T. H., & Amrullah, M. H. F. (2022). Development of application-based badminton material teaching media for middle school level PJOK teachers. Journal of Science and Education (JSE), 3(2): 82-93. https://doi.org/10.56003/jse.v3i2.162 This is an open access article under CC-BY-SA license. This is an open access article under CC-BY-SA license. INTRODUCTION According to Dasupang & Pane (2017) learning is a process to regulate the environment of students or students so that they can encourage and create a learning atmosphere for students. Learning is an attempt to transfer knowledge or information using certain methods in order to achieve the desired goals (Don, et al. 2018). Of course, a systematic learning process must originate from KI-KD in the curriculum. This expression is in line with Kristin (2016) in his journal which reveals that the process of learning activities will run and get maximum results if there is a good relationship between students, teachers and the existing curriculum. Whatever the level of education, of course the learning material delivered must be in accordance with the KI-KD in the curriculum. There are many levels of education from elementary to university. At the junior high school education level, for example, there are many subjects including PJOK or physical education which contain a lot of material, one of which is badminton. According to Yuliansyah, et al (2021) physical education is a learning activity that focuses on the emotions and movements of students. Badminton is a sport that is very popular and popular in Indonesia and the lessons taught in physical education subjects in Indonesia (Lismadiana, et al, 2020). According to Arwih, Fitria & Saifu (2020) badminton is a game that is stiffened by hitting the ball This is an open access article under CC-BY-SA license. This is an open access article under CC-BY-SA license. 82 Kurniawan, Wiguno, & Amrullah – Development of application-based badminton … 83 … 83 83 or shuttlecock using a racket to be able to cross the net with the aim of attacking and positioning the ball so that it cannot be reached by opposing players. According to Cendra & Gazali (2017) in their research journal said that the game of badminton is very familiar among students because it has been included in the O2SN competition. In Permendikbud Number 37 (2018) specifically on badminton material students are required to master basic competencies and core competencies gradually, starting from class VII given basic badminton technique material, class VIII material variations on basic badminton techniques, until class IX enters variations and combinations of badminton games. INTRODUCTION However, PJOK learning activities, especially badminton material in Kediri Regency, are still not going well and still do not refer to the curriculum because PJOK teachers still often generalize the provision of badminton material and do not focus on the existing curriculum so that the learning objectives are not optimally achieved. According to Asro & Muna (2019) the achievement of learning objectives is largely determined by how the learning process is carried out. In addition to selecting material, the use of media elements in delivering will also greatly affect the quality of learning (Yanto, 2019). Learning media is anything that is used by students in providing subject matter to students (Dewi, 2017). According to Kurniawan, et al (2021) the use of attractive and efficient media will foster students' interest and enthusiasm for learning. This statement is in line with Maimunah (2016) which states that the use of interesting and not monotonous media will increase motivation and new stimulation to students. According to Lestari (2018) teachers must innovate themselves more in honing themselves to utilize technology in order to create a fun and not boring learning atmosphere. The increasingly rapid development of the times in terms of technology followed by the development of the characteristics of students or students where the need for teaching requires new innovations to improve innovative ways of learning to adapt technological developments in learning activities according to the curriculum including physical education lessons (Belmonte, et al. 2019). In this era of globalization, many new learning media have grown which are more interesting and can be used to facilitate learning activities. Among them is learning media based on Android technology smartphone among students, application development as teaching media will be very useful and effective (Arista & Kuswanto, 2018). Application Android is software that can be accessed using smartphone-based Linux that is widely used by people in all walks of life (Kuswanto & Radiansah, 2018). The more students who have Android smartphones there will be many opportunities to develop an effective and efficient application-based learning to support learning activities (Astuti. et al., 2017). According to Herlina & Suherman (2020) innovative learning development innovations are needed during the Covid-19 to overcome boredom and increase interest in learning and to achieve learning objectives in PJOK subjects. In previous research conducted by Firlando. INTRODUCTION et al (2020) by creating soccer learning media using a java at SMPN SMP Ar Risalah Lubuklinggau, the result is an increase in student learning motivation because it uses learning media that is efficient and can be carried out anywhere and anytime. In previous research, according to Artanayasa, Dewi & Suwiwa (2018), interactive multimedia applications used in learning basic 84 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 84 badminton techniques at Singaraja State Middle School received good marks after being tested on experts and students so that they can be used as an effective and efficient learning resource. Based on the data from the needs analysis, learning activities for badminton material in Kediri Regency still use print learning media and YouTube, and there is no development of effective and efficient application-based interactive learning developed by teachers in badminton material. Based on the results of initial observations from the collection of needs analysis data for SMP-level PJOK TEACHER in Kediri Regency conducted by researchers on April 10, 2021 through a direct questionnaire filled out by 40 PJOK MGMP teachers, the result was that 100% of the teachers had given badminton material, there was 100% of teachers have provided badminton material using print media such as books, lesson plans or worksheets, 90% of teachers have provided badminton material using learning media based on video and powerpoint, 100% of teachers have cellphones or smartphones, 100% of teachers have never developed teaching media for badminton material based on android application-based learning development is held android an effective and efficient with a load of badminton game material. And based on the interviews that the researchers conducted with the Head of the Kediri Regency MGMP, the results were obtained, namely that there were only 1-2 meetings of badminton material learning in the learning process which was carried out with field practice but there was a lack of emphasis on basic competencies and core competencies and there was no development of teaching media applications-based badminton material android in Kediri Regency. With this problem, the researchers tried to solve the problem by developing learning applications using Java which contained badminton game material for Middle School PJOK Teachers in Kediri Regency. INTRODUCTION According to Ali (2019) Java is a type of programming language with very rapid development and can be run in many applications or web-shaped application devices and can be run on many platforms including using the Android, Windows, and Linux platforms. By using the Android, there are several aspects why this research is important to do, namely, to make it easier for teachers to convey subject matter and make it easier for students to understand the material presented both with online and offline. The application also displays material material for badminton games which are supported by clear images, video and audio and there are evaluation questions that can be accessed easily. This research has the goal of developing badminton material learning products containing pictures, material, video (audio-visual) with animations that are made as attractive as possible so that the objectives of learning badminton material for PJOK Teachers at the Middle School Level in Kediri Regency can be achieved. METHOD This study refers to the development research steps of Borg & Gall (1983) in Sugiyono (2016) using a modified procedure as follows (1) Potential & problems, namely analyzing problems by conducting interviews with the Head of MGMP SMP Kediri Regency, (2) Data collection, namely collecting data called needs analysis and conducting interviews with PJOK Teachers in Kediri Regency, (3) Product Design is the Kurniawan, Wiguno, & Amrullah – Development of application-based badminton 85 stage of developing an application product that contains badminton material for junior high school students by taking videos, recording audio, entering material, editing applications and so on, (4) Design validation, namely the stage of testing the feasibility of the product before testing. In this stage there are 3 experts, namely Learning Experts, Badminton Game Experts and Media Experts. (5) Design revision, namely the stage of evaluating and revising the product based on expert input at the validation stage. (6) Product Trial, namely the product testing stage involving 40 PJOK Teachers of Kediri Regency, divided into 2 groups, namely 10 teachers in small groups and 30 teachers in large groups. (7) Product revision, namely the final revision stage from the previous trial stage input. (8) Final Product at this stage the product is ready to be used by PJOK Teachers in Kediri Regency to provide badminton game material to students. Figure 1. Procedure for Developing Application-Based Badminton Game Learning Media for PJOK Teachers in Kediri Regency Figure 1. Procedure for Developing Application-Based Badminton Game Learning Media for PJOK Teachers in Kediri Regency This research obtained qualitative and quantitative data. Qualitative data were obtained through evaluations of learning experts, badminton game experts, media experts and the results of interviews with the Head of the MGMP PJOK SMP Kediri Regency and the results of a questionnaire distributed to PJOK teachers in Kediri Regency. Meanwhile, quantitative data is the result of numerical analysis from product tests by PJOK teachers in Kediri Regency. The variable used is in the form of learning media for badminton material in the form of applications with the Java. This Development Research has several data collection instruments including initial observation for needs analysis in the form of a questionnaire filled out by PJOK Teachers of SMP Kediri Regency, expert validation questionnaires namely learning experts, media experts, badminton game experts and product testing. METHOD The technical stages of analyzing the research data are in the form of descriptive statistics technique for measuring in the process of collecting data uses a Likert, with the aim of measuring the attitudes or views of individuals or groups towards the phenomena that occur (Sugiyono, 2016). The results of data analysis are used to improve the product being developed scale instrument Likert has category aspects ranging from very positive to negative. Table 1. Rating Scale for Positive Statements No. Explanation Answers Positive Score 1. Strongly Agree A 4 2. Agree B 3 3. Undecided C 2 4. Disagree D 1 Source: Sugiyono (2016) 86 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 The formula for data processing is in the form of descriptive quantitative percentage analysis from Akbar & Sriwiyana (2011), as follows: Description: Description: V : Validity. TSEV : Total empirical score of the validator. S-max : Maximum expected score. 100% : Constant number. : Total empirical score of the validator. : Maximum expected score. Next according to Akbar & Sriwiyana (2011) to process and conclude the results of the analysis in the form of percentages so that they are categorized as below: Table 2. Product Quality Criteria Criteria Description Meaning 75.01%-100.00% Very Valid Used without revision 50.01%-75.00% Valid Enough Used with minor revisions 25.01%-50.00% Invalid Cannot be used 00.00% -25.00% Very Invalid Forbidden to use Source: Akbar & Sriwiyana (2011) RESULTS AND DISCUSSION Source: Akbar & Sriwiyana (2011) RESULTS AND DISCUSSION RESULTS AND DISCUSSION There are seven main contents in this badminton learning application, namely KI-KD, Learning Materials, Learning Videos, Facilities and Infrastructure, Evaluation Questions, References and Developer Profile. The entire material and use of the application program have been prepared so that it can be used effectively and efficiently and is displayed in an attractive design so that learning objectives can be more easily achieved. Figure 2. Main Display of Application Products Figure 2. Main Display of Application Products Kurniawan, Wiguno, & Amrullah – Development of application-based badminton … … 87 87 The following is a discussion of product development, presentation of data consisting of needs analysis instruments, expert validation, product trials and product revisions. The following is a discussion of product development, presentation of data consisting of needs analysis instruments, expert validation, product trials and product revisions. Table 3. Results of Data Analysis of Learning Experts No. Aspect % Category 1. Attractiveness 91% Very Valid 2. Ease 82% Very Valid 3. Conformity 100% Very Valid 4. Accuracy 82% Very Valid Validity 87% Very Valid Figure 3. Learning Expert Research Diagram Table 4. Revision from Learning Experts No. Before Revision After Revision 1. Learning videos have not been grouped and made according to KI-KD as a whole. Learning videos are made according to KI-KD and are presented in more detail. 2. There are no supporting images on the learning material menu. Learning materials have been added pictures to facilitate student understanding. 3. There are no examples of motion sequence material on the class IX learning material menu. Added material for a series of motion variations on the basic techniques of badminton on the class IX learning material menu. 4. There are several writing errors on the evaluation question menu. All text on the evaluation question menu is neat and written correctly. 91% 82% 100% 82% 87% 0% 20% 40% 60% 80% 100% Attractiveness Ease Conformity Accuracy Validity Learning Expert Learning Expert Table 4. Revision from Learning Experts No. Before Revision After Revision 1. Learning videos have not been grouped and made according to KI-KD as a whole. Learning videos are made according to KI-KD and are presented in more detail. 2. There are no supporting images on the learning material menu. Learning materials have been added pictures to facilitate student understanding. 3. There are no examples of motion sequence material on the class IX learning material menu. RESULTS AND DISCUSSION Added material for a series of motion variations on the basic techniques of badminton on the class IX learning material menu. 4. There are several writing errors on the evaluation question menu. All text on the evaluation question menu is neat and written correctly. The first data is a presentation of quantitative data obtained from the evaluation of learning experts regarding application products for learning badminton material using the Java for Middle School PJOK Teachers in Kediri Regency with a level on the attractiveness aspect of 91% which is classified as very valid, the convenience aspect is 82% classified as very valid, the aspect 100% suitability, which is classified as very valid and the accuracy aspect of 82%, which is classified as very valid. Based on the results of the data on all aspects, it can be concluded that the results of the analysis of this product development obtain a validity of 87% so that it can be interpreted as very valid. 8 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 88 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 88 Table 5. Results of Badminton Game Expert Data Analysis No. Aspect % Category 1. Clarity 83% Very Valid 2. Attractiveness 83% Very Valid 3. Accuracy 92% Very Valid Validity 84% very valid Figure 4. Badminton game expert assessment diagram Table 6. Revision of Badminton Experts No. Before Revision After Revision 1. The learning video for holding a racket that is presented is still lacking in detail in its implementation. The learning video for holding a racket has been presented correctly and clearly. 2. The learning video for variations of footwork or footwork has not been presented in detail from the side of singles and doubles games. The learning video for variations of footsteps or footwork has been presented correctly and clearly. 3. The backhand learning video is still not quite right in its implementation. The backhand learning video has been presented correctly and clearly. 4. The variety of smash strokes is still incomplete. The smash punch video has been presented completely and clearly. h d d i h i f i i d b i d f h l i f b d i 83% 83% 92% 84% 0% 20% 40% 60% 80% 100% Clarity Attractiveness Accuracy Validity Badminton Game Expert Badminton Game Expert Table 5. Results of Badminton Game Expert Data Analysis No. Aspect % Category 1. Clarity 83% Very Valid 2. Attractiveness 83% Very Valid 3. Accuracy 92% Very Valid Validity 84% very valid Figure 4. Badminton game expert assessment diagram Table 6. Revision of Badminton Experts No. Before Revision After Revision 1. The learning video for holding a racket that is presented is still lacking in detail in its implementation. The learning video for holding a racket has been presented correctly and clearly. 2. The learning video for variations of footwork or footwork has not been presented in detail from the side of singles and doubles games. The learning video for variations of footsteps or footwork has been presented correctly and clearly. 3. The backhand learning video is still not quite right in its implementation. The backhand learning video has been presented correctly and clearly. 4. The variety of smash strokes is still incomplete. The smash punch video has been presented completely and clearly. 83% 83% 92% 84% 0% 20% 40% 60% 80% 100% Clarity Attractiveness Accuracy Validity Badminton Game Expert Badminton Game Expert Table 6. Revision of Badminton Experts No. Before Revision After Revision 1. 88 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 The learning video for holding a racket that is presented is still lacking in detail in its implementation. The learning video for holding a racket has been presented correctly and clearly. 2. The learning video for variations of footwork or footwork has not been presented in detail from the side of singles and doubles games. The learning video for variations of footsteps or footwork has been presented correctly and clearly. 3. The backhand learning video is still not quite right in its implementation. The backhand learning video has been presented correctly and clearly. 4. The variety of smash strokes is still incomplete. The smash punch video has been presented completely and clearly. The second data is the presentation of quantitative data obtained from the evaluation of badminton game experts regarding product application learning material for badminton using the Java for Middle School PJOK Teachers in Kediri Regency with an aspect level of clarity of 83% classified as very valid, an aspect of attractiveness 83% classified as very valid, an aspect of accuracy 92% classified as very valid. Based on the results of the data on all aspects, it can be concluded that this development product obtains a validity of 84% so that it can be interpreted as very valid. Table 7. Results of Media Expert Data Analysis No. Aspect % Category 1. Clarity 96% Very Valid 2. Attractiveness 84% Very Valid 3. Ease 100% Very Valid 4. Accuracy 100% Very Valid Validity 93% Very Valid Table 7. Results of Media Expert Data Analysis Kurniawan, Wiguno, & Amrullah – Development of application-based badminton … Figure 5. Media Expert Assessment Diagram Table 8. Revision from Media Experts No. Before Revision After Revision 1. There are no supporting images on the learning material menu. All infrastructure materials have been added with pictures to make it easier for students to understand. 88 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 96% 84% 100% 100% 93% 0% 20% 40% 60% 80% 100% Clarity Attractiveness Ease Accuracy Validity Media Expert Media Expert Media Expert 100% After Revision Before Revision The third data is the presentation of quantitative data obtained from the evaluation of media experts regarding application products for learning badminton material using the Java for Middle School PJOK Teachers in Kediri Regency with an aspect level of clarity of 96% classified as very valid, the attractiveness aspect of 84% is classified as very valid, the aspect of convenience is 100 % is classified as very valid and the accuracy aspect is 100% which is classified as very valid. Based on the results of the data on all aspects, it can be concluded that this development product obtains a validity of 93% so that it can be interpreted as very valid. Table 9. Small Group Trial Analysis Results No. Aspect % Category 1. Clarity 79% Very Valid 2. Attractiveness 79% Very Valid 3. Ease 79% Very Valid 4. Accuracy 78% Very Valid Validity 79% Very Valid Figure 6. Small Group Trial Assessment Diagram 79% 79% 79% 78% 79% 0% 20% 40% 60% 80% 100% Clarity Attractiveness Ease Accuracy Validity Small Group Trial Small Group Trial Table 9. Small Group Trial Analysis Results Small Group Trial Figure 6. Small Group Trial Assessment Diagram Fourth data is the presentation of quantitative data through 10 SMP PJOK teachers regarding badminton material learning application products using the Java for SMP PJOK Teachers in Kediri Regency with a level of clarity of 79% classified as very valid, 79% for attractiveness aspects classified as very valid, 79% for convenience aspects. classified as very valid and the accuracy aspect of 78% is classified as very valid. ournal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 90 Based on the results of the data on all aspects, it can be concluded that this development product obtains a validity of 79% so that it can be interpreted as very valid. Based on the results of the data on all aspects, it can be concluded that this development product obtains a validity of 79% so that it can be interpreted as very valid. Table 10. Results of Analysis of Large Group Trials No. Aspect % Category 1. Clarity 83% Very Valid 2. Attractiveness 84% Very Valid 3. Ease 83% Very Valid 4. 88 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 Accuracy 83% Very Valid Validity 83% Very Valid Figure 7. Large Group Trial Assessment Diagram 83% 84% 83% 83% 83% 0% 20% 40% 60% 80% 100% Clarity Attractiveness Ease Accuracy Validity Large Group Trial Large Group Trial Large Group Trial Figure 7. Large Group Trial Assessment Diagram The fifth data is the presentation of quantitative data through 40 Middle School PJOK Teachers regarding product application learning material for badminton using the Java for Middle School PJOK Teachers in Kediri Regency with an aspect level of clarity 83% classified as very valid, 84% for attractiveness aspects classified as very valid, 83% for convenience aspects classified as very valid and the accuracy aspect of 83% was classified as very valid. Based on the results of the data on all aspects, it can be concluded that this development product obtains a validity of 83% so that it can be interpreted as very valid. The overall suggestions from each expert on application products have been improved to make the product even more perfect. The final product of this research is learning media in the form of an application using Java which contains badminton game material for PJOK Teachers at SMP Kediri Regency. According to Arliando, Sallaby & Utami (2015) in his journal said that Java is a programming language commonly used in making applications that can be accessed using smartphones android with functions including so that the display is more attractive. According to Irsan (2015) library in Java is very diverse and complete, which has been provided and contains a collection of programs to facilitate the process of making the product the programmer wants. Java is a basic level coding language but can produce attractive and attractive application results according to the programmer's wishes (Nugroho & Rusli, 2017). According to Chotimah (2018) in his journal states that a media or learning device that is packaged in an interesting and varied way really helps students understand the material. Based on the data from the needs analysis, among others, it was stated that 100% of teachers agreed that if effective and efficient badminton learning media were developed, of course Kurniawan, Wiguno, & Amrullah – Development of application-based badminton 91 … 91 this would be in line with the existence of Java which were arranged with effective material coverage and an attractive appearance to achieve learning objectives. 88 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 Applications android as learning support has the potential to succeed in seeing the increasingly widespread use of android smartphones in junior high school students. The use of interesting learning media will certainly provide a positive stimulus to students in participating in learning activities (Karo & Rohani, 2018). Sourced from expert validation data and trials that the badminton material learning application products that have been developed by researchers have very valid results in product attractiveness. The game of badminton is very popular with the community and can be played by anyone (Majid, et al. 2021) The game of badminton is a game that requires a very complex combination of movements, starting from holding a racket, hitting the ball, moving your feet, to producing a punch that can kill the opponent's defense (Mauludy & Sartono, 2017). According to Sholeh (2017) badminton is a game played by hitting the shuttlecock to the opponent's area. Badminton is one of the many PJOK materials contained in the curriculum from elementary to high school with certain learning stages (Sukarini, 2020). In Permendikbud Number 37 (2018) specifically on badminton material students are required to master basic competencies and core competencies gradually, starting from class VII who is given basic badminton technique material, class VIII material variations on basic badminton techniques, until class IX enters variations and combinations of badminton games. Based on expert validation data, product testing and revision, it is obtained from research data that the scope of material in learning applications has very valid accuracy results. There are several menus and material content displayed in the form of images, text to learning videos that are presented as attractively as possible in this application. According to Yudianto (2017) videos can help direct students' concentration and attention. According to Kamlin & Keong (2020) video media really helps students understand learning material because the understanding process becomes easier with the right display and interesting material. Based on research data from experts and trials, the results show that the presentation of video material has a very valid level of accuracy and attractiveness. The results of developing application-based badminton teaching media products using the Java programming language for SMP PJOK TEACHER, Kediri Regency have gone through several revision stages to obtain product results that are very valid and feasible to use. REFERENCES Akbar, S. & Sriwiyana, H. (2011). Pengembangan Kurikulum dan PembelajaranIlmu Pengetahuan (IPS). Yogyakarta: Cipta Media. Ali, A. F . (2019). Rancang Bangun Aplikasi Penjualan Barang Berbasis Java Programimming. Jurnal SIMTIKA, 2(1), 8–17. Arista, F. S & Kuswanto, H. (2018). Virtual Physics Laboratory Aplication Based On The Android Smartphone To Improve Learning Independence And ConceptualsUnderstanding. International Journal Of Instruction, 11(1), 1-16. Arliando, Y., Sallaby, A. F., & Utami, F. H. (2015). Aplikasi Widget Berbasis Java. 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Aplikasi Pembelajaran Teknik Dasar Sepak Bola Berbasis Android. Jurnal Teknologi Informasi Mura. 12(02), 166-172. Herlina & Suherman M. (2020). Potensi Pembelajaran PJOK Ditengah Pandemi Corona Di Sekolah Dasar. Journal Sport Sciense And Physycal Education, 8(1) 1-7. Irsan, M. (2015). CONCLUSION The results of research on the development of teaching media for application-based badminton materials with Java programming can be stated to have clarity, convenience, attractiveness, suitability and accuracy which are very valid according to the results of validation by several experts and product testing by PJOK teachers at SMP Kediri Regency. Thus this application product is suitable for use in badminton learning activities. 92 Journal of Science and Education (JSE), Vol. 3, No. 2, Special Issue 2022, pp. 82-93 REFERENCES Rancang bangun aplikasi mobile notifikasi berbasis android untuk mendukung kinerja di instansi pemerintahan. 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https://openalex.org/W4285533297	https://zenodo.org/records/6822674/files/D3.4%20Empowering%20children_FINAL.pdf	English	null	Empowering children: childcare services in a rights-based comparative perspective	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	31,185	Empowering children: childcare services in a rights-based comparative perspective Authors Leonie Westhoff Francesco Corti Izabela Grabowska Cinzia Alcidi Agnieszka Chłoń-Domińczak EuSocialCit working paper July 2022 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 870978 This working paper is published as part of the EuSocialCit project, which has received funding from the European Commission’s Horizon 2020 Research and Innovation programme under Grant Agreement No 870978. This publication reflects the authors’ view only. The European Commission is not responsible for any use that may be made of the information it contains. Westhoff, L., Corti, F., Grabowska, I., Alcidi, C., Chłoń-Domińczak, A. (2022), Empowering children: childcare services in a rights-based comparative perspective, EuSocialCit Working Paper, July 2022. Doi: 10.5281/zenodo.6822674. Leonie Westhoff is Associate Researcher in the Jobs & Skills Unit at CEPS. Francesco Corti is Postdoctoral researcher at University of Milan. Izabela Grabowska is Assistant Professor at the Warsaw School of Economics. Cinzia Alcidi is Director of Research, Head of the Economic Policy and Jobs & Skills Unit at CEPS. Agnieszka Chlon-Dominczak is the head of the Institute of Statistics and Demography at the Warsaw School of Economics. This publication is licensed under the Creative Commons CC BY-NC. This information may be freely used, copied and adapted, provided that the source is acknowledged. inform Keywords: Childcare, European Union, Social Investment Summary This paper analyses early childhood education and care policy in Italy, Germany and Poland. Drawing on the capability approach and building on the power resources analytical framework, it maps policy design features in these three countries. Although the three childcare regimes fall under the umbrella of ‘familialism’, they exhibit some important differences. The paper finds that legal entitlement alone, which only exists in Germany, is not a necessary or sufficient condition to guarantee access for all children. In addition, it appears that similar funding structures do not affect all children and their families equally and quality of services can vary significantly. Instrumental resources, which are expected to help families to access childcare and reduce informational barriers to take-up, are underdeveloped in the three countries. Where they do exist, it is at local level. Finally, enforcement resources, that is, legal channels for individuals to claim their entitlement to childcare, only exist in Germany, where children have the legal entitlement, and seem to provide families with additional leverage in claiming a childcare place. Besides the specific results, the paper provides an original analytical grid that for a broader assessment of childcare policies. Keywords: Childcare, European Union, Social Investment Keywords: Childcare, European Union, Social Investment 3 3 14 July 2022 Deliverable 3.4 - Empowering children: childcare services in a rights-based comparative perspective Project name The Future of European Social Citizenship Project acronym EuSocialCit Grant Agreement ID 870978 Deliverable number D3.4 Lead partner CEPS Work package EuSocialCit is an interdisciplinary research project aiming to support the EU in strengthening social rights and European social citizenship. It evaluates the current state of social rights in Europe and their relationship to social inequalities, gender inequalities, poverty, and precariousness, and diagnoses the shortcomings of current policies and institutions at the level of individual countries and the EU. The EuSocialCit project focuses on three domains in which social rights are important: the empowerment of citizens (e.g. education and activation), fair working conditions, and social inclusion. Each of these domains are respectively studied as part of WP3, WP4 and WP5. This report is produced as part of WP3, which is entitled Empowerment through social investment. WP3 focuses on social rights that are associated with the aspiration of empowerment: rights that enable citizens not only to fully share in the social heritage, but also to further develop this heritage (Marshall, 1950). Summary Hence, WP3 examines policies that are commonly captured by the notion of social investment. Web address For more information about the EuSocialCit project, please visit www.eusocialcit.eu. EuSocialCit’s output can also be found in its community on Zenodo: https://zenodo.org/communities/eusocialcit. The EuSocialCit project focuses on three domains in which social rights are important: the empowerment of citizens (e.g. education and activation), fair working conditions, and social inclusion. Each of these domains are respectively studied as part of WP3, WP4 and WP5. This report is produced as part of WP3, which is entitled Empowerment through social investment. WP3 focuses on social rights that are associated with the aspiration of empowerment: rights that enable citizens not only to fully share in the social heritage, but also to further develop this heritage (Marshall, 1950). Hence, WP3 examines policies that are commonly captured by the notion of social investment. For more information about the EuSocialCit project, please visit www.eusocialcit.eu. EuSocialCit’s output can also be found in its community on Zenodo: https://zenodo.org/communities/eusocialcit. 1. Introduction Over the past three decades, the notion of ‘social investment’ has gained purchase as a novel welfare policy compass to address economic and social change in the 21st century. Its key objective is to provide a mix of policies that empower individuals and societies to respond to the changing nature of post-industrial social risks and labour markets. While policymakers at both national and European level became increasingly interested in ‘social investment’ from the mid-1990s (Hemerijck and Corti, 2022), the pandemic crisis has further strengthened the demand not only for inclusive income support policies but also for (gendered) life-course transition and human capital-enhancing policies (Hemerijck and Huguenot-Noël, 2022). With social investment gaining increasing political attention, the academic focus in comparative welfare state research predictably shifted from explaining change-resistant welfare states (Pierson, 1994, 1998) towards a better understanding of how welfare states do change over time (Ferrera Hemerijck, 2003; Ferrera, Hemerijck, Rhodes, 2000). Understanding trajectories of welfare recalibration became thus a key object of investigation. In our first EuSocialCit deliverable (see Baiocco et al., 2021) we attempted to identify social investment strategies across EU countries and explain their evolution between 2004 and 2018, namely before, during and after the global financial crisis. By using cluster analysis on expenditure variables, we identified different groups of countries that have diversified over time in a progressively complex way. Since the financial crisis, three main strategies have emerged in Europe. They do not overlap with canonical welfare state models, nor have a clear-cut geographical connotation. The strategies are distinct because of different levels of overall expenditure in social investment but also different degrees of life-course orientation. A first group of countries, including continental and Scandinavian welfare states, is characterised by what we called a ‘balanced strategy’, that is, high spending on all main social investment areas, with comparatively higher spending on family policies, in particular on childcare services. A second group of countries, including mostly centre-eastern European welfare states, Spain and Greece, which we call a ‘bent strategy’, is characterised by a medium to low overall expenditure in social investment policies with a particularly high spending in early life-course stage, especially through in-cash parental leaves. Table of contents 1. INTRODUCTION .................................................................................................................... 6 2. THE CAPABILITY APPROACH AND A RIGHTS-BASED APPROACH TO CHILDCARE POLICIES ......... 9 3. CASE SELECTION AND METHODOLOGICAL APPROACH ......................................................... 15 4. RESULTS ............................................................................................................................. 18 4.1. ECEC IN GERMANY ......................................................................................................................... 18 4.2. ECEC IN ITALY ................................................................................................................................ 24 4.3. ECEC IN POLAND ............................................................................................................................ 28 CONCLUSION ............................................................................................................................. 33 APPENDIX – COUNTRY SHEETS ................................................................................................... 37 A1 ECEC IN ITALY ..................................................................................................................................... 37 A2 ECEC IN GERMANY ............................................................................................................................... 44 A3 ECEC IN POLAND ................................................................................................................................. 56 REFERENCES .............................................................................................................................. 68 5 5 14 July 2022 1. Introduction 1. Introduction Finally, a third group of countries, including Italy, Cyprus, Portugal and Ireland, which we call a ‘basic strategy’, is characterised by an overall low-medium to low spending on social investment policies, with higher spending on later life-course stage, especially university and compulsory education, and low expenditure in family policies. 6 14 July 2022 6 14 July 2022 One of the key findings of our previous working paper is how the role of policies for early life stages, namely child and family policies, defines social investment strategies. This is not surprising, as the lynchpin of the new social investment paradigm is the idea of work-family life course (Kuitto, 2016), which requires state intervention over the life course to break the cycle of disadvantage and to smooth life transitions by facilitating women’s participation in the economy. An abundant scholarship demonstrates the benefits of early childhood education and care for children, families and society. Investment in children is thought to lead to long-term benefits for children by enhancing their human capital and learning outcomes, as well as their longer-term social and labour market prospects (Van Lancker and Ghysels, 2016). The first three years of life indeed represent a phase of particular sensitivity in which the possibilities for the acquisition of fundamental skills are maximised (Cavioni and Zanetti, 2015). As such, the quality of care and education has strong preventive implications for later life, because it allows the neuro-psychological system of a child to better structure itself, in cognitive but also socio-emotional and relational terms, developing children’s (skills) resilience (Del Boca et al., 2020; Cefai et al., 2015; Harbach, 2019). Studies by James Heckman and his collaborators (Heckman, 2006; Heckman and Masterov, 2007) have shown that investment (by families and the education system) in the very first years of life is crucial for individual cognitive development. At the same time, a wide literature has provided empirical evidence of how increasing the supply of public places in early childhood education and care (ECEC) services, which reduces the childcare costs borne by families, has a positive impact on the participation of mothers in the labour market (Morrissey, 2017). In particular, the literature shows that it is usually mothers with a lower level of education that benefit the most from the provision of public childcare services, with positive externalities in terms of social integration and the reduction of poverty (Alleanza per l’Infanzia, 2020). 1. Introduction Other studies have shown the positive impact of early childhood investment on society overall, where enrolment in childcare and pre-primary school has an impact in terms of occupation and salaries. Non- cognitive skills acquired at an early age are remunerated in the labour market (Bennett, 2008; Duncan and Te One, 2012). At the same time, a wide literature has provided empirical evidence of how increasing the supply of public places in early childhood education and care (ECEC) services, which reduces the childcare costs borne by families, has a positive impact on the participation of mothers in the labour market (Morrissey, 2017). In particular, the literature shows that it is usually mothers with a lower level of education that benefit the most from the provision of public childcare services, with positive externalities in terms of social integration and the reduction of poverty (Alleanza per l’Infanzia, 2020). externalities in terms of social integration and the reduction of poverty (Alleanza per l’Infanzia, 2020). Other studies have shown the positive impact of early childhood investment on society overall, where enrolment in childcare and pre-primary school has an impact in terms of occupation and salaries. Non- cognitive skills acquired at an early age are remunerated in the labour market (Bennett, 2008; Duncan and Te One, 2012). An in-depth analysis of ECEC policy is therefore key from a social investment perspective. This is certainly not a novelty. As illustrated above, there are plenty of studies that investigate the impact of childcare attendance on children’s educational attainment, female employment, and other societal outcomes. Similarly, the political science literature has broadly investigated the politics behind the introduction of childcare policies over time (Häusermann, 2018; Blome, 2017). Yet what is missing in the literature is a comparative analysis of early childhood and education services from a capability perspective, that is, by looking at how different institutional settings impact on the concrete potential for children to enhance their capabilities by attending childcare. 7 7 7 14 July 2022 14 July 2022 While in the first EuSocialCit deliverable we briefly sketched institutional variation in early childhood education and care policy, the aim of this working paper is to examine the institutional features of these social investment policies in detail. To analyse cross-country variation, we take insights from the traditional defamilialisation (Leitner, 2003; Saraceno, 2016; Korpi, 2000) and degenderisation (Saxonberg, 2013; Kurowska, 2018) approaches to child and family policies. 2. The capability approach and a rights-based approach to childcare policies Based on increasing empirical evidence, childcare policy has undergone rapid change and expansion in many European countries since the 1990s, with the dual ambition of increasing investment in children and furthering work-care reconciliation, particularly for mothers. Simultaneously, substantial advancements and improvements in childcare policies were further incentivised by the changing legal landscape, notably at the EU level1, with the emergence of non-discrimination and equal rights’ cases that obliged employers to accommodate workers’ care obligations (Yerkes and Javornik, 2019). Accordingly, scholarly attention has focused on developing analytical frameworks and tools for categorising different models of childcare policy across countries. Most prominently, the literature on (de)familialisation (e.g. Leitner, 2003; Saraceno, 2016) has focused on the extent to which individuals’ welfare is dependent on their family and, conversely, the extent to which families are responsible for providing welfare to other family members. Key policy dimensions examined in this field include the provision of parental leave, formal childcare and care provision for the elderly (Leitner, 2003). Parts of this literature have also explicitly focused on degenderisation as an analytical lens, examining to what extent policy provisions reinforce gender divisions in society, including the division of labour and care (Saxonberg, 2013). While the familialism perspective is a valuable tool for comparative childcare policy analysis, it tends to focus on the family unit, particularly parents, and on the description of policy design. Conversely, less attention has been paid to children and their personal development. To this end, we use an alternative but increasingly applied analytical approach to childcare policy: the capability approach, originally developed by Sen (1992). The central aim of the capability approach is to assess what individuals are effectively able to achieve in life (their ‘capabilities’), depending on the outcomes (or ‘functionings’) they value, and to remove obstacles to enable them to achieve these ends (Yerkes and Javornik, 2019; Gladstone et al., 2021). The capability approach does not define a specific outcome as desirable, but argues for enabling individuals to access a plurality of options depending on what they most value (Yerkes and Javornik, 2019). In this context, a range of individual, institutional and society dimensions may influence the capabilities individuals are able to achieve (Hobson, 2018). The capability approach is not to be considered as a contrast to the literature on defamilialisation and degenderisation, but rather as a valuable tool for refining this (Kurowska, 2018). 1. Introduction Contrary to traditional literature that looked at childcare policies from a family or female perspective, however, we are principally interested in comparing different welfare regimes based on the extent to which they improve children’s capabilities, that is, whether they are accessible, available, adequate, and quality systems. To do so, we look at childcare policies from a rights-based perspective. Since children cannot directly claim for their dues, a rights-based approach to childcare policies means looking at the resources provided to parents and carers to claim for and help their children access high-quality childcare facilities that contribute to the enhancement of their capabilities. Comparing childcare regimes from a rights-based perspective can thus be an original and useful viewpoint from which to understand which resources are concretely provided to parents and carers and ultimately which capabilities the children can benefit from. The rest of the paper is organised as follows: section 2 illustrates our rights-based approach and sets out the main elements of the analytical grid. Section 3 presents the case selection and the methodology used for the analysis. Section 4 presents the results for Italy, Germany and Poland. Section 5 concludes. 8 8 14 July 2022 1 Three recommendations are particularly relevant: the 2013 Council Recommendation on Investing in children: breaking the cycle of disadvantage; the 2019 Council Recommendation on High-Quality Early Childhood Education and Care Systems; and the 2021 Council Recommendation establishing a European Child Guarantee. 2. The capability approach and a rights-based approach to childcare policies In particular, it allows 1 Three recommendations are particularly relevant: the 2013 Council Recommendation on Investing in children: breaking the cycle of disadvantage; the 2019 Council Recommendation on High-Quality Early Childhood Education and Care Systems; and the 2021 Council Recommendation establishing a European Child Guarantee. 9 9 14 July 2022 14 July 2022 the perspectives of children to be taken into account more thoroughly, and to distinguish between policy regimes based on how these strengthen children’s concrete capabilities (Hobson, 2018; Kurowska, 2018). As stressed above, to analytically study childcare regimes and how they enhance, in principle, children’s capabilities, we adopt a rights-based approach. Building on Vandenbroucke et al. (2021), we define rights as a bundle of power resources that confer to individuals the possibility of obtaining conformity from other individuals (horizontal power) and from public authorities (vertical power). Applying the power resources framework to childcare policies might not appear appropriate at first sight. Children do not and cannot claim for something. It is their parents or carers who, both as workers and representatives of the interests of their children, mobilise for childcare policies. Similarly, the public administration does not interact with young children, but rather with parents. In this respect, one might say that parents are ultimately empowered and provided with resources to claim for childcare services. Yet capabilities are developed by children and the fact that parents can claim for a childcare service that is free of charge or not, that is guaranteed from the age of 1 or not, that is provided part-time or full-time, that is subject to high-standard educational guidelines or not, ultimately affects the capabilities of children. This is why adopting the lenses of the power resources approach, zooming in on what parents or carers can claim for, is an original and interesting analytical angle to identify variation in childcare policies across countries and ultimately to understand how this can affect children’s capabilities. The traditional power resource theory (Korpi, 1974) has concentrated mainly on the vertical upward dynamic, which led from individual discontent to collective mobilisation to the evolution of the various welfare regimes and the outputs (benefits and services) and outcomes they have generated (dependent variables). Yet the power resource theory has largely ignored the vertical downward dynamic leading from the formal institution of social rights (macro level) to the concrete improvement of life chances and relational power at the individual (micro) level. 2. The capability approach and a rights-based approach to childcare policies Vandenbroucke et al. (2021) argue that the presence of institutionalised social rights (their content and production), despite being a necessary condition, is not sufficient for the real improvement of people’s life chances. Indeed, what counts is the effective access of individuals to their rights, which the norm itself, however, cannot guarantee. The concrete access to a right requires in fact a direct encounter between the owner of a 10 14 July 2022 10 14 July 2022 right and the administration that provides it. Through this encounter, rights-holders obtain what is due to them: a transfer of money, an exemption, a service2. Building on Vandenbroucke et al. (2021), we argue that the concrete use of a service depends on three sets of normative, instrumental and enforcement resources that parents or carers can be entitled to. First, normative resources are the ensemble of legislative acts that provide a detailed and operational definition about entitlements to a service, as well as the scope of any entitlement and the responsible authority for provision. There are several dimensions of childcare provision that are affected by such legislative provision, including availability, accessibility, affordability and quality (Yerkes and Javornik, 2019). In the first place, there is the question of whether any legal entitlement to childcare exists in a given country, and if so, who is covered by it. A policy that guarantees a childcare place empowers parents to legitimately claim for their children access to childcare from the legal age. The more expansive and universal such a legal entitlement is, the greater the positive effect on children’s capabilities. When a child can be refused a place based on preferential criteria – such as parental employment, income or siblings – service provision may be more restricted (Yerkes and Javornik, 2019). The existence of a right to childcare alone does not necessarily guarantee access to high-quality childcare services; other resources also play an important role. First, normative resources typically encompass funding arrangements for childcare. Depending on the fee structure set out in legislation, affordability of and therefore access to childcare may be influenced. While in some countries, funding support is often means-tested and mostly consists of tax concessions or cash benefits for parents with children in non-parental care, other countries provide more direct funding for childcare provision (OECD, 2020). 2 To explain this idea, the authors explicitly invoke the capability literature and Amartya Sen (1999), who argues that the actual enjoyment of the ‘what’ that is foreseen by a formal right can be made difficult and/or impeded by many obstacles. During a famine, for example, the right to subsistence by means of in-kind benefits (such as the distribution of food parcels), can remain a dead letter if the state is unable to guarantee access to the aid needed in the area concerned. 2. The capability approach and a rights-based approach to childcare policies In some cases, fees are directly set or capped by the government, sometimes based on a sliding-fee scale, with discounts for certain groups (Yerkes and Javornik, 2019). These arrangements will have different effects on the net costs of childcare, and ultimately on the affordability of early childhood education and care for parents, which then might be desensitised to enrol their pupils. A further aspect of childcare policies influencing its quality is the means of provision, which may be specified in legislation. In this respect, four main logics underlie the provisions of services or goods: 2 To explain this idea, the authors explicitly invoke the capability literature and Amartya Sen (1999), who argues that the actual enjoyment of the ‘what’ that is foreseen by a formal right can be made difficult and/or impeded by many obstacles. During a famine, for example, the right to subsistence by means of in-kind benefits (such as the distribution of food parcels), can remain a dead letter if the state is unable to guarantee access to the aid needed in the area concerned. 11 14 July 2022 11 14 July 2022 market provision (for profit), state provision, provision by association (formal private not-for-profit organisations), and private family provision (Brennan et al., 2012). Public and market provision of childcare may have different impacts on the availability and quality of childcare. For instance, in the absence of government involvement, profit childcare may be associated with lower quality, higher costs and rationing (Van Lancker and Ghysels, 2016). Increased marketisation may also favour families with more financial resources, who have advantages in acquiring information about options and navigating the system, so that inequality among service users may be exacerbated (Brennan et al., 2012). Overall, higher levels of government involvement in the form of public or subsided childcare places, legal entitlements and lower net costs have been shown to be related to lower levels of inequality in childcare use (Van Lancker and Ghysels, 2016). Conversely, where structural constraints in childcare provision are more prevalent, children growing up in disadvantaged circumstances tend to be particularly impacted (Pavolini and Van Lancker, 2018). Legislative provision can also address aspects relating to the quality of childcare. Quality can be operationalised both in structural terms (e.g. maximum group size, child-to-staff ratios and the educational level of childcare staff and their working conditions) and procedural terms at the organisational/staff level (e.g. 2. The capability approach and a rights-based approach to childcare policies what happens in the setting: the play and learning environment, child– teacher, quality management and child–child interaction) (Yerkes and Javornik, 2019). Given the poor information available on procedural quality, in this paper we focus on structural quality. All aspects of the legislation should be considered, as it is shown that benefits for children associated with childcare provision are conditional on quality in terms of teachers’ qualifications, child ratios, group sizes and curricula. By contrast, low-quality services may lead to detrimental outcomes in terms of child development relative to home care (Van Lancker and Ghysels, 2016). If codified in the national or regional legislation, the child ratio, group size, curriculum and a minimum qualification for educators can constitute a power resource and a guarantee for parents to claim for compliance and thus enhance the quality of the childcare service and ultimately strengthen their children’s capabilities. To access any service, however, the existence of normative resources is not enough. As shown in the literature on take-up of social rights, many obstacles can prevent the holder of an entitlement from accessing it, such as the complexity of application procedures, insufficient information and fragmentation among levels of administration (Daly, 2002). Therefore, next to normative resources, we also focus on instrumental resources, that is institutional and non-institutional channels that are available to facilitate parents’ access to childcare (Vandenbroucke et al., 2021). In the case of childcare services, families might face several barriers to access, such as lack of knowledge about the existence of a service, or lack of familiarity with application procedures for places and funding. Furthermore, as various studies have illustrated, sometimes barriers to accessing childcare facilities are not only of an 12 12 14 July 2022 14 July 2022 economic nature, but can also be cultural (e.g. scepticism about the opportunity to enrol children too early) that ultimately affect the decision to enrol children (Milan et al., 2020). Instrumental resources include informational channels that may reduce such cultural barriers. In addition, instrumental resources may particularly benefit vulnerable groups within the population, for whom it may be more difficult to access childcare. For instance, an increasing number of studies emphasise migrants’ lack of knowledge about their childcare rights as a barrier to their childcare usage (Seibel, 2021). 3 Each item of expenditure is expressed in 2005 constant prices and converted in PPS for EU-27, to ensure comparability both among countries and across time by discounting for differences in price levels and price fluctuations over time. 2. The capability approach and a rights-based approach to childcare policies Evidence shows that migrant parents tend to acquire knowledge about their childcare rights either via formal information platforms, such as governmental webpages, childcare information brochures, or through informal networks (ibid.). Finally, access to childcare services can be related to a public administration’s lack of compliance. Enforcement resources are channels providing avenues for individuals to enforce the supply of a service in the case of non-provision or disputes (Vandenbroucke et al., 2021). Where children are entitled with a legal right to childcare services, their families may then claim and call for enforcement channels, such as civil trials. As demonstrated by recent disputes in Germany (DW, 2016), the existence of a legal entitlement to childcare gives children and their families additional resources to legitimately claim for the provision of a service and thus further increase, in principle, the accessibility of childcare services. Indeed, in Germany, where some local authorities failed to provide access to childcare facilities, families took the case to court and received a pecuniary compensation for the non- service. Table 1 summarises the dimensions we selected to operationalise the three sets of resources outlined above. 13 13 14 July 2022 Table 1. Indicators used to analyse childcare policies from a rights-based approach Type of resource Dimension Operationalisation Normative resources Existence of legal entitlement to childcare Yes/No Scope of legal entitlement to childcare Universal/Restricted to specific groups based on preferential criteria/Not applicable Main providers of childcare Public/Private for profit/Private non-profit/Mixed provision Funding mechanisms Public funding/Public funding with parental fee contributions/Mixed/No public funding Support policies Tax deductions/Allowances/None Funding provisions for disadvantaged groups None/Staggered fees for disadvantaged groups/Free provision for disadvantaged groups Main providers of childcare Public/Private for profit /Private non-profit/ Mixed provision Regulation on structural quality provision None/Regulation on child-staff ratio/Group sizes/Regulation on staff qualification/Regulation on hours of provision/Educators’ salaries Extent of regional variation in provision Low/Medium/High Instrumental resources Existence of resources for improved access to childcare National programmes/Some regional provision/Local services/Private initiatives/None Existence of specific initiatives to reach out to groups with lower access to childcare National programmes/Some regional provision/Local services/Private initiatives/None Enforcement resources Existing judicial procedures for claiming childcare Yes/No Source: Authors’ composition. 14 14 14 July 2022 Figure 1. Budgetary effort (per capita, in euro PPS) in childcare facilities over time (lhs panel) and enrolment rate 2005- 2018 (rhs panel) Figure 1. Budgetary effort (per capita, in euro PPS) in childcare facilities over time (lhs panel) and enrolment rate 2005- 2018 (rhs panel) Figure 1. Budgetary effort (per capita, in euro PPS) in childcare facilities over time (lhs panel) and enrolment rate 2005 2018 (rhs panel) Source: Authors’ composition. 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 IT DE PL 2004-2008 2009-2013 2014-2018 0 5 10 15 20 25 30 35 40 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Proportion (%) Germany Italy Poland Figure 1. Budgetary effort (per capita, in euro PPS) in chi 2018 (rhs panel) Source: Authors’ composition. 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 IT DE PL 2004-2008 2009-2013 2014-2018 0 5 10 15 20 25 30 35 40 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Proportion (%) Germany Italy Poland Source: Authors’ composition. Traditionally, the three countries considered have been grouped as part of the same explicit familialism regime, as they were lagging behind in the development of family policies and suffered from a modernisation deficit in this field. The varieties of familialism literature labelled Germany, Poland and Italy as welfare regimes that strengthen the role of families in caring for children through highly generous income support measures, but ones that lack provision of alternative policies that unburden families from their caring responsibilities, such as childcare services (Szelewa, 2017; Saraceno, 2016). While this is true for the early 2000s, all three countries undertook a series of important reforms in the 2000s and after the Great Recession in the direction of strengthening the provision of childcare services and degenderising family policies (see next section for more details). In Germany, the first significant development in childcare policy had already occurred in the 1990s, while the first major interventions were in the 2000s. In Poland, a first order change with respect to childcare services occurred only in 2011. Finally, in Italy, a paradigmatic change occurred only between 2015 and 2017. Against this background, in what follows, we are interested in exploring how concretely parents have been empowered to claim for childcare services and consequently how children’s capabilities have been strengthened. Our purpose is descriptive and comparative and not explanatory. 3. Case selection and methodological approach We apply our analytical grid to three case studies: Germany, Italy and Poland. Based on the results of Baiocco et al. (2021), these countries figure in three different clusters of social investment strategies, and indeed their budgetary efforts in early childhood education differ significantly in the time periods 2004-2008, 2009-2013, and 2014-2018 (see Figure 1, left panel). The budgetary effort variable is expressed in per capita terms, scaled down to the target population, and measures the general government public spending per possible beneficiary, that is, in the case of childcare, the total spending per children aged 0-33. Germany is the country that invests the most in early childhood education per number of pupils aged 0-3, with a significant increase between the early 2000s and the post-Great Recession period. Next is Poland, which experienced a net increase of spending per targeted population in the immediate period after accession to the EU. Italy has the lowest budgetary effort in childcare, which remains on average the same over the three periods before, during and after the financial crisis. We juxtapose these figures with data on enrolment rates for children under 3 years in the three countries over the same period. Though it is notable that none of the three countries achieve the Barcelona target of 33 % enrolment for children under 3 – with Germany coming closest – there has been a significant increase in enrolment rates in Germany and Poland. Yet the most noticeable trend is that of Italy, where enrolment rates appear comparatively high compared to the other two countries and relatively stable with some ups and downs. 3 Each item of expenditure is expressed in 2005 constant prices and converted in PPS for EU-27, to ensure comparability both among countries and across time by discounting for differences in price levels and price fluctuations over time. 15 14 July 2022 4. Results In the previous sections, we have set out our analytical framework, detailing how the capabilities approach provides a normative framework for focusing on policy to increase children’s access to early childhood education and care. We have also used the power resources framework to derive and operationalise indicators of the design of ECEC, consisting of three essential dimensions: normative resources, instrumental resources, and enforcement resources. We now apply this analytical grid to three country case studies - Germany, Italy and Poland – drawing on desk research and the interviews with national experts. Our analysis first provides a brief description of the management of ECEC in each country and then zooms in on the three types of resources. We focus in particular on ECEC for children under 3, which has been an area of policy focus in the three countries in recent years. The Appendix provides further detail on the three case studies in the text. Figure 1. Budgetary effort (per capita, in euro PPS) in childcare facilities over time (lhs panel) and enrolment rate 2005- 2018 (rhs panel) We do not aim to explain how the design of early childcare policies in the three countries can ultimately explain children’s take-up of these services. Neither do we want to explain the interaction between institutional inputs and budgetary outputs. The relationship between childcare spending effort (explored in our EuSocialCit deliverable D3.1) and power resources inputs (explored in this deliverable) 16 14 July 2022 14 July 2022 and their interplay impact on childcare take-up, that is, the outcome, will be explored in our forthcoming paper (EuSocialCit D3.5). Methodologically, in this paper, the analysis makes use of a range of qualitative and quantitative data. First, for each country, we carry out an evaluation of the ‘state of the art’ of the current set of power resources. Official documents and national reports concerning the progress of reforms, and the laws and regulations associated with these, are the object of our investigations. Data are collected from various national administrative and statistical sources: Bertelsmann Stiftung, the Italian National Institute of Statistics (ISTAT), the Polish Labour Force Survey, the Polish Central Statistical Office, Eurydice Comparative Reports and the MISSOC databases. In addition, 10 semi-structured interviews were carried out with country experts: 4 experts were interviewed in Italy, 4 in Germany and 2 in Poland. 17 17 14 July 2022 4.1.1. Normative resources As described above, normative resources define the entitlements to early education and care set out in national legislation, and as such, may have crucial influence on children’s educational capabilities. The first step towards the right to childcare in Germany was made in 1996, when a right to ECEC for children aged 3 and older was first codified into legislation (West, Blome and Lewis, 2020). The Daycare Expansion Act (Tagesbetreuungsausbaugesetz, or TAG) from 2005 was the first significant step towards expansion of ECEC for children under 3, as the first legislative effort to determine concrete goals for the number of childcare places for children (Scholz et al., 2019). A very significant development occurred in 2008, when the Child Support Law (Kinderförderungsgesetz, or KiföG) established a universal legal right to ECEC for all children from the age of 1, to officially come into force on 1 August 2013 (BPB, 2021). In addition to the universal right to ECEC from age 1, the 2008 law also laid down a gradual expansion of supervision and care offers for children under the age of 3 to meet the increased demand (Eurydice, 2021). In addition to the existence of a right to childcare, the mix of providers and design of funding structures can hold central importance in influencing access. Funding structures for ECEC in Germany are very complex and characterised by regional variation due to decentralisation, though they generally rely on a mix of public and private sources (FiBS, 2016; Scholz et al., 2019). The municipality is usually responsible for most of the funding for ECEC, with limited involvement from the regional and, particularly, federal government. This means that funding, and therefore access and quality, depends strongly on place of residence, as highlighted by experts. Though there is a universal entitlement to a place in care, ECEC is generally not free of charge (Eurydice, 2021). The level of contributions depends, for instance, on the region, parents’ financial circumstances, the number of children and attendance time (ibid.). In some Länder, there are general subsidies for all children or free amounts for some years or families with several children, while elsewhere, provision is completely free of charge, at least for some hours (Bertelsmann Stiftung, 2021). There are also varying regional funding arrangements for lunch provision and additional services (ibid.). 4.1. In Germany, ECEC is part of the public welfare system, where responsibility is shared between the federal government, the 16 state governments and the municipalities (Linberg, Baeumer and Rossbach, 2013). The governance structure of ECEC in Germany is complex and decentralised, relying on a system of multilevel governance (Oberhuemer, 2014). At national level, the German ECEC system is the responsibility of the Federal Ministry for Family Affairs, Senior Citizens, Women and Youth (BMFSFJ) (OECD, 2016), with some responsibility for the Ministry of Education and Cultural Affairs (Eurydice, 2021). However, the Federation only sets the general framework for ECEC, with the national legislative framework on ECEC contained within the Child and Youth Services Act in Book Eight of the Social Code (Achtes Buch Sozialgesetzbuch) (Scholz et al., 2019). While the national level sets binding framework conditions and guidelines for childcare services and defines the key tasks of ECEC, no concrete content requirements beyond general guidelines are provided (ibid.). Rather, the 16 German Länder are responsible for adapting the framework conditions set by federal legislation into state-specific ECEC laws (Oberhuemer, 2014) and have regulatory competence, meaning that there are 16 state-specific frameworks for the funding and licensing of ECEC, as well as for setting standards and developing curricula (Scholz et al., 2019). Finally, the responsibility for the planning, organisation and provision of ECEC lies at the local level of municipalities, who also have responsibility for securing funding for ECEC (Scholz et al., 2019). 18 14 July 2022 ECEC in Germany is provided in daycare centres (Kindertageseinrichtungen) for children under 3 years (Krippen), in Kindergärten for 3 to 6 year-olds or in mixed-age facilities (BPB, 2021). Increasingly, ECEC in Germany is provided in daycare centres (Kindertageseinrichtungen) for children under 3 years (Krippen), in Kindergärten for 3 to 6 year-olds or in mixed-age facilities (BPB, 2021). Increasingly, 18 18 14 July 2022 14 July 2022 Kindergärten are expanding their services to younger children so that the traditional formal distinction between Krippen and Kindergärten no longer applies in many cases, and the mixed-age model is more common (Linberg, Baeumer and Rossbach, 2013). In addition, there is the option of Kindertagespflege, home-based child-minding services by qualified individuals, which is particularly common for children under 3 (Eurydice, 2021). 4.1.1. Normative resources Fees remain a barrier to take-up of ECEC (BPB, 2021) – as highlighted in interviews, fees vary enormously, even across municipalities in the same region, and are sometimes very high. The median cost of care for children under 3 in Germany is EURO 214 per 19 14 July 2022 14 July 2022 month, while that for children over 3 is EUR 100 per month (DJI, 2021). However, fees for under 3s can vary from little to no fees to more than EUR 300 per month, depending on the region (ibid.). Fees are a particularly significant barrier to entry for socioeconomically disadvantaged families. 31 % of German families with children under 3 and with a low net equivalent household income indicate that fees are a barrier to accessing ECEC (DJI, 2021). Through the 2019 Good Day-care Facilities Act (KiQuTG, ‘Gute-Kita-Gesetz’), since August 2019, parental fee contributions are required to be staggered according to social criteria, including parental income (Eurydice, 2021). Nevertheless, the precise determination of how fees are staggered depends on the Land-specific legislation. Over time, progressively more regulations to make ECEC affordable have been introduced, and in some Länder, care is already completely free, depending on age and scope of care (ibid.). As a result of the increased investment in provision and more generous entitlements, take-up of ECEC has increased significantly in Germany in recent years (Appendix, Figure A2.1). Attendance for children aged 3-6 is almost universal. Moreover, between 2006 and 2015, the proportion of children aged 1 and 2 in care (daycare centres and childminding services) – that is, children covered by the legal entitlement to care – increased from 29.4 % to 48.5 %, with further increases between 2015 and 2020. However, a lack of places for children under 3 remains an issue in some Länder, with provision particularly low in the West (Appendix, Figure A2.2). Survey data indicate that the demand for childcare places by parents remains significantly higher than available supply, particularly in Western Germany (Anton, Hubert and Kuger, 2020). On average across Germany, the gap between indicated demand (49 %) and actual take-up of places (34.3 %) amounts to 15 percentage points (DJI, 2021). In addition to regional inequalities, there are socioeconomic disparities in access. Parents of children under 3 from lower income families remain less likely than those from other families to use ECEC (West, Blome and Lewis, 2020). 4.1.1. Normative resources In principle, the diversity in providers was evaluated as positive by experts, as it increases choice and options. Yet studies have shown that social segregation can already be observed among young children in ECEC, as care providers exhibit marked differences with regard to, for instance, the proportion of children coming from poor families or children with native languages other than German (Hogrebe, Mierendorff and Nebe, 2021). Though research in this area is still developing, there is some evidence that these segregational tendencies could be linked to the German provider structure, as groups of providers differ with regard to the access criteria they prioritise, which include the family situation and age of the child, but also subjective criteria such as alignment with the provider institution’s values or pedagogical concept (ibid.). Moreover, the heterogeneity of quality in provision across providers is a concern. While large associations of providers have a large infrastructure and can provide high-quality training for staff, for instance, this is not the case for small providers. Indeed, in addition to normative resources that affect access to care, legal provisions on the quality of ECEC are of importance. The central motif in the German case is that of regional variation. While the Social Code does not define a specific time entitlement to care, 10 of the 16 Länder define a minimum number of hours per day, with additional variation at regional level (Bertelsmann Stiftung, 2021). Entitlements tend to be more generous in Eastern Germany, where childcare use was historically more prevalent than in the Western part of the country. There are also no national requirements regarding the child-staff ratio, which exhibits large regional variation according to Länder-specific regulation (BPB, 2021). However, it should be noted that there are efforts to improve the quality of childcare across Germany, with the national government increasingly involved. In 2019, the Good Day-care Facilities Act committed to federal support of EUR 5.5 billion from 2019 to 2022 to implement measures in 10 qualitative fields (which the Länder can select from), such as decreasing the specialist-child ratio, in addition to decreasing and socially staggering fee burdens. As regards quality provisions directed at children from socioeconomically disadvantaged backgrounds, the focus is mainly on fee staggering, but there may also be other entitlements. 4.1.1. Normative resources Moreover, although there have been large increases in the proportion of children with a migration background in ECEC for under 3s in recent years (Olszenka and Riedel, 2020), they continue to be under-represented in German ECEC relative to other children (Bertelsmann Stiftung, 2021), even in families that indicate that they are in need of a place (Anton, Hubert and Kuger, 2020). In addition to fees, the mix of childcare providers may influence access. In Germany, the provision of ECEC is strongly governed by the subsidiarity principle – that is, public authorities are only obliged to provide social services if non-governmental agencies are not in a position to do so (Oberhuemer, 2014) – and by the principle of diversity of providers (Scholz et al., 2019). Only about one-third of children in Germany are in publicly provided care, with the majority provided for by not-for-profit private organisations (Bertelsmann Stiftung, 2021). In particular, non-profit private organisations active in 20 20 14 July 2022 14 July 2022 childcare provision are churches and welfare organisations, while private for-profit providers are very rare (West, Blome and Lewis, 2020). Providers have to have public recognition but have freedom in designing the content and method of care that they provide (BPB, 2021), within the broad objectives set at national level and the framework specified by the Land (Eurydice, 2021). In principle, the diversity in providers was evaluated as positive by experts, as it increases choice and options. Yet studies have shown that social segregation can already be observed among young children in ECEC, as care providers exhibit marked differences with regard to, for instance, the proportion of children coming from poor families or children with native languages other than German (Hogrebe, Mierendorff and Nebe, 2021). Though research in this area is still developing, there is some evidence that these segregational tendencies could be linked to the German provider structure, as groups of providers differ with regard to the access criteria they prioritise, which include the family situation and age of the child, but also subjective criteria such as alignment with the provider institution’s values or pedagogical concept (ibid.). Moreover, the heterogeneity of quality in provision across providers is a concern. While large associations of providers have a large infrastructure and can provide high-quality training for staff, for instance, this is not the case for small providers. 4.1.1. Normative resources In some Länder, entitlements are expanded, for instance for younger children or additional hours, depending on the position of the parents or if the children have any special requirements (Bertelsmann Stiftung, 2021). In addition, there has been increased investment in the development of language diagnostics and support in daycare to improve linguistic competence, particularly for children with a migrant 21 21 14 July 2022 14 July 2022 background or with deficiencies in language development (Eurydice, 2021). Nevertheless, there remain issues relating to quality in ECEC for children from socioeconomically disadvantaged groups, including children from families with lower financial means and children with a migrant background. These socioeconomic disparities appear to have been intensified, rather than weakened, in the course of the large childcare expansion Germany has undergone in recent years (Stahl, 2015). Evidence indicates that children from migrant families tend to attend facilities with lower performance in structural and procedural quality indicators and a less privileged social composition, though more research on this is necessary (ibid.). Moreover, in practice, the pedagogical approach in daycare seldom devotes space to multilingualism and may even marginalise children with a multilingual background (Olszenka and Riedel, 2020). Finally, there are national standards regarding the qualification of personnel in ECEC. The qualification to become a state-recognised pre-primary educator takes three years of post-secondary vocational training, as well as either a course as a childcare assistant or training and work experience in the field (OECD, 2016). Though there has been a move towards the academisation of ECEC qualifications, this process has been slow (Rauschenbach and Riedel, 2016), and the number of staff with tertiary education remains marginal (Oberhuemer, 2014). In interviews with experts, the lack of qualified staff was highlighted as a major issue for the provision and quality of ECEC in Germany, which will likely be exacerbated in future years. In staff surveys, one in five leaders of educational institutions name staff shortages as a barrier towards providing high quality education and care (OECD, 2018). Expert interviews showed a concern about a decreasing trend in quality of provision even while maintaining the number of places, leading to a situation where the educational role of ECEC is no longer fulfilled. In particular, the lack of entitlements relating to quality at national level results in significant heterogeneity across regions, so that the quality that children are de facto experiencing depends on where they live. 4.1.2. Instrumental resources In addition to normative resources – the legal provisions setting out the right to ECEC for children and what exactly it entails – instrumental resources may be essential in increasing access by providing children and their families with the necessary information and assistance to gain access to care. As stated previously, in Germany, the concrete implementation of measures in ECEC, including measures to facilitate delivery and access, depend on the state and local level. Generally, when parents are looking for a place in (centre-based) daycare, the first step – often during pregnancy – is to seek out local daycare centres, where they can find information and be placed on a waiting list (Schettler, 2016). In addition, in most Länder, regulation specifies that the need for a space has to be registered 22 14 July 2022 14 July 2022 with public authorities, usually the youth and welfare services, who may then provide information on how best to access a place in ECEC (ibid.). Increasingly, there are online tools to facilitate the process of parents registering their need for a place in ECEC, which can increase parents’ access to information and understanding of the process; however, this is not available everywhere (ibid.). Additional measures to raise awareness and provide information on quality are location dependent. As was also pointed out in interviews, there is currently little evidence on the existence of informational and other instrumental resources. Several regional examples of efforts to facilitate access to ECEC do exist, for instance the Kita-Navigator provided by the city state of Berlin4, where parents can search for available places and filter according to their preferences. However, systematic initiatives to provide information and increase access are generally lacking on a national level. In general, it can be concluded that a systematic approach to helping parents decrease barriers to access is lacking. Rather, the onus is on parents to seek out the public authorities, where they can then receive information. In a context where demand for ECEC continues to outpace supply, this lack of systematic information provision can be an impediment to accessing the right to ECEC. Informational barriers related to access to ECEC may be particularly pronounced for children from migrant families or families where parents do not speak German. 4.1.2. Instrumental resources As highlighted by experts, parents in these families may often not be aware of their entitlements to childcare or may not know how to access these entitlements. Here, a greater effort has been made to increase outreach. For instance, there is the federal programme, Daycare Entry: Building bridges for early education (Guter Kita- Einstieg: Brücken bauen in frühe Bildung), which initially targeted mostly refugee children but has since been opened up to other groups, such as families that are economically at risk or in precarious living situations5. The aim of the programme is to provide information on ECEC to these families and thereby facilitate access. There are also local initiatives to increase access for disadvantaged groups, though this is not the case everywhere. 5 For more information see https://www.bmfsfj.de/bmfsfj/service/publikationen/bundesprogramm-kita- einstieg-bruecken-bauen-in-fruehe-bildung--118650. 4 For more information see kita-navigator.berlin.de. 4 For more information see kita-navigator.berlin.de. 4.1.3. Enforcement resources Finally, enforcement resources may play a role in influencing access to ECEC, in that they enable legal avenues for families to take in case their right to childcare is denied. As stated previously, in Germany, the legal right to a place in ECEC is codified in national legislation from the age of 1, and local maintenance bodies are therefore obliged to provide a place for all children. If the legal entitlement 4 For more information see kita-navigator.berlin.de. 23 14 July 2022 g 5 For more information see https://www.bmfsfj.de/bmfsfj/service/publikationen/bundesprogramm-kita- einstieg-bruecken-bauen-in-fruehe-bildung--118650. 5 For more information see https://www.bmfsfj.de/bmfsfj/service/publikationen/bundesprogramm-kita- einstieg-bruecken-bauen-in-fruehe-bildung--118650. 5 For more information see https://www.bmfsfj.de/bmfsfj/service/publikationen/bundesprogramm-kita- einstieg-bruecken-bauen-in-fruehe-bildung--118650. 14 July 2022 to care cannot be honoured, then parents have the right to sue these authorities for a place (Schettler, 2016; Eurydice, 2021). In October 2016, the German Constitutional Court (Bundesgerichtshof) ruled that parents may be entitled to compensation for lost earnings from their local authority if a place in daycare for their child cannot be provided (Spiegel, 2016). However, there may be specific cases where the municipality can justify the lack of places, for instance if agreed contracts were unfulfilled by contracting partners. Nevertheless, while the right to sue for a place in ECEC exists, in practice, few families actually make use of this avenue, as pointed out by experts. Currently, around 1 000 court cases relating to the legal entitlement §24 SGB VIII are listed6. As stated by experts, however, this does not reflect the overall number of complaints, as not all are accepted by courts because they may be legally insecure, or because local authorities find a place before the legal process commences. There are barriers that explain parents’ reluctance to sue, such as an unwillingness to cause a confrontation in the local community, perceived costs, time until the decision, and the chance of losing. Moreover, the families that do sue tend to be those in socioeconomically advantaged positions, who can afford to invest the time and other resources needed for such an endeavour. Hence, suing for a childcare place should not be seen as an effective avenue to increase access to childcare. At the same time, the existence of such a legal avenue may have contributed to increased provision of places. 4.1.3. Enforcement resources As laid out in the expert interviews, many municipalities increased their provision in anticipation of the establishment of the right to childcare for children under 1, and the potential suits associated with it. Hence, while enforcement resources do not in themselves appear as an effective way of accessing the entitlement to ECEC, they can have an indirect effect by increasing the bargaining and legal power of families relative to public authorities. 4.2. In Italy, crèches have traditionally been under the responsibility of local and regional authorities since 1971, when they were first defined as social services of public interest (Law No 1044). In recent years, however, a significant change occurred when an integrated system for early childhood education and care for children aged 0 to 6 years was introduced for the first time (Law No 124/2015 and Legislative Decree No 81/2017). The new system consists of two main pillars: the nurseries or crèches (nidi di infanzia) and the kindergarten (scuole d’infanzia). Importantly, this legislative change constituted the first time that the State, particularly the Ministry of Education (MIUR), assumed responsibility for services for children under 3. Although not directly involved in service management, the MIUR 24 14 July 2022 6 https://dejure.org/dienste/lex/SGB_VIII/24/1.html 6 https://dejure.org/dienste/lex/SGB_VIII/24/1.html 24 14 July 2022 exercises an important role in planning and coordination, as well as financing for the system. While the governance of the system remains decentralised – as will be set out in the analysis of power resources – there is evidently a move towards homogenising the quantity and quality of childcare services offered across the Italian regions. Around the same time, legislation was also introduced to formally recognise nurseries and supplementary services as services of public interest (Law 107/2015 and the subsequent Legislative Decree No 65/2017). Overall, it is evident that early childhood education and care is increasingly recognised as an important policy field within the Italian context, with the national government gradually assuming more responsibility. 4.2.1. Normative resources 4.2.1. This figure, which is equal to one-fifth of the expenditure incurred to provide these services, varies greatly in the different areas of the country: it is highest in the Centre-North, where it is around EUR 1 600-1 700, with a peak of almost EUR 1 900 in the North-East, while it drops to around EUR 600- 700 in the South. Research indicates that despite the availability of financial support measures, 12.8 % of families who do not use nursery services cite financial barriers as a reason not to take up care services. The fragmented governance and funding of childcare services across Italian regions means that the enrolment rate for children aged 0-3 is relatively low, at 29.6 %. In addition, there is significant variation in enrolment rates across regions (Appendix, Figure A1.1). While the North-East (34.5 %) and the Centre (35.3 %) have a relatively high coverage, rates are lower in the North-West (31.4 %) and particularly in the South (14.5 %) and the Islands (15.7 %). Coverage also tends to be higher in urban areas. In addition, financial barriers may be particularly significant for children from socioeconomically disadvantaged families. However, in Italy there are no particular provisions, including financial support, for children from disadvantaged backgrounds. At the same time, evidence shows that these children do indeed have significantly reduced access to childcare services relative to those in more economically advantaged positions. Recent studies by ISTAT (2020) indicate that for children belonging to the poorest brackets within the population (first and second quintile of income distribution), the enrolment rate in nurseries remains just below 14 %, while in the third and fourth quintiles it increases to 20 % and to 26 % respectively, reaching 35 % in the top quintile (ISTAT, 2020). Hence, substantial inequality in access to childcare across income groups remains in Italy. Inequality in access to childcare may also be influenced by the structure of providers. Both public and private nurseries exist in Italy, with public nurseries run directly by municipalities in accordance with regulation defined at local level. Alongside these facilities, there are also services run by private not- for-profit or for-profit organisations, which may receive municipal funding, as well as fully private offers, mainly financed through parental fees. Across Italy, services remain roughly equally split between private and public providers, though there is significant regional variation (Appendix, Figure A1.1). 4.2.1. As set out in the theoretical framework, a very significant part of the policy configuration in ECEC are normative resources, the legislative acts that define the entitlement to a service and the scope of such entitlements. Within the context of ECEC, the first important dimension of normative resources is whether a legal entitlement to ECEC exists. In Italy, there is no formal entitlement to a place in education and care. Rather, to access a place, families submit a request of admittance to childcare services at the relevant local office responsible for the organisation of ECEC services. Depending on availability in their region, in principle, families have freedom of choice of the facilities. Considering requests and admittance requirements established at local level, municipalities make priority lists based on criteria defined at local level in order to regulate admissions, and then allocate places. Given the lack of formal entitlement, a place in ECEC is not guaranteed for children. Nevertheless, there are other significant provisions under the umbrella of normative resources. First, as discussed in the analytical framework, the funding structure of ECEC is significant in affecting access to childcare services. Prior to the 2017 reform, the state intervened in ECEC only with extraordinary funding, but has become more involved in funding provision for ECEC since then. As part of the funding structure for ECEC, different types of financial support measures to help families in financing ECEC are available in Italy. These include a 19 % deduction of the cost of nursery school fees, up to EUR 632 per year per child, and the bonus asilo nido (nursery school bonus), which consists of an annual voucher of EUR 1 000 (increased to EUR 1 500 in 2019) to reimburse the cost of attending a nursery school. In 2019, 38 % of families’ expenditure on nurseries was covered by the bonus and another 5 % by deductions. However, the cost of childcare services to parents remains substantially high (Appendix, Table A1.1). 25 14 July 2022 25 14 July 2022 Moreover, there is significant regional variation, as the setting of fees is left to municipalities’ discretion. On average, in Italy, families pay EUR 1 581 per year for each of their children enrolled in nursery. 4.2.1. Research suggests that the high share of private providers affects the affordability of childcare services (Alleanza per l’Infanzia, 2020), therefore constituting a barrier to access. In addition to normative resources, particularly funding provisions, that influence access to childcare, regulation on the quality of childcare services is very important. Again, there is significant regional variation. Each municipality is responsible for the organisation of the educational services for early 26 14 July 2022 14 July 2022 childhood, in compliance with regional regulations, and also develops its own educative project. These plans are then implemented by nurseries within the framework set by the municipality. Regulation on important aspects of quality, including the child-staff ratio and group sizes, is established at regional level. Finally, a further dimension of normative resources on quality of ECEC are regulations regarding staff qualifications. In contrast to other dimensions of quality, national regulation exists in this respect. With the legislative decree 65/2017, a requirement for educators to have a university degree was introduced for both public and private nurseries. However, in expert interviews, the bad working conditions and wages of early childhood educators was highlighted as a cause of shortages in qualified teaching personnel. Working conditions of educators change significantly depending on whether they are employed in publicly managed services or in private ones (Neri, 2016). Leon et al. (2019) show that in 2015 29.3 % of the teachers in childcare had non-standard contracts and 30.3 % worked part-time, despite having stayed with the same employer for 12.3 years on average. In terms of wages, there is a significant monthly wage gap between childcare teachers and their pre-primary colleagues of around 15 %. While quality regulation related to the qualification of educators therefore does exist, the staff shortages within the ECEC sector pose a significant challenge for maintaining quality in the future. 4.2.2 Instrumental resources Instrumental resources constitute the second type of resource we consider. These include targeted or universal support channels, which facilitate individuals’ access to social rights. In the absence of an individual entitlement to access to childcare or pre-primary school, as is the case in Italy, instrumental resources consist in those procedures, information and support channels that support families in overcoming the barriers that still hamper the access to the service. Significant barriers to access to childcare do exist in the Italian case. In addition to financial barriers and regional inequalities, as set out above, these also include informational barriers, and in some cases cultural barriers. Instrumental resources could play an important role here in increasing access to information about ECEC and therefore incentivising take-up. Despite this, we generally find no evidence of systematic provision of such resources in Italy. There are some local or regional initiatives, for instance, the Emilia-Romagna region has a dedicated website and service, Informa Famiglie e Bambini, where support is provided to families who want to access to ECEC services. A particularly good example is that of Reggio Emilia, where the municipality informs new parents, even before they give birth, about the opportunity to enrol their children in nursery. A childcare information kit is sent to the new parents with support for the administrative procedures. As stressed by our interviews, however, this ‘door-to-door’ information service is a peculiarity of few 27 27 14 July 2022 14 July 2022 municipalities, mostly in the Centre (Bologna) and North of Italy (Milan). Other information activities are carried out by the organisations that manage the private not-for-profit childcare services. For instance, the FISM (Federazione Italiana Scuole Materne) raises awareness around the educational and social services provided by the kindergarten. Nevertheless, overall, there is little systematic provision of information about access to ECEC. Informational barriers may be particularly prevalent for children from socioeconomically disadvantaged families. For instance, as observed by Favaro (2013), the enrolment of pupils aged 3-5 in pre-primary school reduces significantly among non-Italian citizens (80.2 %, compared with 93.6 % of Italians). 4.2.3 Enforcement resources As no legal entitlement to childcare exists in Italy, there are no remedy procedures in place for cases where children do not have access to services. 4.2.2 Instrumental resources This discrepancy can be attributed to factors affecting both the family (an underestimation of the importance of this educational stage, or the organisational difficulties and the physical distances between home and school, especially for the those that live in isolated situations) and the reception context (the costs of canteen service being unsustainable for all, the difficulties of integrating the children of immigrants, especially where available places are limited or where, in a discriminatory manner, the intention is to discourage the attendance of foreign children). At the moment, however, there are neither institutional nor non-institutional channels to facilitate access and support non- Italian citizens in accessing childcare services. 4.3. In Poland, ECEC is not a uniform system but is rather based on two pillars: the childcare system supervised by the Ministry of Family and Social Policy (children aged 0-3 years) and the pre-primary education system that falls under the Ministry of Education and Science (children aged 4-6/7 years). For children aged 0-3, centre-based childcare is provided in crèches (żłobki) and, since 2011, kids clubs (kluby dziecięce), as well as home-based provision by daycare providers or childminders (opiekun dzienny) and nannies (niania). It is regulated by the Act of 4 February 2011 on the Care for children aged up to 3. For children aged 4-6/7 years, pre-school education is provided in nursery schools (przedszkole), pre-school classes (oddziały przedszkolne) in primary schools and other pre-school education settings. It is regulated by the Act of 14 December 2016, the Law on School Education. 28 14 July 2022 28 14 July 2022 ECEC includes three functions: educational, upbringing, and care provision. However, there is a clear division of competences between particular ministries (the Ministry of Family and Social Policy for 0- 3 year-olds and the Ministry of Education and Science for 4-6/7 year-olds). As regards ECEC for children up to 3, the Ministry of Family and Social Policy is responsible for general monitoring of the implementation of the Act on the Care for Children up to the age of 3, but real implementation of the provisions takes place in communes (local municipalities). Local authorities are responsible for running ECEC facilities for children up to 3 years’ old and supervising the whole system of ECEC facilities (private and public) on its territory. Hence, overall implementation is highly decentralised. 4.3.1 Normative resources The first aspect to consider when analysing the normative resources in childcare is the existence of a right to care. In Poland, children aged 3-5 are entitled to a place in a pre-primary setting in their locality, with a year of compulsory pre-school at age 6. However, for children under 3, there is no legal guarantee of a place in ECEC. There is no single national rule for crèche or kids club recruitment. It depends on the institution and can be found on the website of a given institution, city or commune office. As such, access to a place cannot be guaranteed and will depend on local provision and guidelines. However, in addition to legal entitlements to a place in childcare, the design of other normative resources can also significantly influence access and quality. Funding structures are significant. In Poland, funding for care services for children aged up to 4 comes primarily from the budgets of communes and other local government units, targeted state grants and EU structural funds. Generally, nurseries receive funding from the local governance unit for each child in the facility. Targeted state grants (e.g. the Maluch+ Programme) can be allocated for either the creation of new facilities, the creation of new places in existing nurseries and the subsidising of existing places to reduce fees. Moreover, there are specific financial support measures for parents. From 1 April 2022, parents have been able to apply for co-financing of their children’s places at institutional care of up to PLN 400. Previously, such direct support for parents was available through the Maluch+ Programme, but it depended on the availability of the funds within the programme. This subsidy will cover a significant part of the cost of childcare for children aged 0-3 but a portion of the cost will still be borne by parents. In two-thirds of public institutions, fees are PLN 600 or lower, meaning that two- thirds or more of the cost of care will be covered by the subsidy. In contrast, among private institutions, the share of institutions with relatively low fees is smaller and fees can exceed PLN 1 000, so the subsidy may be less effective (Table A3.2). 29 14 July 2022 29 14 July 2022 There are also regional disparities in financial support. 4.3.1 Normative resources Parents can be supported by vouchers or subsidies organised by communes, but this is not a formal obligation; it depends on the particular commune. Big and affluent communes provide better access to institutional care for children under 3 by introducing special programmes, for example the Warsaw crèche voucher. Additionally, employed parents can apply for co-financing from the company’s social benefits fund. However, it is not the case that these funds are open and available in every case – the rules of the company’s social benefits fund are the deciding factor here. There may also be further regional financial support, depending on the commune. There are no specific financial support provisions for children from socioeconomically disadvantaged families. As a result of public policy aimed at improving the access to ECEC, 12.4 % children aged 0-2 were covered by ECEC services in 2019, compared with 2 % in 2004 and 4.8 % in 2013 (Appendix, Figure A3.2). For children aged 0-3, the coverage rate rose to 15.4 % in 2019 and 17.5 % in 2020. Nevertheless, demand for care is not met, even though the number of children is decreasing. Moreover, the design of the Polish ECEC system exhibits significant regional inequalities in access. The coverage rate in 2020 for children 0-3 years’ old varies greatly by regions. It ranges from 11.3 % in the Świętokrzyskie Province, 12.2 % in Warmia and Masuria, and 13.1 % in the Lubelskie Province, to 25.6 % in Lower Silesia, 21.4 % in the Opolskie Province and 21 % in Mazovia (Statistics Poland 2022). There are still communes where no places of institutional care for children up to 3 exist. There are also strong differences in access to ECEC for children up to 3 between rural and urban areas (especially big cities). In some rural communes, there are no places of institutional care for children up to 3. Costs are also a more significant barrier for disadvantaged groups. In general, women with low and medium education indicate higher barriers than those with tertiary education, especially in the case of the cost of childcare, though a reduction in these barriers has been observed in recent years (Appendix, Figure A3.1). In terms of the type of providers for childcare, creches and kids clubs are mostly non-public. There are approximately three times as many private creches as public institutions, and the dimensions are similar for kids clubs. 4.3.1 Normative resources The differences are less pronounced when it comes to number of places, but private provision still dominates (Table A3.1). Among children aged 4-7, the majority of institutions are run by local municipalities, but 45.1 % of institutions remain privately run (Statistics Poland, 2020). Creches and kids clubs that operate as private companies or are run by a non-government organisation have to comply with standards set in the Act of 4 February 2011 on the Care for children up to the age of 3. They can apply for public grants from the Maluch+ Programme, and after fulfilling the requirements they are generally granted support. In private care settings, financing is based mainly 30 14 July 2022 14 July 2022 on fees from parents, supplemented by other sources. It is not an obligation, but communes can grant subsidies to private care settings, after meeting some specific conditions set by the commune. Generally, private institutions operate mostly in urban areas and demand higher fees from parents than public institutions (Table A3.2). As a result, the access to these institutions is constrained by place of residence and income. In addition to funding structures and the mix of providers, we also examine resources relating to the quality of childcare. In Poland, some national regulations on quality-of-service provision do exist. At national level, there are regulations on group sizes in crèches (Act on Care for children up to the age of 3), set at a maximum of 8 children cared for by 1 childminder in a crèche or kids club. The maximum number is reduced to 5 when a group includes a child with a disability, a child requiring special care or a child under 1 year of age. Crèches with more than 20 children employ at least 1 nurse or midwife. However, the Act on the Care for Children up to the age of 3 does not specify the number of working hours per week for childcare institutions. Working hours in crèches and kids clubs are determined by their organisational regulations, taking into account parents’ preferences. Crèches and kids clubs provide care for up to 10 hours per day. In special cases, at parents’ request, the duration of the stay can be extended for an additional fee. For a daycare provider (home-based provision), working hours are set in an agreement between the provider and the employing entity. 4.3.2 Instrumental resources As set out previously, instrumental resources consist of those procedures, information and support channels that support families in overcoming the barriers that continue to hamper access to childcare services. There are substantial barriers to take-up of childcare services in Poland (Magda, 2020). These include the lack of availability of high-quality childcare services, the difficulties mothers face in finding flexible working arrangements, and financial incentives to stop working, given the cost of childcare. Cultural pressure and social norms also influence decisions about childcare take-up. Instrumental resources could play a role in increasing information about access to childcare services. In Poland, activities that facilitate the delivery and access to ECEC are undertaken locally, mainly in big cities. Many communes create separate websites for educational and care issues. For example, in Warsaw, each woman in hospital receives a layette for the baby she is delivering. This includes small practical gifts and a book with all the necessary information. Moreover, Warsaw provides a separate website (https://zlobki.waw.pl/) with information on crèches and a Warsaw crèche voucher. In the case of nursery schools, there in an electronic system for recruitment in Warsaw. There are also provisions that facilitate the access of children with disabilities to ECEC, although mainly for children from 3 years’ old. Some crèches or kids club offer places for younger children with disabilities or special care needs, but it does not create a coherent structure and is dependent on a particular unit. In Poland there are two distinct groups of NGOs supporting parents with different services, as well as legal organisations. One group of NGOs is aimed at the general population of families with children, and the second integrates parents of children with disabilities. However, they do not have a common structure, so the support provided by them is fragmented. In conclusion, access to instrumental resources is rather fragmented and locally based, and this can be an impediment to accessing ECEC. 4.3.1 Normative resources Overall, the Act on Care for children up to the age of 3 sets basic requirements, leaving much space for improvement. Each facility can decide on additional activities or introduce higher quality standards than required by law. However, it depends on each facility or its leading authority. There are no additional formal/legal requirements on quality at regional level. Additional quality standards are usually introduced by private entities and are connected to higher fees paid by parents. Higher quality entities have been established mostly in cities, where the competition between institutions (mostly private) is higher, and parental demand and financial capabilities are larger. As regards staff qualifications, different types of qualifications are accepted in crèches and kids clubs: nurses, midwives, childminders, pre-school teachers, early school education or pedagogical specialists, and those who have completed a degree in areas relating to early childhood education and care. If employees have completed a specialist child first aid course less than 2 years before undertaking employment, they can also have completed any type of higher education which includes aspects of childcare and development (in addition to 80 hours of training) or secondary or vocational secondary education with at least 1 year’s experience of work with children aged under 3 (or 280 hours of training). Hence, staff employed in ECEC have diverse profiles. Childminders can also have several qualifications. Childminders and child carers employed in creches and children’s clubs are 31 14 July 2022 14 July 2022 employed on the basis of general employment regulations (Labour Code). Recruiting young people to work in institutional forms of childcare is a growing problem. 4.3.3 Enforcement resources As there is no right to childcare for children under three, there is consequently no access to enforcement resources in Poland. 32 14 July 2022 32 32 14 July 2022 Conclusion This paper provides an analysis of early childhood education and care policy in three EU countries: Italy, Germany and Poland. By drawing on the capability approach, and building on an original power resources analytical framework that defines relevant policy dimensions in ECEC policy from a rights- based perspective, we map policy design features in the three countries. Table 2 presents an overview of the relevant indicators in Germany, Italy and Poland. As can be seen, there are both similarities and differences in policy design. Table 2. Summary of childcare power resources in Italy, Germany and Poland Type of resource Dimension Italy Germany Poland Normative resources Existence of legal entitlement to childcare No Yes Yes Scope of legal entitlement to childcare Not applicable Universal from age 1 Universal from age 3 Funding mechanisms Public funding with parental fee contributions Public funding with parental fee contributions Public funding with parental fee contributions Support policies Tax deduction and child bonuses (applied universally) Subsidies depending on Bundesland Allowances Funding provisions for disadvantaged groups None Staggered fees for disadvantaged groups None Main providers of childcare Public and private for profit Public and private not-for profit Public and private for profit Regulation on structural quality provision Regulation on child- staff ratio/group sizes at regional level Regulation on hours of provision at regional level Regulation on staff qualification at national level Regulation on child- staff ratio/group sizes at regional level Regulation on hours of provision at regional level Regulation on staff qualification at national level Regulation on child- staff ratio/group sizes at national level Regulation on staff qualification at national level Extent of regional variation in provision High High Low Table 2. Summary of childcare power resources in Italy, Germany and Poland 33 14 July 2022 Instrumental resources Existence of resources for improved access to childcare Some municipal provision Some municipal provision Some municipal provision Existence of specific initiatives to reach out to groups with lower access to childcare Some local initiatives Some national initiatives and regional provision Local services and private initiatives Enforcement resources Existing judicial procedures for claiming childcare No Yes No Source: Authors’ composition. Source: Authors’ composition. The first aspect of normative power resources examined in detail was the existence of a legal entitlement to childcare. Within the three countries examined, only one country – Germany - has established a legal entitlement to a place in childcare for the under 3s’ age group. The establishment of such a legal entitlement, and the accompanying expansion in childcare places to guarantee the fulfilment of this right, certainly had a positive impact on the ability of children and families to access childcare in Germany, as evidenced by the significant positive trend in take-up of places in the past decade. In the absence of an entitlement to a place, as is the case in Italy and Poland (for children under 3), there may be limited incentives for public administrations to invest significantly in childcare expansion and limited opportunities for families to claim for places in childcare. However, this does not mean that the existence of a legal entitlement to a place in childcare alone is a necessary or sufficient condition to guarantee access for all children. Indeed, for an entitlement to childcare to be effective at increasing access to childcare, it must be accompanied by further resources to reduce the cost of such care for families. In Germany, we find that there is a large regional variation in funding structures, which results in very high fees for care in some regions. This can significantly curtail access to care, even where a place is guaranteed. In contrast, in Italy and Poland, there are more concerted efforts to specifically reduce the cost of childcare through universal subsidies. In Poland, there has been a marked increase in take-up of childcare – albeit starting from very low levels – even without the existence of a legal right to childcare, whereas levels in Italy have been somewhat erratic, though not significantly lower than in Germany. A common finding across the three countries is that funding structures do not affect all children and their families equally. 33 First, the existence of measures for cost reduction and funding support is particularly significant for children from socioeconomically disadvantaged families. Inequality in access to childcare for children from these families continues to be an issue in all countries examined. The development of measures, including financial measures, targeted to vulnerable groups is an 34 34 14 July 2022 14 July 2022 important avenue to consider. Second, there is significant regional inequality in funding support for childcare, which affects both access and quality within facilities. Since funding for childcare services often relies on funding from local municipalities, the quality of childcare provision can strongly depend on children’s place of residence. Stronger financial support measures at national level to equalise funding for childcare across regions could be an important policy measure in this context. Some measures in this direction have already been taken in the countries examined, but they could be further accelerated. Specifically, policy should explicitly seek to channel funding for childcare services towards regions with comparatively lower financial means. Beyond funding structures, the mix of childcare providers may also play a significant role. In principle, having a range of childcare providers is a positive thing, as it increases choice for parents. Yet it may also be associated with inequality in access to and the quality of childcare services. In the two countries where private provision of childcare plays a significant role - Italy and Poland - this tended to be associated with a higher cost of care for parents, and therefore reduced access. Beyond structural factors affecting access to childcare, quality is a significant issue. In two of the countries examined - Italy and Germany - regulation on staff-child ratios and hours of provision for childcare services falls to the regional level, while only Poland has regulation on staff-child ratios at national level. In the absence of national standards on the quality of care, regional inequalities will continue to persist. While the management and administration of services at local level – a common theme across all countries – may be appropriate in the context of the service being delivered, it should be accompanied by central (national/federal) action to reduce regional inequalities, not only in access but also in quality. In all three countries, regulations on staff qualifications in ECEC services exist at national level, though the type of qualification required differs. 33 Putting greater emphasis on high-quality training and education for care staff in ECEC facilities emerged as one of the most significant policy measures to ensure continuing high-quality provision of care services in the country examined. However, all three countries face issues in recruiting and retraining staff in ECEC, particularly highly qualified staff, and this was highlighted as a major issue for the future. In this respect, taking measures to increase working conditions and pay within the ECEC sector is crucial to counteract the issues in staff recruitment. In addition to the formal entitlements, the analysis also examined the role of instrumental resources, that is, institutional and non-institutional channels to aid families in accessing childcare and reduce informational barriers to take-up. In all three countries examined, these resources tend to be underdeveloped and, if anything, are available at local level. Yet instrumental resources may greatly 35 35 14 July 2022 14 July 2022 help in increasing access to childcare services, particularly for children from particular backgrounds, such as families who do not speak the native language in the country of residence. Experience does suggest that specific programmes aiming to ease their access to education and care services can significantly increase take-up. This illustrates the role that instrumental resources could play in improving access to ECEC, particularly for disadvantaged groups. Finally, the empirical research also investigated the role of enforcement resources, that is, legal channels for individuals to claim their entitlement to childcare. In Germany, the only country where children under 3 have a right to childcare, such channels do exist. In practice, enforcement resources appear of lower relevance in the context of childcare policy, as only a few families – those with sufficient resources – use legal channels to claim for their place in childcare. However, the existence of enforcement resources may have an indirect effect, in that the threat of a potential suit puts pressure on administrations to invest in the development of facilities in order to meet their legal obligations. In this sense, enforcement resources do provide families with additional leverage in claiming for a childcare place. Overall, the analysis allowed us to shed light on the variation in the institutional design of three childcare regimes that, based on traditional literature, are grouped under the umbrella of familialism. 33 Even though they exhibit different levels of budgetary effort in childcare policies, they are all characterised by a performance below the Barcelona targets in terms of enrolment of children under the age of 3. We provided an original analytical grid that links the rights-based approach with insights from the capability approach literature. The resources-based approach that draws on Vandenbroucke et al. (2021), and is here operationalised, can serve as a grid for the assessment of the quality of childcare policies. In the context of the EuSocialCit project, an forthcoming working paper links the institutional design of childcare policies with the public budgetary effort, and studies their interaction and the impact on the final take-up. The aim is to investigate the extent to which institutional design and public spending affect the use of these services. 36 36 14 July 2022 Appendix – Country sheets A1 ECEC in Italy With the adoption of Law No 124/2015 and then the Legislative Decree No 81/2017, Italy introduced Type of resource Dimension Normative resources Existence of legal entitlement to childcare No Scope of legal entitlement to childcare No legal entitlement but universal free of charge from age 3 Funding mechanisms Public funding with parental fee contributions Support policies Tax deduction and child bonuses (applied universally) Funding provisions for disadvantaged groups None Main providers of childcare Municipalities (0-3 years) Private for profit (0-3 years) State (3-5 years) Private non-profit (3-5 years) Regulation on structural quality provision Regulation on child-staff ratio/group sizes at regional level Regulation on staff qualification at national level Regulation on hours of provision at regional level Extent of regional variation in provision High Instrumental resources Existence of resources for improved access to childcare Some municipal provision Existence of specific initiatives to reach out to groups with lower access to childcare Some local initiatives Enforcement resources Existing judicial procedures for claiming childcare No A1 ECEC in Italy at regional level Extent of regional variation in provision High Instrumental resources Existence of resources for improved access to childcare Some municipal provision Existence of specific initiatives to reach out to groups with lower access to childcare Some local initiatives Enforcement resources Existing judicial procedures for claiming childcare No With the adoption of Law No 124/2015 and then the Legislative Decree No 81/2017, Italy introduced for the first time an integrated system for early childhood education and care from 0 to 6 years’ old. 33 It is composed of two main pillars: the nurseries or crèches (nidi di infanzia) and the kindergarten (scuole d’infanzia). With this new provision, the State and, in particular, the Ministry of Education (MIUR) take on the responsibility for childcare services for the first time. Although not directly involved in the management of these services (unlike the kindergartens), the MIUR is called on to exercise a very important role of the direction, planning, coordination, and financing of the entire 0-6 system, 37 37 37 37 37 14 July 2022 14 July 2022 with the aim of gradually homogenising the offer in terms of quantity and quality. The governance of the system remains decentralised (see below for details), but the assumption of responsibility by the MIUR is evident and represents the main challenge of the decree. In what follows, we look at the individual power resources that are provided to children under the age of 5 in Italy to access both early childcare services and pre-primary school. Normative legal resources Formally in Italy there is no legal entitlement to early childhood education and care (ECEC) Formally in Italy there is no legal entitlement to early childhood education and care (ECEC). Initially developed as health and welfare services within the company to help working mothers (Royal Decree No 718 of 1926), crèches were defined as social services of public interest by Law No 1044 of 1971, under the responsibility of local and regional authorities. In more detail, public nurseries are run directly by the municipalities, in accordance with general criteria defined at regional level, either directly or indirectly. Alongside services with mainly municipal funding (both managed directly by the municipalities and given in agreement to private non-profit or for-profit organisations, and private but affiliated with the municipalities), there is a private offer in Italy, mainly financed with fees paid by families, and in some cases by companies (public or private). From a regulatory perspective, childcare services were initially recognised as services of individual interest, which means without any duty from the state to guarantee access to them. In general, families submit a request of admittance to childcare services at the relevant local office responsible for the organisation of ECEC services. Where more than one service is available in the municipality, families have freedom of choice, and they may also indicate more than one option. Considering requests and admittance requirements established at local level, municipalities make priority lists based on criteria defined at local level in order to regulate admissions in case there are fewer places available than requests. At the national level, the educational purpose of 0-3 services has definitively established itself since 2001, with the reform of Title V, which defines the competences of the state, regions and municipalities, and the various sentences of the Constitutional Court that accompanied it, such as judgment no 467 of 2002. 37 Yet the formal recognition occurred only in 2015 with Law 107/2015 and the subsequent Legislative Decree No 65/2017, which brought nurseries and supplementary services for early childhood back to the educational sphere rather than to the welfare sector, and defined them as ‘services of public interest’. The 2015 law and the 2017 decree also introduced the ‘integrated education and training system from birth up to six years’, directed and coordinated by the Ministry of Education, University and Research, with the aim of ensuring the continuity of the educational and scholastic path from birth up to 6 years of age. Before 2017, the state intervened only with ad hoc extraordinary funding, as in 2007 with the Extraordinary Plan for the development of the integrated system of socio-educational services for early childhood. This was financed by the 2007 Budget Law (Law No 296 of 2006) and in 2011, with the Cohesion Action Plan (PAC), a state intervention that financed, among other things, the strengthening of early childhood services in four southern regions - Calabria, Campania, Puglia and Sicily. In the 1990s, Italy acknowledged various types of supplementary services for early childhood, such as playgrounds, parent-children centres and childminders as well as the micro-nurseries, defined as smaller crèches with greater flexibility, sized according to individual regional regulatory provisions. 38 14 July 2022 14 July 2022 In 2007, spring sections were created in pre-primary schools, which welcome girls and boys between 24 and 36 months of age and favour the continuity of the educational path from 0 to 6 years of age. In terms of quality of the service provided, each municipality is responsible for the organisation of the educational services for early childhood, in compliance with regional regulations. Therefore, the offer varies throughout the country. In general, nurseries admit children aged from 3 to 36 months. Municipalities may also offer services for children aged less than 3 months. Each municipality draws up a general educative project that is applied by each nursery through its own educational plan. The school plan details the activities carried out. The children/educator ratio, as well as minimum and maximum group size, are legally established at regional level. Since nurseries are managed at local level, it is not possible to provide detailed information on the organisation of time. 37 Coverage of 0-2 childcare services in Italy by region (2019) g g y y g ( ) Source: Authors’ composition based on ISTAT. 0 5 10 15 20 25 30 35 40 Public Private Target Source: Authors’ composition based on ISTAT. The high share of private providers affects the affordability of childcare services (Alleanza per l’Infanzia, 2020). To support families in taking up childcare services, the Italian state offers two types of support for the costs incurred by families: a 19 % deduction of the cost of nursery school fees, up to EUR 632 per year per child, and the bonus asilo nido (nursery school bonus), which consists of an annual voucher of EUR 1 000 (increased to EUR 1 500 in 2019) to reimburse the cost of attending a nursery school. In 2019, 38 % of families’ expenditure on nurseries was covered by the bonus and another 5 % by deductions. There are other forms of homecare assistance (Forme di supporto presso la propria abitazione) for disabled children. In this case the bonus asilo nido is granted under the form of home nursing for children under 3 years of age suffering from serious chronic diseases. Despite these measures, as shown in Table 1.1, the costs for parents remain quite high, thus undermining children’s overall take-up of the service. Indeed, municipalities have the discretion to set the amount and methods of calculating these fees. On average, in Italy, families pay EUR 1 581 per year for each of their children enrolled in nursery (see Table A1.1). This figure, which is equal to one-fifth of the expenditure incurred to provide these services, varies greatly in the different areas of the country: it is highest in the Centre-North, where it is around EUR 1 600-1 700, with a peak of almost EUR 1 900 in the North-East, while it drops to around EUR 600-700 in the South. Such amounts are quite significant if we consider that according to the Ministry of Education, 12.8 % of the families who do not use nurseries report having made this choice because they can’t afford the expense. 37 The high fragmentation in the governance of childcare services as well as the fragmentation in the funding scheme means Italy has been characterised by a low coverage, with a national average of 26.9 %, with significant disparities across regions (24.6 % for childcare services, and 2.3 % for integrative services). While the North-East and the Centre of Italy have a coverage level above the European target (34.5 % and 35.3 % respectively), the North-West present levels a little below the target (31.4 %). The South (14.5 %) and the Islands (15.7 %) are still far from the target. On a regional level, Emilia Romagna (36.4 %) has the highest coverage of childcare services (nurseries, micro-nurseries, spring sections), followed by various regions in the Centre (Umbria, Toscana, Lazio), Aosta Valley, and the autonomous province of Trento, all of which are above the European target. Southern regions, notably Campania and Calabria, show the worst results. Such percentages slightly increase if we account also for the integrative services, such as game space, childminders and child-parent centres, which cover at national level respectively 1.3 %, 0.4 % and 0.6 % of children aged 0-2. Game spaces are particularly important in Aosta Valley (7.1 %), Umbria (4.6 %) and Piemonte (3.1 %), while childminders are present in the autonomous province of Bolzano (7.1 %) and Trento (3.7 %). The province capitals have a coverage rate of 34.8 %. All the remaining municipalities have an average of 23.7 spots per 100 people under the age of 3. The cities of Florence, Bologna, and Rome have more than 45 % coverage among the central municipalities of the central-northern metropolitan areas, followed by other metropolitan cities and, at a significant distance, those of the South and the Islands, where coverage does not exceed 20 % (with the exception of Cagliari). Some urban cities, such as Bologna, Florence, Milan, and Genoa, are able to provide adequate coverage even in outlying municipalities. The Rome metropolitan area’s peripheral municipalities, however, deviate significantly from the area’s centre, with a coverage of 23.6 % (ISTAT, 2021). In terms of providers, as shown in Figure A1.1, childcare services remain substantially split between public and private sector, with significant cross-regional differences. 39 39 14 July 2022 Figure A1.1. Coverage of 0-2 childcare services in Italy by region (2019) Figure A1.1. 37 Recent studies by ISTAT (2020) show that for children belonging to the poorest brackets (I and II quintile of income distribution), the frequency of nursery school remains just below 14 %, while in the third- and fourth-income classes it rises to 20 % and to 26 % respectively, reaching 35 % frequency in the last fifth (ISTAT, 2020). 40 14 July 2022 Table A1.1 Average expenditure per child Table A1.1 Average expenditure per child Percentage of expenditure paid by parents Average expenditure per child Fee paid by the municipalities (euro) Fee paid by users (euro) North-West 23.0 5.655 1.687 North-East 22.9 6.381 1.892 Centre 17.8 7.546 1.636 South 10.7 5.374 643 Islands 10.7 6.180 739 ITALY 19.8 6.393 1.581 Source: Authors’ composition based on ISTAT. Finally, with respect to the educators, a university degree requirement has been introduced with the legislative decree 65/2017, which is required in both public and private nurseries and corresponds to a 3-year degree in the L-19 class (Educational Sciences). Working conditions of educators change significantly depending on whether they are employed in publicly managed services or in private ones (Neri, 2016). Overall, Italy is characterised by a high share of temporary involuntary contracts. Leon et al. (2019) show that, in 2015, 29.3 % of the teachers in childcare had non-standard contracts, 30.3 % worked part-time, despite the average number of years worked with the same employer being quite high (around 12.3 years). The consequence of such career fragmentation is a high dissatisfaction linked to income discontinuity and uncertainty, which is then translated into lower quality of the service for kids. In terms of wages, there is a significant monthly wage gap between childcare teachers and their pre-primary colleagues of around 15 %. If one considers the hourly wage, the gap between childcare and pre-primary teachers is even higher at around 25 %. Considering the wage dynamics for the level of qualification of teachers and the length of careers, childcare teachers have the lowest wages, regardless of their qualifications and their ages, with the sole exception of older teachers with 3-year degrees and a very long career. As stressed by our interviews, the bad working conditions and wages of early childhood educators in part explains the low attractiveness of this job and therefore the shortages of qualified teaching personnel. 37 In this respect, the fact that the educators are not even recognised at the same level as pre-primary teachers in public schools, and are represented by two different trade union organisations, does not help in supporting the former and explains the low mobility. Instrumental resources Instrumental power resources include targeted or universal support channels, which facilitate individuals’ access to social rights or assist them in access to justice in cases of non-compliance or rights-violation by third parties. In the absence of an individual entitlement to access to childcare or pre-primary school, instrumental resources consist of those procedures, information and support channels that support families in overcoming the barriers that still hamper the access to the service. As observed above, access to pre-primary school is almost universal and barriers in access are limited. Yet, as observed by Favaro (2013), the enrolment of pupils aged 3-5 diminishes significantly among non-Italian citizens (80.2 %, compared with 93.6 % of Italians). The reasons for this absence can be attributed respectively to the family (an underestimation of the importance of this educational stage, the organisational difficulties and the physical distances between home and school, especially for the 41 41 14 July 2022 14 July 2022 those that live in isolated situations) and the reception context (the costs of canteen service unsustainable for all, the difficulties of integrating the children of immigrants, especially where available places are limited, or where, in a discriminatory manner, the intention is to discourage the attendance of foreign children). At the moment, however, there are no institutional or non- institutional channels to facilitate access and support non-Italian citizens in accessing pre-primary schools. The situation is more complex when it comes to early childcare. As observed above, there are various factors that hamper participation. First, there are territorial barriers, as the services are more widespread in the Northern and Centre regions than they are in the South and the Islands. In the southern regions, the available places in public and private nurseries and complementary services do not reach an average of 15 % of the potential catchment area for children up to 3 years of age, compared with the Italian average of 24.7 %. In addition, significant disparities occur within territories, depending on the size of the municipality. The available places in centres with fewer than 10 000 inhabitants are 22.7 %, compared with 26.6 % in cities between 10 001 and 50 000 inhabitants, and 29.9 % in cities with more than 50 000 inhabitants. The second type of barriers are economic barriers. Instrumental resources Educational barriers also play an important role, with the high school diploma and the middle school certificate under-represented among the families that use nurseries (31.8 % and 18.7 % respectively, against 39.5 % and 24.2 % of total families). Finally, cultural barriers play only a minor role in explaining the take-up of childcare facilities. In this case the main reason for not sending a child to childcare is that they are too young to be sent and would be better educated at home. While territorial and economic barriers cannot be addressed via instrumental resources, that is, via strengthening information channels and helping families with the administrative procedures to enrol their children at nursery, educational and cultural barriers can be in principle reduced with instrumental resources. In this respect, however, there is no systematic evidence of the existence of such channels in Italy. Since the childcare system is managed locally with regional guidelines, each region provides a system of information and consultancy to parents or future parents on the services of childcare and pre-primary education. For instance, Emilia-Romagna has a dedicated website and service, Informa Famiglie e Bambini, where support is provided to families who want to access ECEC services. A particularly admirable example is that of Reggio Emilia, where the municipality informs new parents, even before they give birth, about the opportunities to enrol their children in nursery. A childcare information kit is sent to the new parents with support for the administrative procedures. As stressed by our interviews, however, this door-to-door information service is a peculiarity of just a few municipalities, mostly in Centre (Bologna) and North Italy (Milan). Other information activities are carried out by the organisations that manage the private not-for-profit childcare services. For instance, the FISM (Federazione Italiana Scuole Materne) raises awareness of public opinion around the educational and social service provided by the kindergarten. To address the problem in access to information about childcare services, a proposal has been discussed in the Italian Parliament regarding the possibility of creating a home-visiting service, which would consist of the creation of a network of paediatricians to support families in getting the information to apply for the nurseries. Another initiative has created a network of parents to spread information about the advantages of enrolment in childcare services. 42 14 July 2022 14 July 2022 Enforcement resources Since there is no right to childcare, and attendance not compulsory, there are no remedy procedures if a child cannot access childcare facilities. 43 43 14 July 2022 A2 ECEC in Germany Type of resource Dimension Normative resources Existence of legal entitlement to childcare Yes Scope of legal entitlement to childcare Universal from age 1 Funding mechanisms Public funding with parental fee contributions Funding provisions for disadvantaged groups Staggered fees for disadvantaged groups Regulation on quality provision Regulation on child-staff ratio/group sizes Regulation on staff qualification Regulation on hours of provision Main providers of childcare Mixed provision Regulation on quality provision Regulation on child-staff ratio/group sizes Regulation on staff qualification Regulation on hours of provision Extent of regional variation in provision High Instrumental resources Existence of resources for improved access to childcare Some regional provision Existence of specific initiatives to reach out to groups with lower access to childcare Some national initiatives and regional provision Enforcement resources Existing judicial procedures for claiming childcare Yes A2 ECEC in Germany 44 14 July 2022 In Germany, ECEC is part of the public welfare system, where responsibility is shared between the federal government, the 16 state governments and the municipalities (Linberg, Baeumer and Rossbach, 2013). As such, the governance structure of ECEC in Germany is complex and decentralised, relying on a system of multilevel governance (Oberhuemer, 2014). At national level, the German ECEC system is the responsibility of the Federal Ministry for Family Affairs, Senior Citizens, Women and Youth (BMFSFJ) (OECD, 2016), with some responsibility for the Ministry of Education and Cultural Affairs (Eurydice, 2021). However, the Federation only sets the general framework for ECEC, with the national legislative framework on ECEC contained within the Child and Youth Services Act in Book Eight of the Social Code (Achtes Buch Sozialgesetzbuch) (Scholz et al., 2019), which was passed in 1990 (Eurydice, 2021). While the national level sets binding framework conditions and guidelines for childcare services and defines the key tasks of ECEC, no concrete content requirements beyond general guidelines are provided (ibid.). In addition, the national level has ‘stimulatory competence’ regarding childcare issues, for instance through launching initiatives to improve the quality of childcare (Scholz et al., 2019). At regional level, the 16 German Länder are responsible for adapting the framework conditions set by federal legislation into state-specific ECEC laws (Oberhuemer, 2014). Enforcement resources The states have regulatory competence, meaning that there are 16 state-specific frameworks for the funding and licensing of ECEC, as well as for setting standards and developing curricula (Scholz et al., 2019). In addition to a legislative framework, each Land also has its own policy initiatives for access and quality in ECEC (Oberhuemer, 2014). Finally, the responsibility for the planning, organisation and provision of ECEC lies at the local level of municipalities, which also have responsibility for securing funding for ECEC (Scholz et al., 2019). Within the framework set by state law, each municipality can set its own framework for ECEC and the youth welfare offices (Jugendämter) – local agencies responsible for child welfare – have the overall responsibility for planning and implementation (BPB, 2021). ECEC in Germany is provided in daycare centres (Kindertageseinrichtungen) for children under 3 (Krippen), in Kindergärten for 3 to 6 year-olds or in mixed-age facilities (BPB, 2021). Increasingly, Kindergärten are expanding their services to younger children so that the traditional formal distinction between Krippen and Kindergärten no longer applies in many cases, and the mixed-age model is more common (Linberg, Baeumer and Rossbach, 2013). In addition, there is the option of Kindertagespflege, home-based child-minding services by qualified individuals, which is particularly common for children under 3 (Eurydice, 2021). Traditionally, there is no strict distinction between childcare and education in care provision. Rather, institutionalised daycare is defined by a triad of Bildung (education), Erziehung (upbringing) and Betreuung (care) (OECD, 2016; Frindte and Mierendorff, 2017). 45 14 July 2022 14 July 2022 Normative resources As set out in the theoretical framework, normative resources define the entitlements to early education and care set out in the national legislation, and as such, may have crucial influence on children’s educational capabilities. The provision of and entitlements to ECEC have been progressively expanding in Germany in recent decades. As regards the existence of a right to childcare, a first step was made in 1996, when a right to ECEC for children aged 3 and older was first codified into legislation (West, Blome and Lewis, 2020). The Daycare Expansion Act (Tagesbetreuungsausbaugesetz, or TAG) from 2005 was the first significant step towards expansion of ECEC for children under 3, as the first legislative effort to determine concrete goals for the number of childcare places for children (Scholz et al., 2019). It required local authorities to provide enough places for children below 3 to meet demand or, at a minimum, to make places available for children with particular need, for instance where parents were in employment (West, Blome and Lewis, 2020). According to experts interviewed, expansion efforts were only progressing slowly in this period. However, in 2008, the Child Support Law (Kinderförderungsgesetz, or KiföG) established a universal legal right to ECEC for all children from the age of 1 to officially come into force on 1 August 2013 (BPB, 2021). In addition to the universal right to ECEC from age 1, the 2008 law also laid down a gradual expansion of supervision and care offers for children under the age of 3 to meet increased demand (Eurydice, 2021). As a result of the increased investment in provision and more generous entitlements, take-up of ECEC has increased significantly in Germany in recent years, as highlighted in Figure A2.1. Between 2006 and 2015, the proportion of children aged 1 and 2 in care (daycare centres and childminding services) increased from 29.4 % to 48.5 %, with further increases between 2015 and 2020. Overall, it can be concluded that the introduction of a right to childcare for children aged 1 and older, and the associated investment in provision, had a significant impact on access to childcare. Figure A2.1. Proportion of children in care (daycare centres and childminding services), Germany, 2009-2020 Figure A2.1. Proportion of children in care (daycare centres and childminding services), Germany, 2009-2020 Source: Authors’ calculation based on (Bertelsmann Stiftung, 2022). 0 20 40 60 80 100 2009 2011 2013 2015 2016 2017 2018 2019 2020 Proportion (%) Children 1-2 Children 3-6 Source: Authors’ calculation based on (Bertelsmann Stiftung, 2022). 46 14 July 2022 However, while attendance for children aged 3-6 is almost universal, a lack of places for children under 3 remains an issue in some Länder. In particular, as shown in Figure A2.2 provision remains lower in West Germany compared with the East. This can be traced back to historical development, as childcare provision was much more common in Eastern Germany (in the states previously belonging to the GDR), whereas initial levels of provision were much lower in the West. Survey data indicate that the demand for childcare places on the part of parents remains significantly higher than available supply, particularly in Western Germany (Anton, Hubert and Kuger, 2020). On average across Germany, the gap between indicated demand (49 %) and actual take-up of places (34.3 %) amounts to 15 percentage points (DJI, 2021). It should also be noted that the legal entitlement to publicly subsidised daycare may be realised in home-based provision (Eurydice, 2021). That is, not all families who would like access to institutionalised daycare receive a place there. Parents are free to indicate a facility of their preference but are not entitled to receive a place at any particular facility (Eurydice, 2021). Overall, therefore, there have been substantial advancements in Germany in access to ECEC following the introduction of the legal entitlements for children aged 3 in 1996 and aged 1 in 2013. Nevertheless, a discrepancy between demand and supply remains. Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland Source: Authors’ calculation based on (Bertelsmann Stiftung, 2022). 83,4 82,3 81,9 79,1 76,3 69,0 66,3 51,3 48,3 46,5 45,6 43,8 43,4 43,1 43,0 42,8 76,2 45,4 51,1 0 10 20 30 40 50 60 70 80 90 Proportion (%) Source: Authors’ calculation based on (Bertelsmann Stiftung, 2022). Furthermore, as set out previously, in addition to the existence of a right to ECEC, other legal provisions may further lay out what precisely is contained within this right, in turn affecting the access to and quality of ECEC, and therefore the educational capabilities of children. First, funding provisions are of central importance. Funding structures for ECEC in Germany are very complex and characterised by regional variation as a result of decentralisation, though they generally rely on a mix of public and private sources (FiBS, 2016; Scholz et al., 2019). Overall, approximately 0.8 % of GDP in Germany is 47 14 July 2022 14 July 2022 devoted to ECEC, a similar figure to the OECD average (OECD, 2016). While the precise funding arrangements vary by Land, it is generally the case that the municipality is responsible for most of the funding for ECEC, with limited involvement from the regional and, particularly, federal government. The result of this, as found from the expert interviews, is that access to and quality of ECEC for children depends strongly on their place of residence. In municipalities that have lower financial resources, less funding will in turn be available for ECEC. Though there is a universal entitlement to a place in daycare, ECEC is not part of the school system and generally not free of charge (Eurydice, 2021). The level of contributions will depend on several factors, including the region, parents’ financial circumstances, the number of children and attendance time (ibid.). Funding and subsidies for childcare differ by Land. In some, there are general subsidies for all children or free places for some years or families with several children, while in others, provision is completely free of charge, at least for some hours, and there are also varying regional funding arrangements for lunch provision and additional services (Bertelsmann Stiftung, 2021). Fees remain a barrier to take up of ECEC (BPB, 2021). As highlighted in interviews, fees can vary enormously, even across municipalities in the same region, and are in some cases very high. Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland The median cost of care for children under 3 in Germany is EUR 214 per month, while for children over 3 it is EUR 100 per month (DJI, 2021). However, fees for under 3s can vary from little to no fees to more than EUR 300 per month, depending on the region (ibid.). Fees may particularly form a barrier to entry for socioeconomically disadvantaged families. 31 % of German families with children under 3 and a low net equivalent household income indicate that fees are a barrier to accessing ECEC (DJI, 2021). Since August 2019, the Good Day-care Facilities Act (KiQuTG, ‘Gute-Kita-Gesetz’) requires parental contributions to be staggered according to social criteria, including parental income (Eurydice, 2021). Nevertheless, the precise determination of how fees are staggered depends on the legislation set out by the Land. Over time, progressively more regulations to make ECEC affordable have been introduced, and in some Länder, care is already completely free, depending on age and scope of care (ibid.). However, despite these subsidies, parents of children under 3 from lower income families still remain less likely than other families to use ECEC, (West, Blome and Lewis, 2020). Some aspects of ECEC provision may disproportionately benefit socially privileged groups, such as the common prioritisation of double-earning households where both parents are working (BPB, 2021). Further to funding structures, legal provisions on the quality of ECEC are of importance. Again, the central motif in the German case is that of regional variation. As highlighted multiple times in the expert interviews, additional provisions beyond the right to childcare vary by Land, resulting in a high 48 48 14 July 2022 14 July 2022 degree of heterogeneity across the country. For instance, there is a high degree of variation in the number of hours of care children are entitled to. While the Social Code does not define a specific time entitlement to care, 10 of the 16 Länder define a minimum number of hours per day, with some additional variation at regional level (Bertelsmann Stiftung, 2021). Entitlements tend to be more generous in Eastern Germany, where childcare use was historically more prevalent than in the western part of the country. There are also no national requirements regarding the child-to-staff ratio, which exhibits large regional variation according to Länder-specific regulation (BPB, 2021). Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland Finally, there are specific support 49 49 14 July 2022 14 July 2022 measures available for children with disabilities and special education needs (Eurydice, 2021). These children can either attend daycare centres with inclusive care concepts or separate facilities. Most of the children attending a daycare facility who receive integration support or who have special educational needs will be cared for alongside children without disabilities. As regards the specific support provisions, there is a large amount of regional variation. However, issues relating to access and quality in ECEC for children from socioeconomically disadvantaged groups remain, including for children from families with lower financial means and children with a migrant background. Evidence indicates that these socioeconomic disparities have been intensified, rather than weakened, in the course of the large expansion in ECEC Germany has undergone in recent years (Stahl, 2015). For instance, although there have been large increases in the proportion of children with a migration background in ECEC for under 3s in recent years (Olszenka and Riedel, 2020), they continue to be under-represented in German ECEC relative to other children (Bertelsmann Stiftung, 2021), even in families that indicate that they are in need of a place (Anton, Hubert and Kuger, 2020). This is likely related to a variety of barriers to entry on the parts of both institutions and families, including the cost of care, distance to the daycare, lack of information and transparency about access, perceived quality of care, the intercultural and linguistic competencies of staff in daycare and norms and values within the family (ibid.). While the educational plans of many states include support for multilingualism in daycare centres, in practice, the pedagogical approach in daycare seldom devotes space to multilingualism and may even marginalise children with a multilingual background (Olszenka and Riedel, 2020). In addition, evidence appears to indicate that children from migrant families tend to attend facilities with lower performance in structural and procedural quality indicators and a less privileged social composition, though more research on this is necessary (Stahl, 2015). Investment in the quality of ECEC in Germany, and in closing socioeconomic gaps in the quality of education that children receive, therefore remains a significant concern for the future. There are also national standards regarding the qualification of personnel in ECEC. Historically, the concept of public childcare and the professionalisation of the ECEC was contentious in Germany (Rauschenbach and Riedel, 2016). Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland Across Germany, the child-staff ratio for children under 3 in full-time daycare is 3.9, increasing to 8.2 for children aged 3-6 (DJI, 2021). As a result, the de facto quality of childcare that children are entitled to varies immensely across Germany. However, it should be noted that there is more effort being made to improve the quality of childcare across Germany, with the national government increasingly involved. In 2019, the Good Day-care Facilities Act committed to federal support of EUR 5.5 billion from 2019 to 2022 to implement measures in 10 qualitative fields (which the Länder can select from), such as decreasing the specialist- child ratio, in addition to decreasing and socially staggering fee burdens. As such, experts noted that efforts to improve quality and decrease heterogeneity therein are increasing, though progress is slow. While provisions to specifically support socioeconomically disadvantaged families mostly focus on fee staggering in some regions, there may also be provisions relating to other entitlements. In some cases, there may be additional entitlements, for instance for younger children or additional hours, depending on the position of the parents or if the children have special requirements (Bertelsmann Stiftung, 2021). There are also Bundesländer, where daycare centres receive higher funding when they have a higher share of children coming from a migrant background (FiBS, 2016). In addition, there has been increased investment in the development of language diagnostics and support in daycare to improve linguistic competence, particularly for children with a migrant background or with deficiencies in language development (Eurydice, 2021). For instance, the federal programme ‘Language-Kitas: Because language is the key to the world’, launched by the BMFSFJ, promotes the teaching of language education in daycare centres, with a commitment of EUR 1 billion spent between 2016 and 2020 (ibid.). Moreover, a joint initiative to develop language promotion was launched by the Standing Conferences of Ministers for Education and Ministers for Youth, together with the Federal Ministries, in 2012 (ibid.). The resulting programme, ‘Education through Language and Writing’ (2013-2019), scientifically developed the linguistic education of children and assessed measures developed in the Länder for language promotion. In March 2020, a five-year initiative was launched to bring the results of the scientific initiative to a wider network of institutions, funded by the Federal Ministry of Education and Research and the Länder. Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland The qualification to become a state-recognised pre-primary educator takes 3 years of post-secondary vocational training, as well as either a course as a childcare assistant or training and work experience in the field (OECD, 2016). Since 2005, there have also been minimum qualifications for childminders (Eurydice, 2021). Currently, the traditional vocational qualification is placed at Level 6 on the National and European Qualifications Framework, on a par 50 50 14 July 2022 14 July 2022 with a bachelor’s degree, though this issue is contentious (ibid.). As such, although there has been a move towards the academisation of ECEC qualifications, this process has been slow (Rauschenbach and Riedel, 2016), and the number of staff with tertiary education remains marginal (Oberhuemer, 2014). Though staff qualifications are agreed nationally, the state government decides on staff-child ratios, the rate of inspections and training for childminders (West, Blome and Lewis, 2020). In interviews with experts, the lack of qualified staff was highlighted as a major issue for the provision and quality of ECEC in Germany, and one that will likely be exacerbated in future years. In some cases, provision already has to be limited because of the lack of qualified staff. In staff surveys, one in five leaders of educational institutions name staff shortages as a barrier towards providing high-quality education and care (OECD, 2018). Expert interviews showed concern about a decreasing trend in quality of provision even while maintaining the number of places, leading to a situation where the educational role of ECEC is no longer fulfilled. Overall, the assessment of normative resources as relating to the quality of ECEC therefore shows that, while there has been significant progress in access to ECEC through the expansion efforts made in recent years, there are concerns relating to the quality of ECEC. In particular, the lack of entitlements relating to quality at national level results in significant heterogeneity across regions, so that the quality that children are de facto entitled to depends on their place of residence. In interviews with experts, the lack of qualified staff was highlighted as a major issue for the provision and quality of ECEC in Germany, and one that will likely be exacerbated in future years. In some cases, provision already has to be limited because of the lack of qualified staff. Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland Though research in this area is still developing, there is some evidence that these segregational tendencies could be linked to the German provider structure, as groups of providers differ with regard to the access criteria they prioritise, including the family situation and the age of the child, but also subjective criteria such as alignment of the family with the provider institution’s values or pedagogical concept (ibid.) Nevertheless, all providers are subject to guidelines on the content of ECEC. At national level, the overall objectives of ECEC and broad (but binding) guidelines are defined. As specified in the Child and Youth Welfare Act, all ECEC should encourage children’s mental, physical and emotional development to help them become responsible and autonomous members of society, and support families in raising children and reconciling this with employment (Linberg, Baeumer and Rossbach, 2013). These general objectives of daycare also apply for home-based provision of ECEC (Eurydice, 2021). In 2004, the Standing Conferences of Regional (Länder) Ministers for Education and Cultural Affairs and Ministers for Youth adopted the Joint Framework of the Länder for Early Education in Day-Care Centres (Gemeinsamer Rahmen der Länder für die frühe Bildung in Kindertageseinrichtungen), which applies to centre-based ECEC across age groups (Eurydice, 2021). The framework lays down the objectives of ECEC, which include transferring basic skills, developing and fostering personal resources to motivate children and promote their ability to take up and cope with future challenges in learning and life, encouraging children to become responsible members of society, and enabling them to cope with lifelong learning (Linberg, Baeumer and Rossbach, 2013). In 2009, the national working group of the Länder youth welfare services adopted the Specialist Recommendations on the Quality of Education, Upbringing and Care for Children Under Three Years of Age in Day-Care Centres and Child- minding Services (Fachliche Empfehlungen zur Qualität der Bildung, Erziehung und Betreuung der unter Dreijährigen in Kindertageseinrichtungen und Kindertagespflege (Eurydice, 2021). The national frameworks set out guidelines for ECEC (Eurydice, 2021). As regards ECEC for children under 3, the 2009 guidelines state that care must respond to the basic needs of small children, such as loving attention, sympathetic support and safety and security. At this stage, the aim of educational support is to holistically support child development through play, social interaction and communication. Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland In staff surveys, one in five leaders of educational institutions name staff shortages as a barrier towards providing high-quality education and care (OECD, 2018). Expert interviews showed concern about a decreasing trend in quality of provision even while maintaining the number of places, leading to a situation where the educational role of ECEC is no longer fulfilled. Overall, the assessment of normative resources as relating to the quality of ECEC therefore shows that, while there has been significant progress in access to ECEC through the expansion efforts made in recent years, there are concerns relating to the quality of ECEC. In particular, the lack of entitlements relating to quality at national level results in significant heterogeneity across regions, so that the quality that children are de facto entitled to depends on their place of residence. Finally, the provision of ECEC is strongly governed by the subsidiarity principle – that is, public authorities are only obliged to provide social services if non-governmental agencies are not in a position to do so (Oberhuemer, 2014) – and by the principle of diversity of providers (Scholz et al., 2019). Only about one-third of children in Germany are in publicly provided care, with the majority provided by not-for-profit private organisations (Bertelsmann Stiftung, 2021). In particular, non-profit private organisations that are very active in childcare provision are churches and welfare organisations, while private for-profit providers are rare (West, Blome and Lewis, 2020). Providers have to have public recognition but have freedom in designing the content and method of care that they provide (BPB, 2021). Overall, there are many diverse providers in Germany. In principle, the diversity in providers was evaluated as positive by experts, as it increases choice and options. However, there is a concern as regards the heterogeneity of quality in provision across providers. While large associations of providers have a large infrastructure and can provide high- quality training for staff, for instance, this is not the case for small providers. Pay rates for staff can also vary immensely by provider. Moreover, studies have shown that social segregation can already be observed among young children in ECEC, as care providers exhibit marked differences with regard to, for instance, the proportion of children coming from poor families or children with native 51 14 July 2022 14 July 2022 languages other than German (Hogrebe, Mierendorff and Nebe, 2021). 7 For more information see kita-navigator.berlin.de. Figure A2.2. Proportion of children aged 1 and 2 in care (daycare centres and childminding services), Germany, by Bundesland From the age of 3, the 2004 Joint Framework of the Länder sets out further educational areas in order to support the development of children’s intellectual, physical, emotional and social abilities. For instance, educational areas include (ibid.) language and communication, personal development and nature and cultural environments. 52 14 July 2022 At Länder level, while there are no binding curricula, in recent years there has been an increased focus on developing educational plans and language support programmes (Linberg, Baeumer and Rossbach, At Länder level, while there are no binding curricula, in recent years there has been an increased focus on developing educational plans and language support programmes (Linberg, Baeumer and Rossbach, 52 14 July 2022 At Länder level, while there are no binding curricula, in recent years there has been an increased focus on developing educational plans and language support programmes (Linberg, Baeumer and Rossbach, At Länder level, while there are no binding curricula, in recent years there has been an increased focus on developing educational plans and language support programmes (Linberg, Baeumer and Rossbach, 52 14 July 2022 2013). Educational plans (Bildungspläne) further concretise the legal mandate for ECEC by providing a (non-binding) framework for curricula and developing recommendations for personnel and providers of ECEC at local level, covering guiding principles of ECEC, the core objectives of education, and educational areas to be addressed in ECEC (BPB, 2021). Based on the framework specified by the Land, each centre providing ECEC must develop its own pedagogical plan, which must be approved by youth and welfare services and is to be coordinated with primary schools (Eurydice, 2021). Depending on the Land, the responsibility for the licensing of family daycare and childcare services lies with regional/state level authorities or local municipalities (OECD, 2016). 8 For more information see https://www.bmfsfj.de/bmfsfj/service/publikationen/bundesprogramm-kita- einstieg-bruecken-bauen-in-fruehe-bildung--118650. Instrumental resources As highlighted by experts, parents in these families may often be unaware of their childcare entitlements or may not know how to access these entitlements. Here, a greater effort has been made to increase outreach. For instance, there is the federal programme ‘Daycare Entry: Building bridges for early education’ (Guter Kita-Einstieg: Brücken bauen in frühe Bildung), which targeted mainly refugee children at first but has since been opened up to other groups, such as families that are economically at risk or in precarious living situations8. The aim of the programme is to provide information on ECEC to these families and thereby facilitate access. There are also local initiatives to increase access for disadvantaged groups, though this is not the case everywhere. In most municipalities, information in multiple languages is lacking, which is an issue in this context. Informational barriers related to access to ECEC may be particularly pronounced for children from migrant families or families where parents do not speak German. As highlighted by experts, parents in these families may often be unaware of their childcare entitlements or may not know how to access these entitlements. Here, a greater effort has been made to increase outreach. For instance, there is the federal programme ‘Daycare Entry: Building bridges for early education’ (Guter Kita-Einstieg: Brücken bauen in frühe Bildung), which targeted mainly refugee children at first but has since been opened up to other groups, such as families that are economically at risk or in precarious living situations8. The aim of the programme is to provide information on ECEC to these families and thereby facilitate access. There are also local initiatives to increase access for disadvantaged groups, though this is not the case everywhere. In most municipalities, information in multiple languages is lacking, which is an issue in this context. Instrumental resources In addition to normative resources – the legal provisions setting out the right to ECEC for children and what exactly it entails – instrumental resources may be essential in increasing access by providing children and their families with the necessary information and assistance to gain access to care. As stated previously, in Germany, the concrete implementation of measures in ECEC, including measures to facilitate delivery and access, depend on the state and local level. Generally, when parents are looking for a place in (centre-based) daycare, the first step – often during pregnancy – is to seek out local daycare centres, where they can get information and be placed on a waiting list (Schettler, 2016). In most Länder, regulation specifies that the need for a space has to be registered with public authorities, usually the youth and welfare services, who may then provide information on how best to access a place in ECEC (ibid.). Increasingly, there are online tools to facilitate the process of parents registering their need for a place in ECEC, which can increase parents’ access to information and understanding of the process. However, this is not available everywhere (ibid.). Additional measures to raise awareness and provide information on quality are location dependent. As was also pointed out in interviews, the existence of informational and other instrumental resources is an issue on which there is currently little evidence. Several regional examples of efforts to facilitate access to ECEC do exist, for instance the Kita-Navigator provided by the city state of Berlin7, where parents can search for available places and filter according to their preferences. However, systematic initiatives to provide information and increase access are generally lacking on a national level. In general, it can be concluded that approaching parents in order to decrease barriers to access is not systematic. Rather, the onus is on parents to seek out the public authorities, where they can then 53 14 July 2022 7 For more information see kita-navigator.berlin.de. 53 14 July 2022 receive information. In a context where demand for ECEC continues to outpace supply, this lack of systematic information provision can be an impediment to accessing the right to ECEC. Informational barriers related to access to ECEC may be particularly pronounced for children from migrant families or families where parents do not speak German. Enforcement resources Finally, enforcement resources may play a role in influencing access to ECEC, in that they enable legal avenues that families can take if their right to childcare is denied. As stated previously, in Germany, the legal right to a place in ECEC is codified in national legislation from the age of 1, and local maintenance bodies are therefore obliged to provide a place for all children. If the legal entitlement to care cannot be honoured, parents have the right to sue these authorities for a place (Schettler, 2016; Eurydice, 2021). In October 2016, the German Constitutional Court (Bundesgerichtshof) ruled that parents may be entitled to compensation for lost earnings from their local authority if a place in daycare for their child cannot be provided (Spiegel, 2016). The court responded to a suit by three mothers, whose return to work after childbirth was delayed because of unavailability of daycare places. The Constitutional Court ruled that the legal right to ECEC is partially motivated by helping parents balance their employment with caring responsibilities; if authorities fail in this legal mandate, they may be required to pay compensation. However, there may be specific cases where the municipality can justify the lack of places, for instance if agreed contracts were not met by contracting partners. Nevertheless, while the right to sue for a place in ECEC exists, in practice, few families actually make use of this avenue, as pointed out by experts. Currently, around 1 000 court cases relating to the legal 54 14 July 2022 8 For more information see https://www.bmfsfj.de/bmfsfj/service/publikationen/bundesprogramm-kita- einstieg-bruecken-bauen-in-fruehe-bildung--118650. 8 For more information see https://www.bmfsfj.de/bmfsfj/service/publikationen/bundesprogramm-kita- einstieg-bruecken-bauen-in-fruehe-bildung--118650. 14 July 2022 entitlement §24 SGB VIII are listed9. As stated by experts, however, this does not reflect the overall number of complaints, as not all are accepted by courts. This is because they may be legally insecure, or the local authorities find a place before the legal process commences. Nevertheless, it was also pointed out that parents can be reluctant to sue owing to barriers such as an unwillingness to cause a confrontation in the local community, perceived costs, time until the decision and chances of losing. Moreover, the families that do sue tend to be those in socioeconomically advantaged positions, who can afford to invest the time and other resources needed for such a legal endeavour. 9 https://dejure.org/dienste/lex/SGB_VIII/24/1.html Enforcement resources As such, suing for a childcare place should not be seen as an effective avenue to increase access to childcare. At the same time, the existence of such a legal avenue may have contributed to increased provision of places. As laid out in the expert interviews, many municipalities increased their provision in anticipation of the establishment of the right to childcare for children under 1, and the potential suits associated with it. Hence, while enforcement resources do not in themselves appear to be an effective way of accessing the entitlement to ECEC, they can have an indirect effect by increasing the bargaining and legal power of families relative to public authorities. 9 https://dejure.org/dienste/lex/SGB_VIII/24/1.html 55 55 14 July 2022 14 July 2022 A3 ECEC in Poland A3 ECEC in Poland The right to ECEC is codified into the Polish national legislation. ECEC in Poland does not form a single Type of resource Dimension Normative resources Existence of legal entitlement to childcare Yes Scope of legal entitlement to childcare Universal from age 3 Funding mechanisms Public funding with parental fee contributions Support policies Allowances Funding provisions for disadvantaged groups None Main providers of childcare Mixed provision Regulation on quality provision Regulation on child-staff ratio/group sizes Regulation on staff qualification Extent of regional variation in regulation Low Instrumental resources Existence of resources for improved access to childcare Some local services (in some communes) Existence of specific initiatives to reach out to groups with lower access to childcare Local services and private initiatives Enforcement resources Existing judicial procedures for claiming childcare No The right to ECEC is codified into the Polish national legislation. ECEC in Poland does not form a single or uniform system in Poland. It is based on two systems: the childcare system supervised by the Ministry of Family and Social Policy (children aged 0-3 years) and the pre-primary education system that falls under the Ministry of Education and Science (children aged 4-6/7 years). 56 14 July 2022 56 14 July 2022 So, ECEC in Poland is divided into two stages: So, ECEC in Poland is divided into two stages: So, ECEC in Poland is divided into two stages: So, ECEC in Poland is divided into two stages: 1. for children aged 0-3 years: centre-based childcare provided in crèches (żłobki) and, since 2011, kids clubs (kluby dziecięce), and home-based provision by daycare providers or childminders (opiekun dzienny) and nannies (niania). Enforcement resources It is regulated by the Act of 4 February 2011 on the Care for children aged up to 3. Centre-based crèches and home-based provision (daycare providers/child minders and nannies) care for children aged at least 20 weeks, while kids clubs are for children aged 1 year and above. Children can attend those settings until the end of the school year in which they reach the age of 3 or up to the age of 4 in cases where it is difficult or not possible for the child to participate in pre-school education. Attending a crèche is not obligatory; crèches are not part of the education system. 2. for children aged 4-6/7 years: pre-school education provided in nursery schools (przedszkole), pre-school classes (oddziały przedszkolne) in primary schools and other pre-school education settings, including pre-school education units (zespół wychowania przedszkolnego) and pre- school education centres (punkt przedszkolny). This is regulated by the Act of 14 December 2016, Law on School Education. Pre-school education is optional for 3, 4 and 5 year-old children and obligatory for 6 year olds. Every 3, 4 and 5 year old has an entitlement to a place in a pre-primary setting in his/her locality. As of the school year 2016/17 compulsory education in Grade 1 of primary school starts at the age of 7. All 6 year olds have to attend a pre-school institution for 1 year in order to acquire basic skills before they start school. Normative legal resources Normative legal resources Care for children aged up to 3 years is a statutory task of the commune - gmina (the lowest-level local government unit). However, there is no legal guarantee to a place in ECEC for children aged up to 3 years. Creches or kids club can be led by public or non-public institutions that fulfil the proper conditions and requirements. Funding Funding for care provided to children aged up to 3 years at crèches, kids clubs and daycare providers comes primarily from: Funding for care provided to children aged up to 3 years at crèches, kids clubs and daycare providers comes primarily from: • budgets of communes and other local government units (LGUs) in the form of a subsidy. Generally, the only condition for the payment of the subsidy which follows directly from the Act on the Care for Children up to the age of 3 is the child’s place of residence. The subsidy for a day nursery is granted only for children residing in the municipality – unless the municipality adopts a different decision in the resolution. Subsidies may be granted to children residing outside the municipality, but only if there is a resolution permitting it, and secondly, an inter-municipality agreement (i.e. reimbursement from a neighbouring municipality). • state specific-purpose (targeted) grants, such as the Maluch+ Programme. In the case of the Maluch+ Programme, the funds generally can be allocated to three tasks: (1) creation of new facilities; (2) creating new places in existing nurseries; (3) subsidising existing places, thus 57 14 July 2022 reducing fees. The subsidy covers up to 80 % of the total cost. In 2020, the entity receiving funding from the programme is required to ensure the operation of childcare places created or subsidised with funding from the programme for a minimum period of 5 years. Ensuring the operation of childcare places means that at least 60 % of the places indicated in the agreement are used during the required period. The programme is available both for public entities (mostly communes) and private. The programme is financed from the State Budget and the Labour Fund (since 2018 as a measure facilitating parents’ engagement on the labour market). • EU structural funds (as part of regional operational programmes). • EU structural funds (as part of regional operational programmes). Funding The above funding sources can be granted measures to develop and maintain childcare facilities for children up to the age of 3: renumeration of teachers and administration, supply of utilities, rent, administrative charges relating to the premises, costs related to maintenance of cleanliness in the care institution, purchase of hygiene products, indirect costs, such as service costs (management, accounting, legal, human resources, training, recruitment and staff insurance costs, certification and authorisation costs, training and insurance costs for volunteers, child recruitment costs, costs of promotion and information on childcare facilities, bank account management and transfer costs), purchase of real estate (only for local administrative units – communes), construction or conversion of real estate (only for local administrative units – communes), purchase and installation of equipment (including, among other things, furniture, leisure equipment, sanitary equipment, kitchen equipment, toys), purchase of teaching and educational aids, specialised equipment and tools, purchase of aids for carrying out care and educational activities, specialist equipment and tools for diagnosing developmental and educational needs and psycho-physical capabilities of children, supporting development and carrying out therapy for children with special educational needs, with particular attention to these aids, equipment and tools, equipping and installing a playground with a safe surface. Creches and kids clubs are mostly non-public (Table A3.1). There are approximately three times as many private creches as public institutions, and the dimensions are similar for kids clubs. In terms of number of places, the differences are less pronounced, but private provision still dominates. The vast majority of home-based provision is private. Among children aged 4-7, most institutions are run by local municipalities, but 45.1 % of institutions remain privately run (Statistics Poland, 2020). 58 58 14 July 2022 Table A3.1. Distribution of public and private childcare institutions for children aged 0-3, 2022 Sector Creches Kids clubs Home-based provision TOTAL No of institutions No of places No of institutions No of places No of institutions No of places No of institutions No of places Public 1,119 74,019 152 3,185 106 520 1,377 77,724 Private 3,334 111,408 755 14,321 1,784 11,529 5,873 137,258 TOTAL 4,453 185,427 907 17,506 1,890 12,049 7,250 214,982 Source: National Creche Reporting System. able A3.1. Distribution of public and private childcare institutions for children aged 0-3, 2022 Creches and kids clubs operate mostly as private companies or are run by an NGO. 10 Typical fees in public creches vary between approximately PLN 500-800, so the co-financing for parents can amount to more than 50 %. Funding They have to comply with standards set in the Act of 4 February 2011 on the Care for children aged up to 3. At the same time, non-public entities react faster to growing demand. These institutions can apply for public grants from the Maluch+ Programme, and in all cases after fulfilling some requirements they are granted support. In the case of private care settings, financing is based mainly on fees for parents, supplemented by other sources. Communes can (but this is not an obligation) grant subsidies to private care settings, after meeting some specific conditions set by the commune. Another, completely separate, catalogue of subsidies are those that entitle parents to subsidise their children’s places in creches. From 1 April 2022 parents are able to apply for co-financing of their children’s time in institutional care up to PLN 40010 (provided they are not granted the Family Care Capital – another support measure – for a particular child). Such direct support for parents so far has Another, completely separate, catalogue of subsidies are those that entitle parents to subsidise their children’s places in creches. From 1 April 2022 parents are able to apply for co-financing of their children’s time in institutional care up to PLN 40010 (provided they are not granted the Family Care Capital – another support measure – for a particular child). Such direct support for parents so far has been available from the Maluch+ Programme, but it has depended on the availability of its funds. From April 2022 this possibility became a legal right coded in the proper Act (Bill). This subsidy will cover a significant part of the cost of childcare for children aged 0-3 but a portion of the cost will still be borne by parents. As shown in Table A3.2, in two-thirds of public institutions, fees are PLN 600 or lower, meaning that two-thirds or more of the cost of care will be covered by the subsidy. In contrast, among private institutions, the share of institutions with relatively low fees is smaller and fees can exceed PLN 1 000, so that the subsidy may be less effective. been available from the Maluch+ Programme, but it has depended on the availability of its funds. From April 2022 this possibility became a legal right coded in the proper Act (Bill). Funding This subsidy will cover a significant part of the cost of childcare for children aged 0-3 but a portion of the cost will still be borne by parents. As shown in Table A3.2, in two-thirds of public institutions, fees are PLN 600 or lower, meaning that two-thirds or more of the cost of care will be covered by the subsidy. In contrast, among private institutions, the share of institutions with relatively low fees is smaller and fees can exceed PLN 1 000, so that the subsidy may be less effective. 10 Typical fees in public creches vary between approximately PLN 500-800, so the co-financing for parents can amount to more than 50 %. 59 14 July 2022 Table A3.2. Monthly residence fees by type of institution, 2021 Fee Residence fee, monthly Public Private No of institutions No of places No of institutions No of places no fee* 84 4.7 326 5.4 2-300 579 33.8 276 8.9 301-600 543 32.0 1,192 32.0 601-900 34 0.5 1,420 35.3 901-1200 12 0.5 1,301 28.4 1201-1500 12 0.2 823 14.8 1501-1800 0 0 174 2.9 1801-2100 0 0 63 0.9 2101-2400 0 0 17 0.2 2400> 0 0 6 0.07 Total 1,264 71,622 5,598 128,853 Source: Polish Creche Register. Table A3.2. Monthly residence fees by type of institution, 2021 Additionally, parents can apply for co-financing from the company’s social benefits fund (but this possibility is only available for employed parents). In 2009, an amendment concerning labour law and company social benefit funds came into force. At that time, the definition of social activity that can be financed from the company social benefit funds was broadened – as this is what the funds are to be used for – and crèche care was introduced as one such purpose. Therefore, an employer may finance the participation of its employees’ children in creches from the company’s social benefits fund. The second, more complex, option (and therefore rarely used so far) is the possibility of financing the establishment of a company crèche. The employer must meet safety, hygiene, and fire protection criteria appropriate for creches. Even if they create a creche for a small group of their employees, this is quite a barrier. However, it is not the case that these funds are open and available in every case – the rules of the company’s social benefits fund is the deciding factor here. 11 The coverage rate calculated by the Polish Ministry of Family and Social Policy does not include children below 1 year old, because those children are rarely covered by institutional care. The parental leave (with high replacement rate) lasts 1 year and parents usually do not place children below 1 year old in institutional care. Funding Of course, this co-financing should apply to employees who are most in need according to economic and social criteria. But undoubtedly such a source exists and can be used. Parents can also be supported by all kinds of vouchers or subsidies organised by communes, but this is not a formal obligation, and depends on particular communes. However, there are plans to co- finance the access to institutional care for children up to 3 years’ old from the state budget on a regular basis (in the form of the legal act). 60 14 July 2022 14 July 2022 Care provided by nannies is based on a service agreement which, in accordance with the Civil Code, is subject to regulations applicable to civil law contracts (a ‘contract of mandate’). A nanny can be anyone who meets the following three conditions: (1) is not the parent of the child to be cared for, (2) is over 18 years of age, (3) has a sanitary-epidemiological qualification and a certificate of no impediment to work. A nanny can be a grandmother, a sister, a cousin of the child, or someone from outside the family, for example a neighbour, a student, a pensioner, or a person on a pre-retirement benefit. A nanny does not need to have any specialist education or professional experience of working with young children. Neither do they have to undergo special training. Public funding is available for hiring nannies. The National Social Security Institution pays contributions for nannies, including pension, disability pension, and accident and health insurance contributions. The Social Insurance Institution pays contributions on half of the minimum wage (100 % before 2018), the remaining part is paid by a person or persons (mostly parents) hiring nannies (as social security contribution payers). For example: you hire a nanny in January 2021 and give her a salary of PLN 3 000 (the minimum salary in 2021 was PLN 2 800). The Social Insurance Institution pays contributions on half of the minimum wage, i.e. PLN 1 400. You pay contributions on the remaining amount, i.e. on PLN 1 600 (PLN 3 000 minus PLN 1 400 equals PLN 1 600). Accessibility The way the system is constructed results in inequalities in access to institutional care provided for children up to 3 years’ old. Big and affluent communes provide better access to institutional care for this group of children by, for example, introducing special programmes such as the Warsaw creche voucher. According to the Ministry of Family and Social Policy, the coverage rate of institutional care for children aged 1 and 2 years old11 reached 28 % in 2021 (the rate does not take into account children below 1 year old). This is growing, but the demand is still not met. Although the number of children is decreasing there is a demand for new places of institutional care up to 3 years’ old. This suggests that the access to institutional care for children in this age group is not sufficient. There are still communes where no places of institutional care for children up to 3 years’ old exist at all (especially rural). 61 14 July 2022 14 July 2022 Since 2008, the existence of various types of barriers related to access to childcare (cost, distance, quality of care) is monitored in the Labour Force Survey. Figure A3.1 shows the share of women in age group 25-44 years experiencing these barriers by educational attainment. In general, women with low and medium education indicate higher barriers than those with tertiary education, especially in the case of the cost of childcare, but also in distance to childcare institutions. Significant reduction of barriers has been recorded for women with education below tertiary. Since 2008, the existence of various types of barriers related to access to childcare (cost, distance, quality of care) is monitored in the Labour Force Survey. Figure A3.1 shows the share of women in age group 25-44 years experiencing these barriers by educational attainment. In general, women with low and medium education indicate higher barriers than those with tertiary education, especially in the case of the cost of childcare, but also in distance to childcare institutions. Significant reduction of barriers has been recorded for women with education below tertiary. Figure A3.1. Share of women in age group 25-44 years experiencing barriers in access to childcare by educational attainment, 2008-2019 3.1. Share of women in age group 25-44 years experiencing barriers in access to childcare by nal attainment, 2008-2019 Figure A3.1. Accessibility Share of women in age group 25-44 years experiencing barriers in access to childcare by educational attainment, 2008-2019 a. Below tertiary education b. Tertiary education Source: Polish Labour Force Survey data. As a result of public policy aiming to improve access to ECEC, we can note that in 2019, 12.4 % children aged 0-2 were covered by the ECEC care, compared with 2.0 % in 2004 and 4.8 % in 2013 (Figure A3.2). At the same time, there are significant geographical differences - 18.4 % of children in urban areas but only 3.7 % of children in rural areas attend ECEC. 0 5 10 15 20 25 30 35 40 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Share of women in age gropu 25-44 years with below tertiary education distance cost quality 0 5 10 15 20 25 30 35 40 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Share of women in age gropu 25-44 years with tertiary education distance cost quality b. Tertiary education 0 5 10 15 20 25 30 35 40 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Share of women in age gropu 25-44 years with tertiary education distance cost quality b. Tertiary education 0 5 10 15 20 25 30 35 40 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Share of women in age gropu 25-44 years with tertiary education distance cost quality a. Below tertiary education Source: Polish Labour Force Survey data. 0 5 10 15 20 25 30 35 40 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Share of women in age gropu 25-44 years with below tertiary education distance cost quality Source: Polish Labour Force Survey data. As a result of public policy aiming to improve access to ECEC, we can note that in 2019, 12.4 % children aged 0-2 were covered by the ECEC care, compared with 2.0 % in 2004 and 4.8 % in 2013 (Figure A3.2). At the same time, there are significant geographical differences - 18.4 % of children in urban areas but only 3.7 % of children in rural areas attend ECEC. 62 14 July 2022 Figure A3.2. Participation of children in Early Childhood Education and Care in Poland, 2004-2019 Source: Statistics Poland. Scope of services The tasks of crèches and kids clubs are, in particular, to: The tasks of crèches and kids clubs are, in particular, to: • provide care to children in home-like conditions • provide proper nursing and educational care to children through the organisation of play activities with learning elements, while taking into consideration their individual needs • provide proper nursing and educational care to children through the organisation of play activities with learning elements, while taking into consideration their individual needs • organise care, educational and learning activities that are suitable for the age of children and the level of their physical and psychological development. The services provided by crèches include the following elements based on the norms for children of this age: meals; care and nursing; sleep and rest hygiene; indoor and outdoor educational and learning activities; activities preventing diseases and promoting health; corrective activities; and medical first aid. In accordance with the Act on the Care for Children up to the age of 3, a parents’ council can be established and operate in a crèche or kids club. The council may, for example, propose action, put forward suggestions and give opinions to the head and the administering body of a crèche or kids club in all matters relating to its activities, and, in particular its educational and learning activities. However, as it is for pre-school education, there is no national core curriculum. Eligibility criteria Crèches or home-based provision are for children aged at least 20 weeks, and kids clubs for children aged 1 year and above. Children can attend those settings until the end of the school year in which they reach the age of 3, or up to the age of 4 in cases where it is impossible or difficult for the child to participate in pre-school education. There is no single national rule for crèche or kids club recruitment. It depends on the institution and can be found on the website of the given institution, city or commune office. Accessibility 0 10 20 30 40 50 60 70 80 90 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 % of children in age group 0-2 years Figure A3.2. Participation of children in Early Childhood Education and Care in Poland, 2004-2019 0 10 20 30 40 50 60 70 80 90 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 % of children in age group 0-2 years Figure A3.2. Participation of children in Early Childhood Education and Care in Poland, 2004-2019 0 10 20 30 40 50 60 70 80 90 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 % of children in age group 0-2 years Figure A3.2. Participation of children in Early Childhood Education and Care in Poland, 2004-2019 Figure A3.2. Participation of children in Early Childhood Education and Care in Poland, 2004-2019 0 10 20 30 40 50 60 70 80 90 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 % of children in age group 0-2 years 62 14 July 2022 Learning and development The Act on the Care for Children up to the age of 3 does not recommend any teaching methods or aids. Activities in crèches are based on their organisational regulations, which specify the institution's tasks. These include, in particular, the provision of care and education in safe and hygienic conditions that are necessary for children’s development; disease prevention and health promotion; and nursing care. In addition to care and education and nursing activities, crèches and kids clubs organise play activities with learning elements that are suited to children’s individual needs and the level of their physical and psychological development. Staff are free to choose teaching aids, while taking into consideration children’s age and development needs. Organisation of services The maximum number of children cared for by 1 childminder in a crèche or kids club is 8. However, the maximum number is 5 when a group includes a child with a disability, a child requiring special care or a child under 1 year of age. Crèches with more than 20 children employ at least 1 nurse or midwife. Crèches and kids clubs may have volunteers supporting the provision of childcare. Volunteers who do not have the necessary qualifications are required to complete 40 hours of training in baby/child first aid. The Act on the Care for Children up to the age of 3 does not specify the number of working hours per week for childcare institutions. Working hours in crèches and kids clubs are determined in their organisational regulations, taking into account parents’ preferences. Crèches and kids clubs provide care for up to 10 hours per day. In special cases, at parents’ request, the duration of the stay can be extended for an additional fee. For a daycare provider (home-based provision), working hours are set in an agreement between the provider and the employing entity. 63 14 July 2022 14 July 2022 14 July 2022 12 Based on Magda I. (2020), Increasing female labour market participation in Poland, IBS Policy Paper. https://www.researchgate.net/profile/Iga- Magda/publication/338656073_Increasing_female_labour_force_participation_in_Poland/links/5e21cd12a6fd cc1015715c0e/Increasing-female-labour-force-participation-in-Poland.pdf. Assessment The Act on the Care for Children up to the age of 3 does not provide the assessment for children attending crèches or kids-clubs. Crèche staff work with parents, providing on an ongoing basis information about children’s achievements, their problems and any worrying health symptoms. Crèches with more than 20 children employ at least one nurse or midwife. Crèches and kids clubs may have volunteers supporting the provision of childcare. Volunteers who do not have the necessary qualifications are required to complete 40 hours of training in baby/child first aid. The following types of child carers can be employed in crèches and children’s clubs: The following types of child carers can be employed in crèches and children’s clubs: • a person with the qualifications of a nurse, midwife, childminder, pre-school teacher, as well as early school education or pedagogical specialists in early care, social care, early education, and a pedagogical therapist • a person who graduated from a study programme (or a postgraduate study programme) in the following areas: early development support, child development support in the framework of psychological and pedagogical support in crèches and nursery schools, pro-development education, young child pedagogy, child psychology, psychology of support to development and education, care psychology. The persons employed in crèches and children’s clubs can also hold the following qualifications provided they have completed a specialist child first aid course less than 2 years before undertaking employment: The persons employed in crèches and children’s clubs can also hold the following qualifications provided they have completed a specialist child first aid course less than 2 years before undertaking employment: 1. any type of higher education that includes aspects of childcare and child development, and completed 80 hours of additional training, 2. secondary or vocational secondary education with at least 1 year’s experience in work with children aged below 3 or before employment in ECEC has undergone 280 hours of training, including 80 hours of practical training (looking after a young child under supervision). 64 14 July 2022 14 July 2022 A childminder should complete 160 hours of training that includes such topics as early childhood development, stimulating comprehensive development, childminders’ competences, and practical training. Assessment If the childminder has the qualifications of a nurse, midwife, child carer, pre-school teacher, as well as early school education or pedagogical specialists in early care, social care, early education, and a pedagogical therapist or is a person who graduated from a study programme (or a postgraduate study programme) in the following areas: early development support, child development support in the framework of psychological and pedagogical support in creches and nursery schools, pro- development education, young child pedagogy, child psychology, psychology of support development and education, care psychology, they are obliged to undergo 40 hours of training that includes first aid offered to children, stress management, problem solving, and innovation methods of stimulating a child’s development. Child carers employed in creches and children’s clubs and childminders are employed on the basis of general employment regulations (Labour Code). There is a growing problem in recruiting young people to work in institutional forms of childcare. Besides staff requirements there are precise conditions on premises and nutrition. Instrumental resources There are many causes of non-take-up of childcare facilities12: There are many causes of non-take-up of childcare facilities12: • Low availability of high-quality early education and care facilities of different forms (especially in rural areas with high spatial dispersal of children). Although the availability of early education and childcare facilities has greatly improved in Poland over the past two decades, still only 1 in 10 children aged 0-3 has a secured place at a nursery – while more than two- thirds of municipalities in Poland have no available places for children under 3. Alternative, more flexible forms of childcare provision (such as childminders) are growing dynamically, but their availability is still low. Another limitation to ECEC has been not just its unavailability, but its price (particularly when most available forms are private, i.e. not co-funded by municipal authorities). The quality of childcare is also important as well as the still persistent perception of the creche as a ‘child storage’ facility devised for the sole purpose of ‘pushing’ women into the labour market. Another obstacle are the limited hours when childcare is available, which preclude parents from flexibly reconciling work and family obligations. • Low availability of high-quality early education and care facilities of different forms (especially in rural areas with high spatial dispersal of children). Assessment Although the availability of early education and childcare facilities has greatly improved in Poland over the past two decades, still only 1 in 10 children aged 0-3 has a secured place at a nursery – while more than two- thirds of municipalities in Poland have no available places for children under 3. Alternative, more flexible forms of childcare provision (such as childminders) are growing dynamically, but their availability is still low. Another limitation to ECEC has been not just its unavailability, but its price (particularly when most available forms are private, i.e. not co-funded by municipal authorities). The quality of childcare is also important as well as the still persistent perception of the creche as a ‘child storage’ facility devised for the sole purpose of ‘pushing’ women into the labour market. Another obstacle are the limited hours when childcare is available, which preclude parents from flexibly reconciling work and family obligations. • Labour market situations of mothers. Unattractive jobs with non-flexible working hours that require lengthy and costly commuting leads to mothers’ inactivity on the labour market that in turn results in non-take-up of ECEC. This is especially the case of mothers of two or more children. Only a small number of Polish women are free to decide about their working day’s start and finish times, which would enable them to reconcile work with childcare obligations. • Labour market situations of mothers. Unattractive jobs with non-flexible working hours that require lengthy and costly commuting leads to mothers’ inactivity on the labour market that in turn results in non-take-up of ECEC. This is especially the case of mothers of two or more children. Only a small number of Polish women are free to decide about their working day’s start and finish times, which would enable them to reconcile work with childcare obligations. 65 14 July 2022 12 Based on Magda I. (2020), Increasing female labour market participation in Poland, IBS Policy Paper. https://www.researchgate.net/profile/Iga- Magda/publication/338656073_Increasing_female_labour_force_participation_in_Poland/links/5e21cd12a6fd cc1015715c0e/Increasing-female-labour-force-participation-in-Poland.pdf. 65 14 July 2022 This option is available to 12 % of working women, compared with about 40 % in the EU-28 (Figure A3.3). The situation persists despite numerous demands and recommendations to make the labour market more flexible and introduce solutions that would be more conducive to reconciling work and family life. Most women in Poland have no opportunity to decide about their working hours (this share is twice as high than in the EU). Assessment Figure A3.3. Share of women aged 25-49 declaring their ability to use flexi-time due to childcare obligations re A3.3. Share of women aged 25-49 declaring their ability to use flexi-time due to childcare gations Figure A3.3. Share of women aged 25-49 declaring their ability to use flexi-time due to childcare obligations Source: Eurostat. Source: Eurostat. • Money issues. For some women, taking up work is unprofitable. This concerns, in particular, single mothers and women with potentially low wages. The tax and benefit system – set thresholds, amount of benefits and the rules of their withdrawal – means that taking a low- paid job would lower the total income of a household. This difference becomes even greater if we take into account the cost of childcare provision. Consequently, working becomes unprofitable in the short-term cost-benefit analysis (which fails to include long-term benefits of work, such as gaining experience or accumulating pension contributions). Moreover, unprofitability of work is enhanced by the Family 500+ allowance (PLN 500 paid for each child every month). Non-working mothers usually do not take up childcare facilities for children below the age of 5. • Cultural pressure and social norms influencing decisions about individual childcare provision. As a result, some women decide to withdraw from the labour market and devote themselves to household work and childcare, and thus do not use ECEC. Research fails to give a clear answer as to whether the share of women who make this decision in Poland is higher than in other EU countries. On the one hand, Polish society is generally viewed as more conservative. On the other hand, Poland – like other post-socialist CEE countries – traditionally had high women’s employment rates (strengthened by economic necessity). Moreover, a number of 66 14 July 2022 14 July 2022 signals indicate that social norms are evolving and Polish women are ever more frequently declaring their support for equality in terms of women’s labour market participation. signals indicate that social norms are evolving and Polish women are ever more frequently declaring their support for equality in terms of women’s labour market participation. • Family network providing care for children. Around 20 % of households with dependent children receive unpaid care from family members outside the household. In most cases the care provided by relatives can be considered a substitute for institutional care. • Family network providing care for children. Assessment Around 20 % of households with dependent children receive unpaid care from family members outside the household. In most cases the care provided by relatives can be considered a substitute for institutional care. Activities that facilitate the delivery and access to ECEC are undertaken locally, mainly in big cities. Many communes create separate websites for educational and care issues. For example, in Warsaw, each woman delivering a baby in hospital receives a layette for the baby. This includes small practical gifts and a book with all the necessary information. Moreover, for example, Warsaw provides a separate website (https://zlobki.waw.pl/) with information on crèches and a Warsaw crèche voucher. In the case of nursery schools there in an electronic system for recruitment in Warsaw. There are also provisions that facilitate the access of children with disabilities to ECEC. However, it is mainly available for children from 3 years’ old. Some crèches or kids club offer places for younger children with disabilities or special caring needs, but it does not create a coherent structure and is dependent on a particular unit. In Poland there are NGOs supporting parents with different services, and also legal ones. Two groups of such organisations can be distinguished. The first one refers to NGOs aimed at the general population of families with children, the second one consists of NGOs integrating parents of children with disabilities. However, they do not create a common structure, so the support provided by them is fragmented. Enforcement resources Enforcement resources Enforcement resources Public authorities are not obliged to ensure ECEC for children aged 0-3 years, and there are no remedy procedures. 67 67 14 July 2022 References References Alcidi C., Baiocco, S., Corti, F. and Di Salvo, M. (2021), Changing Social Investment Strategies in the EU, JRC working paper series, https://joint-research-centre.ec.europa.eu/system/files/2022- 01/jrc127769.pdf. 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https://openalex.org/W4311610154	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/s12889-022-14849-4	English	null	Conceptualisations of mental illness and stigma in Congolese, Arabic-speaking and Mandarin-speaking communities: a qualitative study	BMC public health	2,022	cc-by	11,885	Conceptualisations of mental illness and stigma in Congolese, Arabic‑speaking and Mandarin‑speaking communities: a qualitative study Shameran Slewa‑Younan1,2, Klimentina Krstanoska‑Blazeska1, Ilse Blignault1, Bingqin Li3, Nicola J Reavley2 and Andre M. N. Renzaho1 Shameran Slewa‑Younan1,2, Klimentina Krstanoska‑Blazeska1, Ilse Blignault1, Bingqin Li3, Nicola J Reavley2 and Andre M. N. Renzaho1 Abstract Background: Australia is an ethnically diverse nation. Research has demonstrated an elevated risk of developing a mental illness in culturally and linguistically diverse (CaLD) communities yet uptake of mental health services is low. To improve mental health treatment seeking and outcomes for CaLD individuals in Australia there is an urgent need to deeply understand barriers to treatment such as stigma. Using an exploratory qualitative approach, the aim of the study was to explore how CaLD communities’ conceptualise and interpret mental illness and associated beliefs and experiences of stigma. Methods: The study focused on three key CaLD groups: the Congolese, Arabic-speaking and Mandarin-speaking communities residing in Sydney, Australia. A series of eight focus group discussions (n = 51) and 26 key informant interviews were undertaken online using Zoom during the period of November to December 2021. Focus group discussions and key informant interviews were digitally recorded, transcribed, and analysed using NVivo software. Results: Three major themes were identified. The first theme related to mental illness terminology used in the three communities. Despite variation in the terms used to refer to ‘mental illness’ all three communities generally distin‑ guished between ‘mental illness’, a more severe condition and ‘mental health problems’, considered to arise due to stressors. The second theme centred on beliefs about mental illness; with all three communities identifying migration- related stressors as contributing to mental illness. Culturally related beliefs were noted for the Congolese participants with the perception of a link between mental illness and supernatural factors, whereas Mandarin-speaking partici‑ pants highlighted lack of inter and intrapersonal harmony and failure to adhere to values such as filial piety as contrib‑ uting to mental illness. The final theme related to mental illness related stigma and the various ways it manifested in the three communities including presence of collectivist public stigma felt across all three groups and affiliate (family) stigma reported by the Arabic and Mandarin-speaking groups. Slewa‑Younan et al. BMC Public Health (2022) 22:2353 https://doi.org/10.1186/s12889-022-14849-4 Slewa‑Younan et al. BMC Public Health (2022) 22:2353 https://doi.org/10.1186/s12889-022-14849-4 © Crown 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/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. *Correspondence: s.younan@westernsydney.edu.au 1 Translational Health Research Institute, School of Medicine, Western Sydney University, Campbelltown, Australia Full list of author information is available at the end of the article Introduction Australia is an extremely diverse nation with the most recent 2021 census data indicating just over half (51.5%) of Australians were born overseas (first generation migrant) or had a parent born overseas (second genera- tion migrant; [1]). The term Culturally and Linguistically Diverse (CaLD) is often used to refer to migrants from a non-English speaking background, excluding those from an Anglo-Saxon, Anglo Celtic, Aboriginal or Torres- Strait Islander ancestry [2]. Within Australia, two of the largest CaLD groups are those with Chinese and Arab ancestry and correspondingly, the two most commonly spoken languages in the home other than English in Aus- tralia are Mandarin (spoken by 2.7%) followed by Ara- bic (1.4%; [1]). Australia has multiple migration streams including Skilled pathway, the Family Stream and the Refugee and Humanitarian Program [3]. With respect to refugee and humanitarian entrants, Iraq and Syria have comprised the top source of individuals granted offshore humanitarian visas granted over the past seven years [4]. Another emerging migrant community in Australia comprises individuals from the Democratic Republic of the Congo (DRC), with Australia increasingly resettling those fleeing the worsening civil unrest and violence [5]. l Due to greater likelihood of exposure to potentially traumatic events such as torture, murder of family mem- bers and friends and other human rights violations, refu- gee humanitarian entrants are a particularly vulnerable subset of CaLD individuals [6]. They are at significantly greater risk of developing posttraumatic stress disor- der (PTSD) and major depressive disorder [7, 8] com- pared to the general Australian public often as result of increased exposure to trauma events. Post-migration, stressors such as family separation, loss of existing sup- ports, racism, socioeconomic difficulties, and language barriers pose further challenges to the mental health of all migrants irrespective of migration pathways [9–11]. Stigma toward people with mental illness is recog- nised as a significant barrier to seeking help and is asso- ciated with low self-esteem and reduced self-efficacy; a “double burden” when experiencing mental illness [24]. Mental illness related stigma is a complex construct and has been extensively researched and elaborated on since Goffman who first categorised it within a social framework as an “attribute that is deeply discrediting” [25]. . Open Access T Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 2 of 13 Conclusions: We found rich diversity in how these communities view and respond to mental illness. Our findings provide some possible insights on both service provision and the mental health system with a view to building effec‑ tive engagement and pathways to care. Keywords: Culturally and Linguistically Diverse, Mental illness, Mental Health, Stigma, Arabic-speaking, Congolese, Mandarin-speaking, Australia, Qualitative to non-CaLD, with individuals from the Middle East and Africa demonstrating low uptake of mental health ser- vices despite high reported rates of psychological distress [14]. Similarly, despite comparable rates of depression among Chinese and non-Chinese patients in Australia, Chinese immigrants have been shown to under-utilise mental health services compared to non-Chinese individ- uals [15]. Although CaLD individuals underutilise mental health services in the community, evidence suggest they are disproportionately admitted to mental health units [16]. Relatedly, there is evidence from studies examin- ing longitudinal data on resettled refugees in Australia to suggest that even when professional help is sought the average number of mental health sessions is less than the recommended treatment guidelines [17, 18]. In addition to structural barriers such as financial constraints, trans- portation difficulties and lack of culturally and linguisti- cally appropriate mental health services [19], differing knowledge and beliefs about mental illness have been suggested to contribute to lower levels of professional help-seeking among CaLD individuals [20]. Researchers have defined knowledge and beliefs about mental illness as well as attitudes toward mental illness as components of ‘mental health literacy’ [21]. Notably, mental illness related stigma has been postulated to play a significant role in CaLD communities engagement with mental health services [19, 22, 23]. Design and study contexth This exploratory qualitative research was couched within a larger study investigating views on mental health help-seeking including that of stigma. All research was performed in accordance with relevant guidelines and regulations of Western Sydney University Human Research Ethics Committee (approval number H14608). All researchers had qualifications in health, or a health‐ related discipline obtained in their country of origin, Australia or in both countries. Six bilingual health workers from the target commu- nities were employed and trained in the recruitment, screening, and interviewing processes. Triangulation of data sources (community members and community lead- ers) and methods (qualitative individual interviews and focus groups for each community group) contributed to rigour and enabled the researchers to explore a broad range of perspectives and make comparisons within and between the three CaLD communities. The COREQ 32 [37] consolidated criteria for qualitative research (Appen- dix A Table 1A) is utilised to report the current study. Current studyh There is limited research into conceptualisations of mental illness and associated beliefs and experiences of stigma, in the Congolese, Arabic-speaking and Man- darin-speaking communities in Australia, and to our knowledge, there is no study to date comparing the views of people in these three communities. Given that Man- darin and Arabic are the two most spoken languages in Australia other than English, and the emerging Congo- lese community, such an investigation is warranted to inform targeted stigma reduction initiatives and other mental health service provision for these specific CaLD communities. ‘What matters most’ framework for understanding culture‑specific aspects of stigmah i This study was informed by ‘What matters most’ frame- work to understand culture-specific aspects of stigma. Yang and colleagues (2014) postulated a theoretical approach for examining stigma across cultures based on ‘what matters most’ to individuals within a cultural group [27]. They argue that fundamental values that ‘matter most’ in cultures are preserved through every- day activities and social engagements. When an indi- vidual member of the group is unable to participate in such activities, it undermines their standing within the local group and, thus, endangers their ‘personhood’. This theory emerged from Yang et al. (2014) when they interviewed 50 Chinese immigrants living in New York City, USA with a lived experience of mental illness. In this study, it was noted that mental illness stigma was related to the degree to which they were able to par- ticipate in work given that the accumulation of finan- cial resources in that specific cultural context was ‘what mattered most’ [27]. Consequently, the Yang argues that ‘What matters most’ framework can assist in identifying culturally specific dimensions of stigma Introduction the form of affiliate stigma (internalised stigma felt by family or other associates) or courtesy stigma (public’s stigma towards family/associates of mental ill person) [25, 27, 28]. Research has demonstrated that although stigma is a universal experience, it can manifest dif- ferently across cultures and moral contexts [29]. For example, as a result of the collectivistic values that are inherent in African [30, 31], Chinese [32, 33], and Arabic-speaking communities [34] decisions are often made at a family or community level. A sense of self is interconnected with family wellbeing and decisions regarding mental health problems are made by the family/elders not just the individual. In societies that uphold collectivistic values, having a mental illness is a reflection on the person’s family and can bring the family shame. For example, affiliate stigma has been noted to be more pronounced in Chinese communities due to cultural values such as ‘face’ and tainted lineage concerns [27, 28]. Other factors that can influence how stigma is manifested across different CaLD groups are culturally related beliefs regarding the aetiology and risk factors for mental illness [35]. For example, the belief that mental illness is caused by demonic or spirit- ual possession commonly held across different African communities can lead to fearful emotional reactions and place solutions within the purview of traditional or religious leaders [36]. Introduction Generally, the term stigma comprises of multiple components including negative stereotypes (beliefs such as a person with mental illness is dangerous or weak in character), prejudice (emotional reactions such as anger with or fearful of person with mental illness), and discriminatory behaviour towards people with mental illness [26]. For a person living with mental ill- ness, when the public’s negative attitudes are internal- ised, this can result in self-stigma [26]. Stigma may also be felt by the family of a person with mental illness, in Mental health conditions and substance use disor- ders were amongst the top five causes of disease burden among Australians in 2018 [12]. There is good evidence to suggest that untreated mental illness and delayed help- seeking results in significant social and economic costs to the individual, health system, and society [13]. Nota- ble disparities exist between how CaLD groups in Aus- tralia engage and utilise mental health services compared Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 3 of 13 the form of affiliate stigma (internalised stigma felt by family or other associates) or courtesy stigma (public’s stigma towards family/associates of mental ill person) [25, 27, 28]. Research has demonstrated that although stigma is a universal experience, it can manifest dif- ferently across cultures and moral contexts [29]. For example, as a result of the collectivistic values that are inherent in African [30, 31], Chinese [32, 33], and Arabic-speaking communities [34] decisions are often made at a family or community level. A sense of self is interconnected with family wellbeing and decisions regarding mental health problems are made by the family/elders not just the individual. In societies that uphold collectivistic values, having a mental illness is a reflection on the person’s family and can bring the family shame. For example, affiliate stigma has been noted to be more pronounced in Chinese communities due to cultural values such as ‘face’ and tainted lineage concerns [27, 28]. Other factors that can influence how stigma is manifested across different CaLD groups are culturally related beliefs regarding the aetiology and risk factors for mental illness [35]. For example, the belief that mental illness is caused by demonic or spirit- ual possession commonly held across different African communities can lead to fearful emotional reactions and place solutions within the purview of traditional or religious leaders [36]. by exploring the interactions between cultural norms, roles, and values that impact personhood. Participants Recruitment was conducted using a combination of pur- posive and snowball sampling [38]. Participants were recruited using the networks and contacts of the rele- vant investigators (AR, BL, SSY) in addition to those of the bilingual health workers. Focus groups were drawn from existing networks and included males and females. In relation to recruitment for key informant interviews, these participants were identified through their involve- ment with the community via relevant community-based organisations. Following the dissemination of translated flyers on the study and via networks, interested par- ticipants were instructed to contact the bilingual health Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 4 of 13 Page 4 of 13 group discussions lasted approximately 90 min and at the end of each main section of the discussion, the bilin- gual health worker summarised the content to ensure the participants’ perspectives were obtained and interpreted correctly, a verification process that enhanced the credi- bility of the findings. The key informant interviews lasted between 20 to 40 min and were undertaken by a bilin- gual health worker using the interview guide. The inter- view guides included open ended questions formulated using a review of literature and input from the primary researchers. The interview guides which formed part of the larger qualitative research project focused on gener- ating an understanding of each community’s conceptuali- sation of mental illness, including beliefs about aetiology and risk, barriers to seeking treatment such as stigma and mental health help-seeking treatment preferences. These interview guides included open ended questions formu- lated using a review of literature and input from the pri- mary researchers, an approach successful used in past [39]. For the purposes of this current study, responses regarding conceptualisation of mental illness and mental illness related stigma are reported upon. All focus group discussion participants received a $30.00 electronic supermarket voucher as reimbursement for their time in participating in the research. workers. Then, bilingual health workers conducted screening procedures and preparation phone calls with interested potential participants. A relationship was established with participants prior to study commence- ment during the screening procedure and preparation phone calls. Participants were informed of reasons for doing the research at screening and at the beginning of the focus group discussions/informant interviews. Data analysis I i Interviews and focus group discussions were transcribed into English by the bilingual health workers for data analysis. The bilingual health workers listened to the audio recordings and subsequently translated the inter- views from the original language to English, with the added task of checking and re-checking the translations as required. Thematic analysis, using NVivo 12 software, was conducted in accordance with published guidelines for interpretative phenomenological analysis (IPA) [40] in order to understand the lived experiences of study partic- ipants and their views on mental illness and help-seeking and barriers including mental illness related stigma. IPA takes a phenomenological and interpretative approach in which it seeks to understand the subjective account of the phenomenon of those researched, but always recognising the centrality of the researcher’s own experiences and theoretical positioning in making sense of that account [41]. Analysis was conducted systematically in that the data from the Congolese group were analysed first, then the Arabic-speaking group, then the Mandarin- speaking group. Analysis was undertaken by KKB, SSY and an experienced research officer and bilingual health worker (YL). Regular weekly meetings were held to con- sider aspects of analysis including that of reflexivity, also addressed through note keeping. The bilingual health workers and AR were consulted continuously through Participants Participants were eligible to be part of focus group dis- cussions if they were Arabic speaking, Mandarin-speak- ing or Congolese individuals with lived experience of mental illness including those of carers, or family mem- bers. All participants were 18 years of age or older, resid- ing in Sydney and had arrived in Australia no more than seven years ago. However, some of the Mandarin-speak- ing community participants had arrived in Australia ear- lier than seven years but due to back-and-forth travel to China had not spent longer than a total of seven years residing in Australia. Participants were eligible to participate in the key informant interviews if they were identified as a leader within the three groups and had informed perspectives regarding their communities. This included both formal or informal roles with examples being community elders, religious leaders or holding role as community worker within an ethnic specific and/or trusted organisation. These participants were also required to be 18 years of age or older and residing in Sydney. Data collection A series of 8 focus group discussions (n = 51) and 26 key informant interviews with Arabic-speaking, Mandarin- speaking (Chinese) and Congolese community members and leaders residing in Sydney were conducted during November and December 2021. The bilingual health workers conducted the interviews or focus group dis- cussions. Due to the COVID-19 Delta outbreak in Syd- ney and restrictions, all focus group discussions and key informant interviews were conducted remotely using Zoom and digitally recorded. Each focus group discus- sion was facilitated by two bilingual health workers. The facilitator was not only central to the discussion and its moderation, but they also created a relaxed and conform- able Zoom environment to better manage relationships between unfamiliar participants. The second person’s role was to take notes in order to complement the audio files, managed the Zoom meeting by ensuring everyone partic- ipated, and to observe non-verbal interactions and group dynamics. Prior to the focus group discussion, sociode- mographic data of consented participants was collected via a preliminary phone call by a bilingual health worker, during which they also ensured the participant has access to Zoom and understood how to operate it. The focus Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 5 of 13 Page 5 of 13 Page 5 of 13 Slewa‑Younan et al. BMC Public Health (2022) 22:2353 the process to clarify transcriptions and words. This pro- cess was not linear but moved back and forth between members of the team who transcribed (bilingual health workers) and those who were conducting analysis. Cod- ing was conducted separately by YL and KKB with con- sistency and discrepancies resolved at weekly supervision meetings with SSY. Inter-rater agreement was assessed using quadratic weighted Gwet’s AC1 [42]. Gwet’s AC1 and percentage of agreement were 0.88 (95%CI: 0.56, 1.00) and 0.89 (95%CI: 0.63, 1.00) respectively, suggest- ing excellent agreement. Initial codes were then grouped into categories. The fourth stage was to search for con- nections between categories thus generating themes and subthemes. This process was then repeated for the next transcript. Once analysis had been completed for each transcript, a final master list of themes was generated. At this final stage, a thematic analysis across the interview types and the community groups was undertaken to gen- erated the final results. Data collection In line with the broad principles for IPA rigour, findings were presented by SSY and KKB at regular meetings with the primary researchers (IB, AR, BL and NR) as a form of peer critique where descriptive validity and transparency of interpretation was sought [41]. Resultsh The socio-demographic characteristics of the participants are presented in Table 1. A total of 77 individuals partici- pated in the study. Three Arabic-speaking focus group discussions with 6 participants in each were conducted. Three Mandarin-speaking focus group discussions, with 6 participants, 7 participants, and 8 participants, respec- tively, were conducted. The majority of focus group discussion participants in the Arabic-speaking and Man- darin-speaking communities were female and fell within the 18 to ≥ 50 age range. Two Congolese focus group dis- cussions with 4 participants and 8 participants, respec- tively, were conducted. Most focus group discussion participants in the Congolese community were male and most fell within the 30 years or younger age range. With regard to key informant interviews, all participants were aged 30 years or older. # YM and TH facilitated the Arabic-speaking focus group discussions, YM facilitated the Arabic-speaking key informant interviews ^ YL and JL facilitated Mandarin-speaking focus group discussions, YL facilitated the Mandarin-speaking key informant interviews a AR and VM facilitated the Congolese community focus group discussions, NZ facilitated the Congolese community key informant int Theme 1: mental illness terminologyh In this case, Mandarin-speaking peo- ple try to protect themselves from others and try not to let other people know they have a mental illness.” (Mandarin FGD2, Female 60) “The experience in Australia, for instance, someone who’s driving and get a fine, they don’t know how to pay, or they lose their driving license, develop men- tal health because you lose employment, you don’t know how to cope, all those things are associated to mental health.” (Congolese Interview, Community Leader, Male, 50) This distinction in the Arabic-speaking participants is important to note because there was a strong belief that mental illness was a hopeless, chronic and an incurable condition. One focus participant stated: “Our supports are very limited. Not only do we need to support our children to go out and come back, but we also need to do chores and our work and every- thing for our career. We could easily get frustrated. But, then, what does it look like in China? There are many other supporting hands under the base hands, including relatives, parents, parents-in-law, and grandparents.” (Mandarin Interview, Mental Health Worker, Female, 30+) “In our society, the mentally ill is considered a hope- less condition and will never be treated or recovered. They will not accept it as a common disease, make it a shameful situation.” (Arabic FGD1, Female, 35) By holding such views, individuals are less likely to see value in seeking help leading to possibility of a worsening in outcomes and prognosis over time and thus reinforc- ing perceptions of incurability. “We are new here in Australia, and we get annoyed because we don’t know the language and peo- ple make fun of you because of it.” (Arabic FGD3, Female, 53) “We do not think the mentally ill person will recover. But we trust that this person once mentally ill, will be forever ill!” (Arabic FGD2, Female, 47) “We do not think the mentally ill person will recover. But we trust that this person once mentally ill, will be forever ill!” (Arabic FGD2, Female, 47) Changes to the traditional family structure and distinct gender roles, which were clearly differentiated in Africa, following migration were described by some Congolese focus group participants as contributing to lowered self- esteem and loss of identity for males. As a result, a sen- timent that mental illness disproportionally affect males more compared to females was expressed. Theme 1: mental illness terminologyh There was considerable variation in mental illness related terminology used across all three communities in both the focus group discussions and key informant inter- views. Participants utilised various terms to refer to ‘mental illness’ and used these interchangeably with no Table 1 Sociodemographic characteristics of participants # YM and TH facilitated the Arabic-speaking focus group discussions, YM facilitated the Arabic-speaking key informant interviews ^ YL and JL facilitated Mandarin-speaking focus group discussions, YL facilitated the Mandarin-speaking key informant interviews a AR and VM facilitated the Congolese community focus group discussions, NZ facilitated the Congolese community key informant interviews b Iraq, Jordan, Egypt, Lebanon, Syria c DRC, Rwanda, Nigeria Arabic-speaking Community# Mandarin-speaking Community^ Congolese Communitya Focus group discussions Key informant Interviews Focus group discussions Key informant Interviews Focus group discussions Key informant Interviews Characteristics n n n n n n Gender Male 5 7 6 2 8 4 Female 13 3 15 6 4 4 Age Group 18-30 3 - 1 - 9 - 30-39 4 - 6 2 2 - 40-49 7 2 3 3 - 4 ≥50 4 8 11 3 1 4 Country of Origin Middle Eastb 18 9 - - - - Australia - 1 - - - - China - - 21 8 - - Sub-Saharan Africac - - - - 12 8 Years in Australia <5 14 - 6 - 7 - ≥5 4 - 15 - 5 - ^ YL and JL facilitated Mandarin-speaking focus group discussions, YL facilitated the Mandarin-speaking key informant interviews a AR and VM facilitated the Congolese community focus group discussions, NZ facilitated the Congolese community key informant interviews b Iraq Jordan Egypt Lebanon Syria Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 6 of 13 Page 6 of 13 Theme 2: cultural health beliefs consistency. However, it was clear that all three com- munities generally distinguished between ‘mental illness’, a more severe condition and ‘mental health problems’ or ‘mental issues’, commonly considered to arise due to stressors. A notion that mental illness manifests when stress exceeds coping ability was discussed by all three commu- nities, with resettlement challenges such as unemploy- ment, loss of support structures, language barriers and so on consistently mentioned as either contributing to the development or worsening of mental illness. “They are afraid that a mental health problem is a mental illness. Theme 1: mental illness terminologyh And you can see that based on the trauma that they had Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 7 of 13 Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Slewa‑Younan et al. BMC Public Health (2022) 22:2353 in the refugee camp, there was not much support. So they carry the trauma and when they arrive to Aus- tralia, in a new country, they’re again traumatized by moving from Congo to the refugee camp, from the refugee camp to Australia, so there is that shock and trauma.” (Congolese Interview, Community Leader, Male, 50) The importance of inter and intra personal harmony and disruptions to such were highlighted as causing men- tal illness amongst the Mandarin-speaking participants. Disharmony in the family unit, and in particular the par- ent–child relationship, was linked to cultural tensions that may arise due to cultural and generational differ- ences. For example, how a child for example adheres to values such as filial piety. Participants noted that seniors especially will attribute their emotional distress to their children not adhering to cultural values rather than label- ling their emotional distress as mental illness. The impact of prolong trauma was also highlighted. “I just want to point out that all the communities are affiliated, like the Iraqi communities, whether they are Muslims, Assyrian, Chaldean, Mandean etc, they live in the same community, the same shell, and almost have the same mental health status. All of such happens as a result of wars consequences, forced displacement effects, the effects of mental stress, and as a result of everything that happened before 2003 and after 2003. These are all accumula- tions of the difficult conditions that have been expe- rienced by this region and its people.” (Arabic Inter- view, Community Worker & Religious Leader, Male, 59) “I once have met a family, and I am very close to that family. The parents have always told me that their children were unfilial and asked them to do many things. Sometimes, they quarrel with each other badly…For young people, " If I have a bad temper, I may have mental health problems". The young people are willing to see a psychologist, but for the seniors, they won’t. The seniors always say, "I am this kind of person. My children are supposed to be kind to me. They are supposed to do this and that". “Whenever this person is distant from his spiritual and religion, the more he will suffer from mental problems” (Arabic Interview, Community Leader, Male, 56) Theme 1: mental illness terminologyh They blame a lot of the problems on their chil- dren and refuse to admit what problems they have. Maybe they are irritable, but they just won’t admit that they may have mental health issues.” (Manda- rin Interview, Community & Organisation Leader, Male, 70+) Beyond migration related stressors, the Congolese and Mandarin-speaking communities discussed the role of other factors in developing mental illness that had more culturally specific origins. Both focus group participants and leaders in the Congolese community acknowledged that when an individual is experiencing mental illness, some people may attribute it to supernatural causes. Words such as ‘mapepo’ (Swahili for demonic posses- sion), witchcraft, and poison were used to refer to super- natural causes. The participants highlighted that such a belief is commonplace not only amongst some Pastors, but also family members and friends. Other more culture-specific beliefs about mental ill- ness were offered again emphasising the importance of harmony, this time within the individual. “In Mandarin-speaking people’s concept, they always think I may be off-balance. They use ’off balance’ to explain many symptoms, whether it’s physical or psychological.” (Mandarin Interview, Mental Health Worker, Female, 30+) “Because when somebody is experiencing something like that, it means the family would start to think it witchcraft rather than thinking it mental illness. Which is a big problem.” (Congolese FGD1, Male, 59) Other attributes applied to those with mental ill- ness seemingly suggested unpredictability and personal responsibility. By contrast, supernatural or spiritual causes were rarely described in the Arabic-speaking focus group discus- sions, which is inconsistent with previous research that has demonstrated its prevalence in Arabic-speaking society [10]. However, disengagement from religion or absence of spiritual comfort was highlighted as contrib- uting to the development or exacerbation of mental ill- ness by some Arabic- speaking leaders. “They want to do one thing, but they will change their mind in a minute. Or they’ve made a decision, but then they will overturn their decision quickly. This decision must be wrong. There are some new problems. In short, they blow hot and cold.” (Manda- rin FGD1, Female, 60+) “Whenever this person is distant from his spiritual and religion, the more he will suffer from mental problems” (Arabic Interview, Community Leader, Male, 56) Theme 1: mental illness terminologyh An interesting tension emerged in the views of the Congolese community with respect to mental illness. Both community leaders and focus group participants discussed the widespread traditional belief that mental illness is taboo and as such does not exist. and yet symp- toms of mental illness were frequently described. “There is this state of suspicion in my community that people first of all don’t recognise that mental health [illness] exists.” (Congolese Interview, Health Care Worker Male 44) “There is this state of suspicion in my community that people first of all don’t recognise that mental health [illness] exists.” (Congolese Interview, Health Care Worker Male 44) “When you get here in Australia, you have this thing where, I don’t think it necessary a bad thing women too can go and work. They can also go and do these things. This is well and good but then the fact that women can do these stuffs, the role of a men start to get degraded” (Congolese FGD2, Male, 21) “When you get here in Australia, you have this thing where, I don’t think it necessary a bad thing women too can go and work. They can also go and do these things. This is well and good but then the fact that women can do these stuffs, the role of a men start to get degraded” (Congolese FGD2, Male, 21) However, symptoms of mental illness, thought to be observable and usually not concealable to others were frequently described. In addition to stressors associated with resettlement, the Congolese and Arabic-speaking participants dis- cussed the additional unique challenges associated with being from a refugee and asylum-seeker background with the role of trauma featuring strongly as a contributing to mental illness. “They [people with mental illness] act different from normal people” (Congolese FGD1, Female, 19). Overall this variation in terminology when discuss- ing mental illness provides valuable insight into pre- ferred expressions of mental distress. In particular, it is clear that there is a strong attachment of stigma to the label of ‘mental illness’ in all three communities. While greater contextualisation is required to identified pre- ferred terms when working within communities, it would seem that ‘mental health problems’ was more digestible to participants. “Congolese migrated to Australia, most of them came through offshore humanitarian settlement, they are refugees, they are already traumatized. Theme 3: stigma and its variationsh The issue of stigma was commonly raised by participants in all three CaLD groups as a significant barrier to help- seeking. Common to all three communities was that Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 8 of 13 openly speaking about or identifying mental illness was considered ‘taboo’. “Maybe they are afraid of losing face. The traditional mindset is that domestic shame should not be made public.” (Mandarin FGD1, Female, 39) “Our misunderstanding that mental health illness is a taboo topic or a secretive issue, no one should know about it or talk to anyone about it because we still consider mental health or psychological issues are madness.” (Arabic Interview, Community Leader, Male, 68) Relatedly, was the belief that mental illness is associ- ated with a loss of occupational or social functioning and therefore threatens social capital and reputation. There- fore, mental illness is often denied because it is associ- ated with losing ‘face’ and reputation in the community. is a taboo topic or a secretive issue, no one should know about it or talk to anyone about it because we still consider mental health or psychological issues are madness.” (Arabic Interview, Community Leader, Male, 68) “You are born you grow up you never hear about a certain something [mental illness], they start telling you about that thing you feel like “no,” they feel like it is something like kind of taboo.” (Congolese Inter- view, Community Leader, Female 41) “In addition, it is not easy for people to speak up about their problems. It’s like a taboo for Chinese, right?” (Mandarin FGD2, Female, 69) “You are born you grow up you never hear about a certain something [mental illness], they start telling you about that thing you feel like “no,” they feel like it is something like kind of taboo.” (Congolese Inter- view, Community Leader, Female 41) “In addition, it is not easy for people to speak up about their problems. Theme 3: stigma and its variationsh It’s like a taboo for Chinese, right?” (Mandarin FGD2, Female, 69) “You are born you grow up you never hear about a certain something [mental illness], they start telling you about that thing you feel like “no,” they feel like it is something like kind of taboo.” (Congolese Inter- view, Community Leader, Female 41) “One important thing is that if someone has been diagnosed with mental illness, they will not tell us, and their children will not tell us either, as many Mandarin-speaking people care about their ‘face.’” (Mandarin Interview, Community Organisation Leader, Male, 70+) Due to the general belief that religion and commu- nity leaders play an important role in addressing men- tal illness in the Congolese community, the importance of trust was highlighted. Some focus group participants who expressed a concern that when mental illness is addressed at this wider network level it may lead to gos- sip and invasion of privacy. However, other more specific manifestation of stigma seemed to be influenced not only by the communities conceptualisation of mental illness but also by their tradi- tional values. In this way the ‘what matters most’ theoret- ical framework [27] is useful to operationalise these more culture-specific aspects of stigma and their contextual environment effect.h “You could be depressed but you will be wondering by yourself who can I talk to, who can I trust? To tell how I am feeling? You know, you are scared?” (Con- golese FGD1, Female, 19) f The importance of family reputation and honour in the Arabic-speaking community is strongly upheld [43]. Consequently, mental illness is seen as leaving a ‘mark’ on one’s identity as well as their family’s identity and thus bringing dishonour and ‘Aár’ (Arabic word for shame related to mental illness)—to the individual and family. Consequently, participants reported families will hide the individual with mental illness or presence of mental ill- ness in the family from the community to maintain repu- tation and status. Thus self-isolation and withdrawal was seen as a pos- sible response to evade stigmatisation. Theme 3: stigma and its variationsh “That is why is so hard to identify people who are mentally ill in the Congolese community, because you won’t know if this person is staying home because they are depressed or because they’re run- ning away from gossip.” (Congolese FGD1, Female, 19) “His family tries to hide it from the community, they are aware of any rumours that could spread because of this and the reputation will be that this person in the family is mentally ill.” (Arabic FGD2, Female, 34) This strong adherence to religion to alleviate mental ill- ness, was also seen by some of the younger focus group participants as minimising and dismissive. Participants alluded to the strong presence of affiliate stigma in the Arabic-speaking community, that is, the extension of stigma to the family members of the person with mental illness, further motivating need for secrecy. “I think people come to you and tell you these things [mental health issues] straight out they don’t they don’t really hide it but I think – this might sound bad I like to be a rational person usually – I think that religion yeah kind of cloud our judgement. How does it cloud our judgement? I come to you and tell you aunty life, life is difficult I am going through all these stuffs and at school I am not doing well. You know our child, God is there go there and there. You know they start making it about prayers...we use religion to dismiss it.” (Congolese FGD2, Male, 21) “The mentally ill person daughters or sisters will lose the opportunity in getting married even his relatives will lose a good chance in getting married” (Arabic Interview, Community Leader, Male, 56) “The mentally ill person daughters or sisters will lose the opportunity in getting married even his relatives will lose a good chance in getting married” (Arabic Interview, Community Leader, Male, 56) A view that disclosing mental illness is associated with losing ‘face’, and that such a ‘domestic shame’ should not be made public was reported by the Mandarin-speaking focus group participants and leaders. Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 9 of 13 Page 9 of 13 Overall, we found support for the role of traditional beliefs regarding the factors thought to contribute or exacerbate mental illness, which was in line with previ- ous research. Discussion Previous research has demonstrated that stigma towards people with mental illness is common in CaLD commu- nities in Australia and this is supported by the findings of the current study in relation to the Arabic-speaking, Mandarin-speaking, and Congolese communities. Our study provided further insights into the conceptualisa- tions of mental illness and how such conceptualisations may be related to stigma. Notably, we found variations in terminology across all three communities when refer- ring to mental illness. However, in spite of this variation of terms used by participants, it was clear that all three communities generally distinguished between ‘mental illness’ and ‘mental health problems’, with the former denoting more severe conditions and the latter term denoting more common conditions such as anxiety and depression, a finding not dissimilar to that noted in the general Australian public [44]. We also found that migra- tion-related stressors were highlighted by all three com- munities as playing an important role in contributing if not causing mental illness. This is consistent with previ- ous research where factors such as separation from fam- ily, limited English proficiency and experiences of racism can be associated with increased psychological distress and vulnerability to mental illness [9–11]. As such stigma reduction initiatives for CaLD communities should be cognisant of the language and terminology used, with our findings suggesting community members found a focus on specific symptoms such as “stress” more digestible than diagnostic labels as it related to their universal expe- riences of migration. However, further research is needed to understand the implications of incorporating a more psychosocial explanation on measures of stigma. This is especially important as evidence is increasingly dem- onstrating the unintended outcome from public health campaigns in the general public where biomedical mod- els to explain mental illness in an effort to reduce blame can lead to increase aversion and perceptions of danger- ousness [45] g g In relation to the role of stigma in the three communi- ties, again similarities and differences emerged. From the Congolese interviews and focus group discussions, pub- lic stigma and internalised stigma was strongly evident, with family stigma less so. A systematic review by Misra and colleagues [50] found that among 18 studies involv- ing Black Americans, internalised and public stigma were more commonly reported compared to family stigma. Furthermore, we found a culture-specific aspect of stigma related to religion and the role it played in care. Theme 3: stigma and its variationsh Similar to the current findings, Chinese migrants living in Melbourne pointed to psychosocial problems such as life stress and interpersonal conflict as causes of depression [46]. An emphasis on the role of being ‘off balance’, in other words, having an imbalance of ‘qi’ (Chinese term for vital energy) as contributing to mental illness was also noted in the Mandarin-speaking community findings, consistent with traditional Chinese medical view of mental illness [47]. Within the Congo- lese participants, the influence of supernatural beliefs such as demonic possession and witchcraft were noted, consistent with previous research where such factors were reported to cause mental illness [48]. Such beliefs were endorsed to a lesser extent by younger participants, who expressed a desire for a broader perspective of men- tal health, which may point to the role of acculturation in changing views. However, the finding that the Arabic- speaking community members did not report supernat- ural or religious causes of mental illness, is inconsistent with previous research which has found that religious and supernatural attributions of mental illness were com- mon among Arabic-speaking individuals and related to higher levels of stigma [43, 49]. While religion is well rec- ognised as playing a significant role in Arabic-speaking communities, individuals differ in levels of religiosity and this may account for our unexpected finding. Con- sequently, our findings suggest that while cultural beliefs play an important role in how CaLD communities per- ceive and conceptualisation mental illness, the rich diver- sity of these communities is evident and should not be neglected when working in this space. Finally, pride and the failure to espouse the traditional roles of African men as being the provider and ‘a strong person’ was highlighted by community leaders as having a significant impact on men not disclosing their mental illness. “Like men, for example, with men feel proud of themselves rather than coming up and say I’m suf- fering from this, they would rather keep quiet, and the more they keep quiet, that particular mental health [illness] keeps eating them up.” (Congolese Interview, Health Care Worker, Male 44) Discussion For example, the belief that men- tal illness is usually not concealable indicates there may be lack of knowledge that concealable mental illness, typically mild or moderate mental illness that does not impede functioning, exists within the Congolese commu- nity. Furthermore, there may be minimal opportunities to learn that most common mental illness is conceal- able. This is because individuals who choose to disclose their concealable mental illness may be dismissed, lead- ing to less contact opportunities with such individuals, and further reinforcing the stereotype that mental illness is usually observable and extreme. Increasing contact with persons with mild and concealable mental illness may help to disconfirm such culturally specific stereo- types [50]. Other factors which need to be considered is the role of religion and how some participants perceived responses such as prayer and spiritual guidance as being dismissive of experiences of mental illness. These are all complex factors that require further research as they will greatly impact the creation and effectiveness of stigma reduction interventions for the Congolese community in Australia. The belief expressed by the Arabic-speak- ing participants that mental illness is hopelessness and incurable can be explicitly addressed by increasing con- tact with individuals who can challenge the stereotype of what recovery and treatment can look like for someone with mental illness and thus contribute to positive attitu- dinal changes [56]. Within the Arabic-speaking community public, fam- ily, and internalised stigma were noted. Importantly, there was a belief that mental illness as a condition is associated with hopelessness and incurability. Sayed [53] noted there is an implicit assumption in the expectations of Arab patients when it pertains to therapeutic doc- tor/patient relationships, leading to a sense of passivity towards treatment and the omniscient perception of the doctor to “cure” one’s illness, which is at odds with the collaborative process which defines much of the West- ern psychotherapeutic approaches and invites the client to be an active agent in their healing. The cultural asso- ciation that mental illness is incurable and hopeless poses questions about the meaning and concept of recovery or healing in the Arabic-speaking community as well as the expectations held towards professional help-seeking sources. The influence of both pre-migration and post- migration experiences that may influence concept of healing and or recovery require further investigation. Discussion In line with Yang and colleagues [27] research summarising culture-specific aspects of stigma in African American groups, the authors note that the value placed on strong ties to faith and self-reliance contributes to the belief that mental illness can be addressed by religious institu- tions and church-based coping or prayer [27, 51]. Such behaviour may also be related to the structural inequities in health care faced by African individuals and low faith in the cultural competency and helpfulness of Western Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 10 of 13 Page 10 of 13 from such is viewed as a “symbolic threat to societal behaviour” [54]. Similarly, the value placed on regulating one’s behaviour to maintain harmony was also evident in aspects of stigma in the Mandarin-speaking participants. The concept of filial piety, and deviation from, played a role in how Mandarin-speaking seniors viewed mental illness. Another culture-specific aspect of stigma related to the Chinese community was the concept of ‘face’ and the relationship between mental illness and loss of repu- tation, a finding that has been previously reported [27].hi mental health services. A sense of disillusionment with the Australian system and loss of traditional roles may also hinder engagement with the broader Australian health system. Recent research into the barriers to men- tal health help-seeking among African migrants in South Australia also found there was a lack of perceived ben- efits of mental health care which was perceived as cultur- ally incongruent [48, 52]. Furthermore, the construct of gender was found to be related to culture-specific aspects of stigma. Challenges to the traditional family structure and the distinct gendered role of being the breadwinner posed as threats to ‘personhood’ and were acutely felt aspects of stigma for some males. tat o , ai d g t at as bee p ev ous y epo ted [ 7]. The current study’s findings have some implications for the creation and implementation of stigma reduction initiatives in these three communities. There is strong evidence to suggest that education campaigns that incor- porate contact with people with lived experience of men- tal illness can help correct myths and misconceptions as well as dispel stereotypes associated with mental illness that impede help-seeking [55]. However, such campaigns require targeted education accompanied with well sup- ported contact opportunities developed to address the specific beliefs and associated manifestations of stigma in the communities. Conclusions O fi d There are several limitations to note when interpreting the current findings. Although we used a triangulation approach to explore broad perspectives, the sociodemo- graphic characteristics of participants should guide inter- pretation of the findings and impact the generalisability of the results. Participants resided in metropolitan Syd- ney and the generalisability of our results to community members residing outside the Sydney region are limited, in addition to individuals residing in Australia for longer periods of time. Although recruitment was enhanced through a combination of purposive and snowball sam- pling using the networks and contacts of the investigators and bilingual health workers, the possibility remains that individuals who are more active members in the com- munity such as those who attend Church groups were recruited as opposed to members less active in commu- nity networks. Participants voluntarily participated in the research knowing it was investigating mental health and stigma. This may mean the subset of people we included in the study are individuals who hold more open attitudes toward mental health and illness and a desire to learn more about mental health or illness or have had more contact with people with mental illness compared to indi- viduals in the community who may not have volunteered. Furthermore, there is a possibility of social desirability bias in the results given key informant interviews were conducted with community and religious leaders whose views are well regarded in their respective community. In terms of methodological limitations, participants did not have the opportunity to review and check transcripts due to time and funding constraints, however interview- ers did summarise the content of what was discussed at Our findings provide crucial insights into how Arabic- speaking, Mandarin- speaking and Congolese commu- nities in Australia view mental illness, including beliefs on risk factors and the role such views have on stigma. Across all three groups, we found migration-related stressors both pre and post arrival to Australia, were commonly reported as contributing to mental illness, while the influence of culturally related health beliefs featured more prominently in the Mandarin-speaking and Congolese groups. Finally, stigma was a commonly reported in the three communities although the manifes- tation and consequences of stigma differed. Understand- ing the richly diverse nuances in how CaLD communities view and respond to mental illness is the necessary first step, service providers and mental health systems require in order to build effective engagement and pathways to care. Discussion The role of family (affiliate) stigma played an important part in motivating the need for secrecy because the value of individual, and by extension, the family’s reputation in Arab culture is impacted by mental illness. There is some suggestion that such secrecy may provide a protective function and buffer against the negative consequences that may arise in case the individual self-discloses infor- mation which can be shameful and damaging to the moral status of the individual and family unit and threat- ens sense of belonging in the community. Future research may investigate the association between authoritarian attitudes and actions toward family members with men- tal illness and independent/interdependent self-construal among Arabic-speaking individuals. Types of public, internalised and family stigma were also reported by the Mandarin-speaking participants. In terms of public stigma, specifically stereotypes and beliefs about mental illness such as notions of personal respon- sibility were evident as well as a component of unpredict- ability of mental illness. Thus the highly regarded cultural values of self-reliance and restraint means that deviation Addressing the challenges of mental illness stigma in CaLD communities requires systems approach with cli- nicians and community organisations playing important role in reducing stigma. For example, service providers Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Page 11 of 13 Page 11 of 13 should be mindful of how they utilise and apply psychiat- ric labels when working with individuals from these three communities. The provision of a psychoeducation session which takes a Western biomedical approach with a focus on psychiatric labels and biochemical causal model may not be salient to their views or may further risk stigma- tising individuals with mental illness when considered in the context of their cultural beliefs and experiences. The finding that participants were reluctant to apply the label ‘mental illness’ when describing mental illness and their personal experiences also needs to be considered when creating stigma reduction initiatives. While avoiding the label mental illness to encourage uptake and acceptabil- ity of stigma reduction interventions carries the risk of further perpetuating stigma, such an approach may need to be employed in the initial stages of stigma reduction initiatives until attitudes start to shift and stereotypes of mental illness (e.g., mental illness is a hopeless and incur- able condition) are challenged. the end of each of the main sections of the interviews to ensure the participants’ perspectives were obtained. Discussion Notwithstanding these limitations, the current study had several strengths. Firstly, the research team and chief investigators were well integrated in the respective communities allowing trust. Secondly, the interviews and focus group discussions were held in the respec- tive language for each community which allowed for the inclusion of newly-arrived individuals who may have otherwise been excluded due to low English proficiency. The use of online technology (Zoom) whilst in the midst of a prolonged COVID-19 related lockdown is another strength and goes towards demonstrating innovation and adaptability of both the research team and participants [57]. Finally, in undertaking community consultations by training bilingual health workers, we were able to build capacity within the CaLD groups and foster advocacy skills. Conclusions O fi d Future research using large scale survey methods are required to examine the extent to which these find- ings apply to target communities elsewhere in Australia including regional locations. Abbreviation CaLD: Culturally and linguistically diverse. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12889-022-14849-4. Additional file 1: Appendix A. Table A1. Additional file 2. Interview Guides. Abbreviation CaLD: Culturally and linguistically diverse. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12889-022-14849-4. Additional file 1: Appendix A. Table A1. Additional file 2. Interview Guides. Abbreviation CaLD: Culturally and linguistically diverse. Funding y 13. The Lancet Global Health. Mental health matters. Lancet Glob Health. 2020;8(11):e1352. https://doi.org/10.1016/S2214-109X(20)30432-0. 13. The Lancet Global Health. Mental health matters. Lancet Glob Health. 2020;8(11):e1352. https://doi.org/10.1016/S2214-109X(20)30432-0. Funding for this research was provided by Mental Health Australia Ltd as part of the Embrace Multicultural Mental Health Project. 14. Boufous S, Silove D, Bauman A, Steel Z. Disability and health service utili‑ zation associated with psychological distress: the influence of ethnicity. Ment Health Serv Res. 2005;7(3):171–9. 14. Boufous S, Silove D, Bauman A, Steel Z. Disability and health service utili‑ zation associated with psychological distress: the influence of ethnicity. Ment Health Serv Res. 2005;7(3):171–9. Acknowledgements 7. Alhasnawi S, Sadik S, Rasheed MO, Baban AL, Al-Alak MM, Othman AY, et al. The prevalence and correlates of DSM-IV disorders in the Iraq Men‑ tal Health Survey (IMHS). World Psychiatry. 2009;8(2):97–109. This study could not have been completed without the community leaders and members from the Congolese, Arabic-speaking, and Mandarin-speaking communities, who generously participated and shared their time and views. The topics discussed were often sensitive and personal and yet their capacity to share, motivated by the desire to help their community, was nothing short of inspiring. We would also like to acknowledge the talented bilingual health workers who assisted with facilitating the interviews and focus groups; Dr Yaser Mohammad, Dr Yiran Li, Noel Zihabamwe, Tahani Husari, Jocelyn Yu and Victoria Mbuto. This study could not have been completed without the community leaders and members from the Congolese, Arabic-speaking, and Mandarin-speaking communities, who generously participated and shared their time and views. The topics discussed were often sensitive and personal and yet their capacity to share, motivated by the desire to help their community, was nothing short of inspiring. We would also like to acknowledge the talented bilingual health 8. Slewa-Younan S, Mond JM, Bussion E, Melkonian M, Mohammad Y, Dover H, et al. Psychological trauma and help seeking behaviour amongst reset‑ tled Iraqi refugees in attending English tuition classes in Australia. Int J Ment Health Syst. 2015;9(5):1–6. 9. Kirmayer LJ, Narasiah L, Munoz M, Rashid M, Ryder AG, Guzder J, et al. Common mental health problems in immigrants and refugees: general approach in primary care. CMAJ. 2011;183(12):E959–67. workers who assisted with facilitating the interviews and focus groups; Dr Yaser Mohammad, Dr Yiran Li, Noel Zihabamwe, Tahani Husari, Jocelyn Yu and Victoria Mbuto. 10. Liddell BJ, Batch N, Hellyer S, Bulnes-Diez M, Kamte A, Klassen C, et al. Understanding the effects of being separated from family on refugees in Australia: a qualitative study. Aust N Z J Public Health. 2022;46(5):647–53. 10. Liddell BJ, Batch N, Hellyer S, Bulnes-Diez M, Kamte A, Klassen C, et al. Understanding the effects of being separated from family on refugees in Australia: a qualitative study. Aust N Z J Public Health. 2022;46(5):647–53. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12889-022-14849-4. Additional file 1: Appendix A. Table A1. Additional file 2. Interview Guides. Page 12 of 13 Page 12 of 13 Page 12 of 13 Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Slewa‑Younan et al. BMC Public Health (2022) 22:2353 Declarations 17. Tomasi A, Slewa-Younan S, Narchal R, Rioseco P. Professional Mental Health Help-Seeking Amongst Afghan and Iraqi Refugees in Australia: Understanding Predictors Five Years Post Resettlement. Int J Environ Res Public Health. 2022;19(3):1896. Consent for publication Not applicable. 19. Byrow Y, Pajak R, Specker P, Nickerson A. Perceptions of mental health and perceived barriers to mental health help-seeking amongst refugees: A systematic review. Clin Psychol Rev. 2020;75:101812. Availability of data and materials The data sets are not publicly available as they contain information that could potentially re-identify individuals but are available from SSY upon reasonable request and with relevant ethical approval. 15. Chan YF, Quine S. 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